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The effect of triamcinolone acetonide on collagen synthesis by human and mouse dermal fibroblasts in… Tan, Elaine Mei Li 1980

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THE EFFECT OF TRIAMCINOLONE ACETONIDE ON COLLAGEN SYNTHESIS BY HUMAN AND MOUSE DERMAL FIBROBLASTS IN CELL CULTURE by ELAINE MEI LI|TAN B. Sc., The Univ e r s i t y of B r i t i s h Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Di v i s i o n of Pharmaceutics and Biopharmaceutics) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA JANUARY 1980 ( ? ) Elaine Mei L i J a n , 1 9 8 0 i i In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements fo an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my written permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date SO, tffd - i i i -ABSTRACT G l u c o c o r t i c o i d s are known to a f f e c t metabolic a c t i v i t i e s of c e l l s . The mechanism of g l u c o c o r t i c o i d actions i n adult human dermal and mouse L-929 f i b r o b l a s t s have yet to be f u l l y a s c e r t a i n e d . This study endeavors to examine the e f f e c t s of one g l u c o c o r t i c o i d , triamcinolone acetonide, on c e l l u l a r p r o l i f e r a t i o n and c o l l a g e n synthesis and to compare such e f f e c t s i n the human and mouse c e l l l i n e s . C e l l u l a r p r o l i f e r a t i o n and c o l l a g e n s y n t h e s i s are analyzed and qu a n t i t a t e d by c e l l counts and s e l e c t i v e d i g e s t i o n of the p r o t e i n by b a c t e r i a l collagenase, r e s p e c t i v e l y . Further a n a l y s i s of c o l l a g e n synthesis i s provided by polyacrylamide g e l e l e c t r o p h o r e s i s . One-tenth triamcinolone acetonide per ml suppresses c e l l u l a r p r o l i f e r a t i o n of mouse L-929 f i b r o b l a s t s . P r o l i n e i n c o r p o r a t i o n i n t o t o t a l and collagenase-s e n s i t i v e p r o t e i n i s enhanced i n the c e l l l a y e r ; that of medium i s a l t e r e d i n c o n s i s t e n t l y . Polyacrylamide g e l e l e c t r o p h o r e s i s of proteins t r e a t e d w i t h pepsin show the a b o l i t i o n of t o t a l and c o l l a g e n a s e - s e n s i t i v e p r o t e i n i n the c e l l l a y e r . Aberrations i n h y d r o x y l a t i o n and/or deformation i n p h y s i c a l s t r u c t u r e of p r o t e i n may confer greater s u s c e p t i b i l i t y to pepsin d i g e s t i o n . C e l l u l a r p r o l i f e r a t i o n and p r o l i n e i n c o r p o r a t i o n i n t o t o t a l and collagenase-s e n s i t i v e p r o t e i n of adult human dermal f i b r o b l a s t s are a f f e c t e d i n c o n s i s t e n t l y by the same dose of triamcinolone acetonide. Except f o r the c o n s i s t e n t suppression of c e l l u l a r p r o l i f e r a t i o n i n the murine L-929 f i b r o b l a s t s by triamcinolone acetonide, a l l observations p e r t a i n i n g to human dermal f i b r o b l a s t s are incompatible w i t h those obtained by other workers. M a n i p u l a t i o n of c u l t u r e c o n d i t i o n s and g l u c o c o r t i c o i d t r e a t -ment d i c t a t e , to a la r g e extent, the k i n d of responses observed. This could account for the wide v a r i a b i l i t y and frequent c o n t r a d i c t o r y f i n d i n g s reported i n the l i t e r a t u r e . - i v -TABLE OF CONTENTS Page ABSTRACT i i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS i x INTRODUCTION 1 LITERATURE REVIEW 2 A. ADVERSE CLINICAL ASPECTS OF GLUCOCORTICOID THERAPY 2 B. STRUCTURE OF VARIOUS TOPICAL GLUCOCORTICOIDS 3 C. INTERACTION OF GLUCOCORTICOIDS WITH CELLULAR MECHANISMS 8 D. RELEVANCE OF COLLAGEN 9 E. BIOSYNTHESIS OF COLLAGEN 10 I. T r a n s c r i p t i o n and T r a n s l a t i o n 10 I I . H y droxylation 11 I I I . G l y c o s y l a t i o n 12 IV. Processing of Procollagen Molecules 12 V. Formation of C r o s s - l i n k s 14 F. PHYSICAL STRUCTURE OF COLLAGEN 15 G. DEGRADATION OF COLLAGEN 15 H. TYPES OF COLLAGEN 16 I. EXOGENOUS FACTORS AFFECTING CELLULAR PROLIFERATION AND COLLAGEN 18 METABOLISM I . Serum 18 I I . Ascorbate 19 I I I . Glutamine 20 J . COLLAGEN METABOLISM OF CELLS IN VITRO 21 I. Mouse L-929 F i b r o b l a s t s 22 I I . Human Dermal F i b r o b l a s t s 22 - v -K. GLUCOCORTICOID EFFECTS ON CELLULAR PROLIFERATION AND COLLAGEN 23 METABOLISM I . In V i t r o Studies 23 (a) c e l l c u l t u r e 23 ( i ) chick embryo tendon f i b r o b l a s t s 23 ( i i ) f o e t a l human dermal f i b r o b l a s t s 26 ( i i i ) adult human dermal f i b r o b l a s t s 29 ( i v ) mouse L-929 f i b r o b l a s t s 31 (b) organ c u l t u r e 32 ( I I ) In Vivo Studies 33 SUMMARY 38 APPROACH 41 MATERIALS AND METHODS 44 A. MATERIALS 44 B. CELL LINES 44 C. CELL CULTURE 45 D. CELL HARVESTING, COUNTING AND SIZING 46 E. ISOLATION OF RADIOACTIVE-LABELLED COLLAGEN AND TREATMENT WITH 47 COLLAGENASE F. PEPSINIZATION OF NATIVE COLLAGEN 48 G. IDENTIFICATION AND QUANTITATION OF PEPSINIZED l^C-LABELLED 48 COLLAGEN IN POLYACRYLAMIDE GELS H. ASSAY OF RADIOACTIVITY OF COLLAGEN 49 I . STATISTICAL ANALYSIS 50 EXPERIMENTAL AND RESULTS 51 A. CELL SIZE AND DISTRIBUTION 51 B. GLUCOCORTICOID EFFECTS ON CELLULAR PROLIFERATION AND PROLINE 56 INCORPORATION: COLLAGENASE-ASSAY STUDY I . Mouse L-929 F i b r o b l a s t s 56 I I . Human Dermal F i b r o b l a s t s 62 (a) short-term incubation w i t h triamcinolone acetonide 62 (b) long-term i n c u b a t i o n w i t h triamcinolone acetonide 67 - v i -C. DETERMINATION OF CONDITIONS FOR EFFICIENT COLLAGENASE DIGESTION 68 D. POLYACRYLAMIDE GEL ELECTROPHORETIC STUDIES 77 I . R a t i o n a l e 77 I I . Methodological Tests f or E f f e c t s of S t a i n i n g and Destai n i n g 77 Polyacrylamide Gels I I I . Recovery of R a d i o a c t i v i t y F o l l o w i n g Pepsin Treatment of 80 P r o t e i n s Containing Collagen IV. G l u c o c o r t i c o i d E f f e c t s on P r o l i n e I n c o r p o r a t i o n i n t o 88 Collagenous P r o t e i n s of Medium and C e l l u l a r Layer F r a c t i o n s  of Mouse L-929 F i b r o b l a s t s : Polyacrylamide Gel E l e c t r o - p h o r e t i c Studies (a) comparison of peaks of medium f r a c t i o n s of c o n t r o l and 88 triamcinolone acetonide-treated samples (b) comparison of peaks of c e l l u l a r f r a c t i o n s of c o n t r o l and 92 triamcinolone acetonide-treated samples (c) comparison of peaks of medium and c e l l u l a r f r a c t i o n s 92 DISCUSSION 93 SUMMARY AND CONCLUSION 101 BIBLIOGRAPHY 103 - v i i -LIST OF TABLES Table I , Parts 1 and 2: The e f f e c t of triamcinolone acetonide on 57 c e l l u l a r p r o l i f e r a t i o n and synth e s i s of c o l l a g e n a s e - s e n s i t i v e p r o t e i n of confluent mouse L-929 f i b r o b l a s t s Table I I , Parts A and B: The e f f e c t of triamcinolone acetonide on 63 c e l l u l a r p r o l i f e r a t i o n and on the i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n by adult human dermal f i b r o b l a s t s Table I I I , P a r t s A and B: The e f f e c t of prolonged exposure to tr i a m - 69 cinolone acetonide on c e l l u l a r p r o l i f e r a t i o n and the synthesis of c o l l a g e n a s e - s e n s i t i v e p r o t e i n of human dermal f i b r o b l a s t s Table IV: The a c t i v i t y of b a c t e r i a l collagenase at d i f f e r e n t concen- 73 t r a t i o n s and temperatures Table V: Concentration and temperature-dependent degradation of 75 ac e t y l a t e d c o l l a g e n by b a c t e r i a l collagenase Table VI: Recovery of r a d i o a c t i v e c o l l a g e n from stained and unstained 78 polyacrylamide gels Table V I I : Recovery of the r a d i o a c t i v i t y of proteins c o n t a i n i n g 82 col l a g e n from polyacrylamide gels Table V I I I : Recovery of r a d i o a c t i v i t y of pooled c e l l u l a r p r oteins from 86 polyacrylamide g e l e l e c t r o p h o r e s i s Table IX: Recovery of r a d i o a c t i v i t y of proteins c o n t a i n i n g c o l l a g e n 89 from polyacrylamide gels - v i n -LIST OF FIGURES F i g . 1:' Cyclopentanoperhydrophenanthrene nucleus 4 F i g . 2: Hydrocortisone 5 F i g . 3: Cortisone 6 F i g . 4: T o p i c a l s t e r o i d tree 7 F i g . 5: measured The d i s t r i b u t i o n of diameters of mouse L-929 f i b r o b l a s t s by micrometer 52 F i g . 6: The d i s t r i b u t i o n of diameters of human f i b r o b l a s t s measured by micrometer 54 F i g . 7: The d i s t r i b u t i o n of diameters of human f i b r o b l a s t s measured i n an e l e c t r o n i c p a r t i c l e counter 55 F i g . 8: Treatment of proteins c o n t a i n i n g c o l l a g e n i n c e l l u l a r l a y e r s f or polyacrylamide gel e l e c t r o p h o r e s i s . Procedure 1 81 F i g . 9: c e l l u l a r phoresis, Treatment of proteins c o n t a i n i n g c o l l a g e n i n medium and pooled f r a c t i o n s f o r a p p l i c a t i o n to polyacrylamide g e l e l e c t r o -Procedure 2 85 F i g . 10: c e l l u l a r Treatment of proteins c o n t a i n i n g c o l l a g e n i n medium and l a y e r f o r polyacrylamide gel e l e c t r o p h o r e s i s . Procedure 3 87 F i g . 11: c o l l a g e n Polyacrylamide g e l e l e c t r o p h o r e s i s of proteins c o n t a i n i n g of the medium f r a c t i o n s 90 F i g . 12: coll a g e n Polyacrylamide g e l e l e c t r o p h o r e s i s of proteins c o n t a i n i n g of the c e l l u l a r f r a c t i o n s 92 - i x -ACKNOWLEDGMENTS I would l i k e to express my g r a t i t u d e to the f o l l o w i n g people whose i n v a l u a b l e support and advice have made t h i s work p o s s i b l e . Mr. Jim Van A l s t i n e : Thank you for the time and care i n counting my c e l l s . Your ever-ready w i l l i n g n e s s to help was accepted and appreciated. Miss Beverly Grimmer: Your help and advice i n the lab were g r e a t l y appreciated but your f r i e n d s h i p was valued above a l l . Dr. R u n i k i s : I could always depend on your support and a s s i s t a n c e when I needed i t . Thank you. Dr. Pearce and Ms. Joyce Mathieson: There are not enough words adequate to express my g r a t i t u d e , respect and a f f e c t i o n f o r you. Your u n f a i l i n g h elp, support, encouragement and, most of a l l , concern have sustained me through many d i f f i c u l t times. My h e a r t f e l t thanks to you both. Joyce: The endless and p a i n f u l hours you spent on r e f i n i n g my t h e s i s have given i t some resemblance of order which otherwise would have l e f t i t i n the c h a o t i c s t a t e that i t was o r i g i n a l l y . Dr. Pearce: From you I've learned more about research than you could imagine. Your calm and cool cucumber-like l o g i c a l approach to problems i s g r e a t l y admired and has set an example for me. Drs. Pearce and R u n i k i s : You gave me a new lease on academic l i f e and my deep g r a t i t u d e to you w i l l remain always. F i n a l l y , to a l l the f e l l o w s who dragged me out to v o l l e y b a l l , you o f f e r e d me a chance to r e g a i n my s a n i t y a f t e r long and o f t e n exasperating hours of academic t o i l . Your endless "torments" w i l l be g r e a t l y missed. - 1 -INTRODUCTION T o p i c a l g l u c o c o r t i c o i d s e f f e c t a l t e r a t i o n s i n the p r o l i f e r a t i o n and col l a g e n metabolism of f i b r o b l a s t s i n v i v o and i n v i t r o (Jablonska et a l . , 1979; Ponec, 1977b). An attempt was made to p r e d i c t and rank the i n t r i n s i c anti-inflammatory a c t i v i t i e s of these s t e r o i d s by examining t h e i r e f f e c t on c e l l u l a r p r o l i f e r a t i o n . Indeed, B e r l i n e r and Ruhmann (1967) and Brotherton (1971) developed and claimed that t h e i r p a r a l l e l l i n e bioassay provided a c o r r e l a t i o n between the anti-inflammatory potencies and the i n h i b i t i o n of mouse L-929 f i b r o b l a s t growth r a t e . This bioassay has since f a l l e n i n disrepute because i t does not agree w i t h c l i n i c a l experience (Winter and Wilson, 1976). G l u c o c o r t i c o i d e f f e c t s on c o l l a g e n metabolism and the a s s o c i a t i o n w i t h dermal atrophy have not been f u l l y a s c e r t a i n e d . Dermal atrophy, thought to a r i s e from a l t e r a t i o n s i n c o l l a g e n metabolism, i s a common adverse phenomenon observed c l i n i c a l l y a f t e r prolonged and intense g l u c o c o r t i c o i d treatment (Snyder and Greenberg, 1977; Wilson, 1978). This work attempts to examine the e f f e c t s of a g l u c o c o r t i c o i d , t r i a m c i n o -lone acetonide, which has been known to cause dermal atrophy (Snyder and Greenberg, 1977), on c e l l u l a r p r o l i f e r a t i o n and c o l l a g e n s y n t h e s i s . Mouse L-929 and adu l t human dermal f i b r o b l a s t s are employed i n t h i s study, the former because g l u c o c o r t i c o i d e f f e c t s on t h e i r c o l l a g e n metabolism have yet to be examined by other i n v e s t i g a t o r s and the l a t t e r f o r th e i r - o b v i o u s relevance to dermal atrophy. - 2 -LITERATURE REVIEW A. ADVERSE CLINICAL ASPECTS OF GLUCOCORTICOID THERAPY During the past quarter century many dermatological d i s o r d e r s have been t r e a t e d w i t h t o p i c a l glucocorticoids. 1' Over the years, s t e r o i d s of i n c r e a s i n g anti-inflammatory potencies have been manufactured, dispensed, and used, freq u e n t l y for prolonged periods. The consequence i s the appearance, with greater frequency, of a wide range of d i v e r s e and adverse s i d e - e f f e c t s . Since the withdrawal of the g l u c o c o r t i c o i d p r e c i p i t a t e s a rebound inflammatory r e a c t i o n , therapy tends to continue, sometimes f o r s e v e r a l years. Occlusive dressings often exacerbate the adverse e f f e c t s (Burry, 1973). For ins t a n c e , d a i l y a p p l i c a t i o n s of hydrocortisone 17-butyrate w i t h o c c l u s i o n produce dermal atrophy ( K i r b y and Munro, 1976). Other adverse e f f e c t s include s t r i a e , t e l a n g i e c t a s i a , epidermal t h i n n i n g , depigmentation e s p e c i a l l y i n the bl a c k p o p u l a t i o n , p e r i o r a l d e r m a t i t i s , purpura, r o s a c e a - l i k e d e r m a t i t i s , u l c e r a t i o n , and erythema (Snyder and Greenberg, 1977). Dermal atrophy i s thought to be produced by an a b e r r a t i o n of c o l l a g e n metabolism (Jablonska, 1979). Undesirable systemic response, such as Cushing's syndrome, does occur r a r e l y . A s i n g l e nonocclusive dose of clobetasone propionate a p p l i e d to normal s k i n suppresses adrenal f u n c t i o n completely w i t h i n nine hours (Ortega et^ a l . , 1975). Although a t r o p h o g e n i c i t y has been associated w i t h f l u o r i n a t e d gluco-c o r t i c o i d s (Saarni and Hopsu-Havu, 1976; Stevanovic, 1972), c e r t a i n n o n f l u o r -inated g l u c o c o r t i c o i d s such as desonide and hydrocortisone 17-butyrate have a l s o been i m p l i c a t e d . Furthermore, betamethasone v a l e r a t e , a potent f l u o r -inated g l u c o c o r t i c o i d , i s le s s atrophogenic than e i t h e r of the foregoing compounds (Kligman and Frosch, 1979). Therefore, the presence of a h a l o -genated atom does not confer any greater atrophogenic a c t i v i t i e s (Winter and Wilson, 1976). - 3 -That the anti-inflammatory and the v a s o c o n s t r i c t i v e p r o p e r t i e s of t o p i c a l g l u c o c o r t i c o i d s are p a r a l l e l i s debatable ( C h i l d , 1968; Zaun, 1966). Although anti-inflammatory potency i s o f t e n associated w i t h the intense side e f f e c t s , the c o r r e l a t i o n i s not c o n s i s t e n t . Furthermore, the frequency and time of onset of these side e f f e c t s do not c o r r e l a t e w i t h the anti-inflammatory potency of the g l u c o c o r t i c o i d s (Kligman and Frosch, 1979). Of most importance i s the observation that there i s no c o n s i s t e n t c o r r e l a t i o n of t h e r a p e u t i c a l l y bene-f i c i a l b i o l o g i c a l responses w i t h the degree of a t r o p h o g e n i c i t y . An e x c e l l e n t example i s given by triamcinolone acetonide which produces an anti-inflammatory response of medium degree i n the human s k i n but causes atrophy comparable i n s e v e r i t y to that of the most atrophogenic g l u c o c o r t i c o i d (Kligman and Frosch, 1979). B. STRUCTURE OF VARIOUS TOPICAL GLUCOCORTICOIDS The b a s i c s t r u c t u r e of the g l u c o c o r t i c o i d i s a cyclopentanoperhydro-phenanthrene nucleus ( F i g . 1). C e r t a i n r a d i c a l s and functions appended to the b a s i c nucleus produce hydrocortisone ( F i g . 2). Such f u n c t i o n s confer a c t i v i t y to the s t e r o i d . The molecule i s rendered i n a c t i v e w i t h the e l i m i n a t i o n of these f u n c t i o n s . Cortisone i s i n a c t i v e t o p i c a l l y because the C - l l ketone i s not converted to C - l l hydroxyl i n s k i n (vide i n f r a ) ( F i g . 3). M o d i f i c a t i o n s of the hydrocortisone molecule to increase the anti-inflammatory potency include double bond at CI to C2, f l u o r i n e atoms at C6 and C9 (alpha p o s i t i o n ) , hydroxyl at C16 (alpha p o s i t i o n ) , acetonide group of the hydroxyls at C16 and C17, and e s t e r i f i c a t i o n of e i t h e r or both hydroxyls i n the C17 side chain ( F i g . 4) (Lee and Rapp, 1975). C e r t a i n r i n g s u b s t i t u e n t s , such as 11-hydroxyl groups, are determinants i n the b i n d i n g c a p a c i t y of the g l u c o c o r t i c o i d molecule to the receptors. R a d i c a l s such as the 21-hydroxyl groups have been known to - A -F I G . 1 : CYCiJLPENTArWPERHYDROWCNANTVIRENE - 5 -CH 2OH c=o PIG.2: HYDROCORTISONE - 6 -CH,OH I 2 716. 3: CORTISONE - 7 -CH 2 OH CO 0 J I JHYDROC CHjOH CO ^ r OH CHjOH HYDROCORTISONE " " \1 JL J - J L ^ * ^ ^ t T ^ ^ y HYDROCORTISONE e T V ^ V j ^ FLURANt C r V ^ V ^ BUTYRATE CHjOC C H 3 C r ^ ^ > T M E T H Y L fLURANDRENOLIDE CHjOH CO CH 2 OH CO C H , METHY PREDNISOLONE C H , A C E T A T E CH 2 OH CO , ^ c e f ^ ^ ^ P R E D N I S O L O N E © ^ v X ^ s ^ D E K t DESONIDE O ^ S s ^ ^ v ^ TRIAMCINOLONE HO. CHjOH CO _ ,CH, | T F T TRIAMCINOLONE f T ^ S ^ S ACETONIDE CHjOH CO .CH, V 0 ^ C H-FLUOCINOLONE ACETONIDE C H 2 CO 0?CH 3 -o-r>c< 3 C H , C H , PLUOCINONIDE ^ CH 2 OH CO C T V ^ N ^ BETAMETHASONE CH 2 OH co 9 - C H , - J L J L J BETAMETHASONE VALERATE BETAMETHASONE BENZOATE FIG. 4: TOPICAL STEROID TREE - 8 -a f f e c t the a l l o s t e r i c e q u i l i b r i u m of the receptors (Baxter and Forsham, 1972). Of the g l u c o c o r t i c o i d s seen i n Figure 4, hydrocortisone i s considered the l e a s t potent and f l u o c i n o n i d e the most potent anti-inflammatory g l u c o c o r t i -c o i d s . Ranking of other t o p i c a l g l u c o c o r t i c o i d s based on anti-inflammatory a c t i v i t y i s d i f f i c u l t . A c t i v i t y i s not merely a f u n c t i o n of chemical s t r u c -ture but includes the concentration and s o l u b i l i t y of the s t e r o i d i n the v e h i c l e , the type of v e h i c l e , and the p a r t i t i o n c o e f f i c i e n t of the s t e r o i d between the v e h i c l e s and the receptor phase which, i n s k i n , would be the stratum corneum (Lee and Rapp, 1975). C. INTERACTION OF TOPICAL GLUCOCORTICOIDS WITH CELLULAR MECHANISMS G l u c o c o r t i c o i d s penetrate the plasmalemma of the target c e l l s and assoc-i a t e w i t h s p e c i f i c , h i g h - a f f i n i t y , cytoplasmic receptor p r o t e i n s evoking conformational changes i n the receptor molecules ( B u l l e r and O'Malley, 1976). G l u c o c o r t i c o i d - s e n s i t i v e c e l l s possess cytoplasmic receptor s i t e s ; unresponsive ones contain few or none (Baxter and Forsham, 1972). Both human dermal and murine L-929 f i b r o b l a s t s possess g l u c o c o r t i c o i d receptors (Groyer et a l . , 1979). Upon a c t i v a t i o n , the g l u c o c o r t i c o i d - r e c e p t o r complexes move to nuclear acceptor s i t e s . A diminution of bi n d i n g receptor s i t e s and g l u c o c o r t i c o i d -receptor complexes i n the cytoplasm occurs concomitantly. G l u c o c o r t i c o i d r e s i s t a n c e has been a t t r i b u t e d to i n s u f f i c i e n t and i n e f f i c i e n t t r a n s l o c a t i o n of cytoplasmic g l u c o c o r t i c o i d - r e c e p t o r complexes to the nucleus. Several thousand s i t e s per c e l l are involved i n the h i g h - a f f i n i t y b i n d i n g of the g l u c o c o r t i c o i d - r e c e p t o r complexes to the chromatin of n u c l e i ( B u l l e r and O'Malley, 1976). - 9 -The b i o l o g i c a l responses e l i c i t e d by the g l u c o c o r t i c o i d s are i n f l u e n c e d by m u l t i p l e f a c t o r s . At those concentrations which would saturate t h e i r s p e c i f i c r eceptors, c e r t a i n g l u c o c o r t i c o i d s do e l i c i t an optimal response; w i t h others, the response i s only submaximal. The l a t t e r could be explained by b i n d i n g of the g l u c o c o r t i c o i d to both a c t i v e and i n a c t i v e conformations of the receptor s i t e s . The g l u c o c o r t i c o i d s showing maximal response bind only to the a c t i v e form of the receptor, thus generating an a l l o s t e r i c s h i f t i n e q u i l i b r i u m which, i n t u r n , binds a d d i t i o n a l s t e r o i d , thereby favouring a maximal response. B i o l o g i c a l l y unresponsive g l u c o c o r t i c o i d s bind only to the i n a c t i v e form of the receptor. At s u f f i c i e n t l y high concentrations, such s t e r o i d s are capable of i n h i b i t i n g c o m p e t i t i v e l y the b i n d i n g of the more a c t i v e g l u c o c o r t i c o i d s (Baxter and Forsham, 1972). D. RELEVANCE OF COLLAGEN Collagen i s the most abundant p r o t e i n i n the body, comprising one-third or one-half of the t o t a l p r o t e i n (Gay and M i l l e r , 1978). Collagen, not e l a s t i c f i b r e s , provides the greatest r e s i s t a n c e to t e a r i n g and deformation ( P i e r a r d and L a p i e r e , 1976). Collagen p a r t i c i p a t e s i n t i s s u e development and remodelling during growth, d i f f e r e n t i a t i o n , ageing, wound h e a l i n g as w e l l as i n p a t h o l o g i c a l d i s o r d e r s such as f i b r o s i s , c i r r h o s i s and v a s c u l a r diseases amongst others and, i n a more s p e c i f i c manner, i n c e l l attachment, chemotaxis and b i n d i n g of antigen-antibody complexes (Gay and M i l l e r , 1978). Although t h i s p r o t e i n i s capable of r e s t o r i n g t e n s i l e strength to a weakened area, over-production hinders t i s s u e m o b i l i t y by generation of excessive scar t i s s u e . The amount and type of c o l l a g e n synthesized, conserved or destroyed - 10 -are r e l a t e d to the extent and form of i n s u l t to the body; genetic make-up may a l s o be involved (Cohen et al., 1977). Collagen i s the p r i n c i p a l s t r u c t u r a l element of the dermis and bone, comprising 34% of the proteins i n the former and 57% i n the l a t t e r . The metabolic turnover i s very low i n the adult ( K l e i n , 1976). E. BIOSYNTHESIS OF COLLAGEN I. T r a n s c r i p t i o n and T r a n s l a t i o n The i n t r a c e l l u l a r b i o s y n t h e s i s of c o l l a g e n begins w i t h the ribosomes of the rough endoplasmic r e t i c u l u m . L i t t l e i s known about the t r a n s c r i p t i o n of the monocistronic mRNA which i s estimated to c o n t a i n 4,500 n u c l e o t i d e s ( F e s s l e r and F e s s l e r , 1979) and has a molecular weight greater than 1.5 x 10 ( K l e i n , 1976; F e s s l e r and F e s s l e r , 1979). U n l i k e other p r o t e i n s , hydroxyproline and hydroxylysine residues are not i n c o r p o r a t e d d i r e c t l y i n t o the nascent pro a chains. G l y c i n e , comprising 33% of the amino a c i d s , occupies every t h i r d p o s i t i o n of the t r i p e p t i d e , glycine-X-Y, the b a s i c amino a c i d sequence of 95% of the a chain. P r o l i n e or l y s i n e i s u s u a l l y p o s i t i o n e d i n the X - p o s i t i o n ; the p r o l i n e i n the Y - p o s i t i o n i s u s u a l l y hydroxylated ( K l e i n , 1976). The molecular weight of each pro a chain i s 1.5 x 10"*. Each chain i s composed of a collagenous p o r t i o n w i t h noncollagenous peptide extensions known as pro-peptides or r e g i s t r a t i o n peptides at the carboxy- and amino-terminal ends. These extensions possess few g l y c i n e , p r o l i n e and hydroxyproline r e s i d u e s , some cy s t e i n e and tryptophan residues and an abundance of a c i d i c amino acids ( M a r t i n et a l _ . , 1975). The carboxy-terminal propetide i s composed 4 of 200 to 300 amino acids and has a molecular weight of 3.5 x 10 . The 4 smaller ammo-terminal propeptide has a molecular weight of 2.0 x 10 and at - 11 -l e a s t 100 amino acids ( K l e i n , 1976; U i t t o and L i c h t e n s t e i n , 1976). The pro a chains, which are 40-50% l a r g e r than the a chains ( K i v i r i k k o and R i s t e l i , 1976), are extruded across the membrane of the endoplasmic r e t i c u l u m i n t o the ci s t e r n a e (Grant and Prockop, 1972; B a i l e y and Robins, 1976; Prockop et^ a l . , 1979). I I . Hydroxylation Collagen requires s e v e r a l post ribosomal m o d i f i c a t i o n s of the primary s t r u c t u r e . P r o l i n e and l y s i n e residues of the completed polypeptide chains are hydroxylated by the enzymes p r o l y l and l y s y l hydroxylase, r e s p e c t i v e l y , l o c a t e d i n the membrane and c i s t e r n a e of the endoplasmic r e t i c u l u m . Molecular oxygen, ferrous i r o n , a reducing agent, p r e f e r a b l y ascorbate, and a-keto-g l u t a r a t e ( K i v i r i k k o and R i s t e l i , 1976) are req u i r e d . One atom of oxygen i s incorporated i n t o hydroxyproline or hy d r o x y l y s i n e . Another atom of oxygen i s incorporated i n t o s u c c i n a t e , a byproduct of the decarboxylation of cx-keto-g l u t a r a t e ( K i v i r i k k o and R i s t e l i , 1976; Lee and Rapp, 1975). The ferrous i r o n c h e l a t o r , a , a ' - d i p y r i d y l , i n h i b i t s h y d r o x y l a t i o n ( K i v i r i k k o and R i s t e l i , 1976). Jackson (1970) b e l i e v e s that most of h y d r o x y l a t i o n occurs f o l l o w i n g the release of the pro a chains from the ribosomes. However, Grant and Prockop (1972) put f o r t h the view that h y d r o x y l a t i o n occurs w i t h i n the membrane of the endoplasmic r e t i c u l u m while the nascent chains are s t i l l lengthening. The current view i s that both h y d r o x y l a t i o n and g l y c o s y l a t i o n are i n i t i a t e d on the ribosomes during the generation of polypeptide chains and continue a f t e r r e l e a s e of the complete polypeptide chains i n t o the c i s t e r n a e of the endo-plasmic r e t i c u l u m (Oikarinen et a l . , 1976). These enzyme-mediated processes terminate upon formation of the t r i p l e h e l i x ( K i v i r i k k o and R i s t e l i , 1976). - 12 -The presence of hydroxyproline i n the procollagen ensures the i n t e g r i t y of the t r i p l e h e l i x (Rao and Adams, 1979) by c o n f e r r i n g thermal s t a b i l i t y to the molecule (Burjanadze, 1979). Hydroxylysine permits c r o s s l i n k i n g of the molecules a f t e r e x t r u s i o n i n t o the e x t r a c e l l u l a r matrix. Non-hydroxylated c o l l a g e n , s u s c e p t i b l e to thermal denaturation and p r o t e o l y s i s , gives r i s e to the weak t i s s u e s c h a r a c t e r i s t i c of scurvy ( K l e i n , 1976). I I I . G l y c o s y l a t i o n A f t e r h y d r o x y l a t i o n , s e v e r a l hydroxylysine residues are g l y c o s y l a t e d to form g a l a c t o s y l h y d r o x y l y s i n e and g l u c o s y l g a l a c t o s y l h y d r o x y l y s i n e . The sugar molecules are attached to the hy d r o x y l y s i n e s . G l y c o s y l a t i o n i s mediated by membrane-bound u r i d i n e diphosphate-galactosyl and u r i d i n e diphosphate-glucosyl t r a n s f e r a s e s ( K i v i r i k k o and R i s t e l i , 1976). Both enzymes re q u i r e manganese to ca t a l y z e the t r a n s f e r of the sugars from the corresponding u r i d i n e diphosphate glycoside to the hydroxyl groups of the hydroxylysine r e s i d u e s . The presence of the sugars i s thought to determine the s i z e and geometry of the c o l l a g e n f i b r i l s . Indeed, an inverse r e l a t i o n s h i p has been suggested between the amount of sugars and the diameter of the f i b r i l s (Grant, 1975). G l u c o s y l -g a l a c t o s y l h y d r o x y l y s i n e r e s i d u e s , more abundant than g a l a c t o s y l h y d r o x y l y s i n e i n human s k i n (Krane, 1975a), increase w i t h age from 30 to 75 years ( K l e i n , 1976). IV. Processing of Pr o c o l l a g e n Molecules Once released from the ribosomes, the pro a chains are converted to procollagen molecules. I n t e r c h a i n covalent d i s u l p h i d e bonding of the carboxy-terminal r e g i s t r a t i o n peptides permits chain a s s o c i a t i o n , proper - 13 -alignment of the pro a chains and the formation of the st a b l e t r i p l e h e l i x . The t r i p l e h e l i x c o n s i s t s of three pro a chains that have wound around each other to form a s u p e r c o i l (Hashimoto, 1978). The almost simultaneous formation of the i n t e r c h a i n bonds and the t r i p l e h e l i x occur before the procollagen molecules migrate from the c i s t e r n a e of the endoplasmic r e t i c u l u m to the G o l g i apparatus. The r e g i s t r a t i o n peptides prevent i n t r a c e l l u l a r f i b r e formation and f a c i l i t a t e the transport of the proc o l l a g e n molecules i n v e s i c l e s from the G o l g i apparatus to the plasmalemma ( K i v i r i k k o and R i s t e l i , 1976). Microtubules are thought to p a r t i c i p a t e i n the movement of pr o c o l l a g e n molecules to the plasmalemma p r i o r to e x t r u s i o n i n t o the e x t r a c e l l u l a r r e g i o n . Agents such as c o l c h i c i n e and v i n b l a s t i n e , which disaggregate microtubules, delay the s e c r e t i o n of the p r o t e i n ( B o r n s t e i n and E h r l i c h , 1973). As e x t r u s i o n occurs, the amino-terminal r e g i s t r a t i o n peptide i s enzym-a t i c a l l y cleaved en bloc by the amino-terminal procollagen endopeptidase to form p c - c o l l a g e n . The remaining r e g i s t r a t i o n peptide i s cleaved separately by the carboxy-terminal endopeptidase to produce tropocollagen. Both enzymes requir e calcium for t h e i r a c t i v i t y . Peptide chains of molecular weight 11,500 or 17,500 are released by amino-terminal peptidase while cleavage at the carboxy-terminus generates t r i m e r i c , d i s u l p h i d e - l i n k e d fragments of molecular weight 75,000. Tropocollagen molecules w i t h a molecular weight of 285,000 are each composed of three c o i l e d chains, each having a molecular weight of 95,000 daltons and a composition of about 1,050 amino acids i n a l i n e a r sequence (Gay and M i l l e r , 1978). - 14 -V. Formation of C r o s s - l i n k s Intermolecular covalent c r o s s - l i n k s between adjacent t r o p o c o l l a g e n monomers and the packing arrangement of tropocollagen i n the f i b r i l confer high t e n s i l e strength. The i n i t i a l step i n the formation of c r o s s - l i n k s requires o x i d a t i v e deamination of the -NR^ group of a s p e c i f i c l y s i n e or hydroxylysine residue to produce the corresponding aldehydes, a l l y s i n e or h y d r o x y a l l y s i n e ( M i l l e r , 1976). The hyd r o x y l y s i n e - d e r i v e d c r o s s - l i n k s are more s t a b l e than those derived from l y s i n e due to the formation of a s t a b l e keto form ( K i v i r i k k o and R i s t e l i , 1976). Aldehyde synthesis i s mediated by l y s y l amine oxidase which requires molecular oxygen, p y r i d o x a l phosphate and copper but i s i n h i b i t e d by copper c h e l a t o r s , p y r i d o x a l phosphate trapping reagents and lathyrogens such as b e t a - a m i n o p r o p i o n i t r i l e ( K i v i k i k k o and R i s t e l i , 1976); p e n i c i l l a m i n e , a copper c h e l a t o r i n h i b i t s p r i m a r i l y by binding to the aldehyde groups ( K l e i n , 1976). Intramolecular covalent c r o s s - l i n k s are formed by a l d o l condensation of two aldehyde groups near the amino-termini of two neighbouring a chains. Intermolecular c r o s s - l i n k s are derived from the condensation of aldehydes with the -NH2 group of l y s i n e or hydroxylysine on neighbouring c o l l a g e n molecules. Dehydrohydroxylysinonorleucine and dehydrodihydroxylysinonorleucine form the major i n t e r m o l e c u l a r c r o s s - l i n k s i n part as g a l a c t o s y l and glucosy-g a l a c t o s y l d e r i v a t i v e s . Dehydrohydroxylysinonorleucine a r i s e s from the condensation of l y s i n e and hydro x y l y s i n e - d e r i v e d aldehyde or of hyd r o x y l y s i n e and l y s i n e - d e r i v e d aldehyde; dehydrodihydroxylysinonorleucine from hydroxy-l y s i n e and hy d r o x y l y s i n e - d e r i v e d aldehyde. L y s i n o n o r l e u c i n e c r o s s - l i n k s a l s o e x i s t but are l e s s s t a b l e ( K i v i r i k k o and R i s t e l i , 1976). - 15 -F. PHYSICAL STRUCTURE OF COLLAGEN The fundamental u n i t of c o l l a g e n i s t r o p o c o l l a g e n , a rod-shaped molecule, measuring about 280 nm i n length and 1.0 to 1.5 nm i n diameter (Hashimoto, 1978). Each molecule i s comprised of three a chains; each chain winds upon i t s e l f to form a left-handed c o i l w i t h a p i t c h of 0.89 nm. A right-handed s u p e r h e l i x of p i t c h 8.7 nm i s generated with the twining of the three a chains (Hashimoto, 1978; V i i d i k , .1979). The aggregation of n a t i v e tropocollagen chains proceeds i n a " q u a r t e r -stagger" manner. Rather than being a l i g n e d so that the amino and carboxy t e r m i n i match between trop o c o l l a g e n molecules, each molecule i s d i s p l a c e d , by about one quarter of i t s length (Hashimoto, 1978; V i i d i k , 1979). G. DEGRADATION OF COLLAGEN Collagen degradation occurs by enzymatic d i g e s t i o n w i t h or without phagocytosis of the p r o t e i n . In v i v o , c o l l a g e n i s degraded by collagenase from n e u t r o p h i l i c granulocytes, f i b r o b l a s t s or macrophages, or by cathepsin B from lysosomes of f i b r o b l a s t s , chondrocytes or macrophages. F i b r o b l a s t s from human s k i n are capable of s y n t h e s i z i n g collagenase (Birkedale-Hansen et a l . , 1976; Bauer, 1977). This enzyme r e q u i r i n g calcium, with optimal a c t i v i t y at n e u t r a l or s l i g h t l y a l k a l i n e pH, cleaves n a t i v e tropocollagen three-quarters of the distance from i t s amino terminal end. The products are denatured at p h y s i o l o g i c a l temperatures and are degraded f u r t h e r by n o n - s p e c i f i c proteases (Birkedale-Hansen et a l . , 1976). Collagenase can be found i n s e v e r a l forms, a proenzyme or zymogen, a free a c t i v e form, or as a complex w i t h an i n h i b i t o r such as alpha 2-macroglobulin. The l a t e n t form of the enzyme i s more prevalent i n t i s s u e s where rat e of - 16 -synthesis i s greater than that of degradation, f o r i n s t a n c e , i n inflammation or remodelling (Krane, 1975b). Often c o l l a g e n i s phagocytosed by f i b r o b l a s t s and other c e l l s ( M i t c h e l l et a l . , 1978) p r i o r to l y s i s by the lysosomal proteases. However, phagocytosis may be a process independent of c o l l a g e n o l y s i s (Svoboda et_ a l . , 1979). More r e c e n t l y , another degradation process has been discovered. W i t h i n minutes of i t s s y n t h e s i s , about 20 to 40% of i n t r a c e l l u l a r c o l l a g e n has been shown to undergo degradation (Bienkowski e£ a l . , 1978a). Such r a p i d destruc-t i o n and the absence of collagenase a c t i v i t y argue against enzymatic or phago-c y t o t i c mechanisms. The newly synthesized c o l l a g e n may not be i n t a c t a or pro a chains. Bienkowski et ajL. (1978b) demonstrated that the s t r u c t u r e of the c o l l a g e n i s a f a c t o r i n i n t r a c e l l u l a r d e s t r u c t i o n as f o l l o w s : A z e t i d i n e -2 - c a r b o x y l i c a c i d , a lower homologue of p r o l i n e , was incorporated i n t o c o l l a g e n , the formation of the normal h e l i c a l conformation being prevented by the a z e t i d i n e r i n g . I n t r a c e l l u l a r degradation was elevated two-fold. Hence, these workers concluded that i n t r a c e l l u l a r degradation regulates not only the amount of p r o t e i n secreted e x t r a c e l l u l a r l y but the s t r u c t u r a l form of that p r o t e i n (Bienkowski, 1978b). H. TYPES OF COLLAGEN Four types of c o l l a g e n , I , I I , I I I and IV have become w e l l e s t a b l i s h e d ( K i v i r i k k o and R i s t e l i , 1976). Several new types, A, B, C, D, E ( M i l l e r , 1979) have been discovered r e c e n t l y ; l i t t l e i s known about types C, D and E. At l e a s t ten d i f f e r e n t genes are i m p l i c a t e d i n the synthesis of c o l l a g e n . Each type i s d i f f e r e n t i n amino a c i d content and sequence, the degree of h y d r o x y l a t i o n and the q u a n t i t y of carbohydrate. Type I , [ctl(I) ] , i s composed of two a 1 chains and one a 2 chain which d i f f e r s i n i t s amino a c i d composition from that of a l . Although u b i q u i t o u s , i t predominates i n adult human s k i n , bone and tendon ( U i t t o and L i c h t e n s t e i n , 1976). A trimer of type I , [al(l)]y n a s been found r e c e n t l y and i s ass o c i a t e d with inflammed t i s s u e s , l a t h y r i t i c chick embryo tendons and c a l v a r i a , and p o s s i b l y normal human s k i n ( U i t t o , 1979). Type I I , [ a l ( l l ) ] g , c o n s i s t s of three i d e n t i c a l a chains g e n e t i c a l l y d i s t i n c t from a l ( l ) and a2 and i s found i n h y a l i n e c a r t i l a g e ( U i t t o and L i c h t e n s t e i n , 1976) and more r e c e n t l y , v i t r e o u s body (Schmut £t a_l., 1979). L i k e type I I , type I I I , [ a l ( l l l ) ] . j > i s also composed of three i d e n t i c a l a chains with high contents of hydroxyproline, g l y c i n e and c y s t e i n e . I t i s ass o c i a t e d w i t h e x t e n s i b l e t i s s u e s . Although type I I I accounts f o r less than 20% of the t o t a l c o l l a g e n i n adult human s k i n , more than h a l f of the t o t a l c o l l a g e n i n human f o e t a l s k i n i s type I I I ( U i t t o and L i c h t e n s t e i n , 1976). The remainder i s type I . Type IV [ a l ( l V ) ] g 5 i s al s o composed of three s i m i l a r a chains and with A, B, C, D and E i s e x c l u s i v e to the basement membranes ( M i l l e r , 1979). Types I and I I I have few hydroxylysine and carbohydrate residues while types I I and IV possess a large q u a n t i t y of both. Types I and I I , u n l i k e types I I I and IV, do not have cysteine residues to form i n t e r c h a i n d i s u l p h i d e bonds ( V e i s , 1975). Type I e x i s t s as densely packed r o p e - l i k e f i b r i l s i n a h i g h l y ordered array. The f i b r i l s form r e a d i l y and provide high t e n s i l e strength (Hance and C r y s t a l , 1975). Type I I I i s composed of f i n e , p l i a b l e , randomly dispersed f i b r i l s a s s o c i a t e d c l o s e l y and l o o s e l y w i t h other components of connective t i s s u e (Perez-Tamayo, 1979); some i s present as r e t i c u l i n . - 18 -I . EXOGENOUS FACTORS AFFECTING CELLULAR PROLIFERATION AND COLLAGEN METABOLISM I. Serum Serum i s known to co n t a i n poorly c h a r a c t e r i z e d f a c t o r s e s s e n t i a l for c e l l u l a r movement, v i a b i l i t y and p r o l i f e r a t i o n . The mouse c e l l l i n e , L-929, appears to be an exception to most c e l l types i n that the generation times of these transformed f i b r o b l a s t s are unaltered by exposure to 10% (v/v) f o e t a l c a l f serum. However, these f i b r o b l a s t s synthesize 20 to 30% more p r o t e i n i n the presence of 10% f o e t a l c a l f serum than i n i t s absence (Nolan et a l . , 1978). Studies using normal g i n g i v a l f i b r o b l a s t s (Narayanan and Page, 1977) showed that the presence of serum can e f f e c t a four and seven-fold increase i n the i n c o r p o r a t i o n of l y s i n e and p r o l i n e r e s p e c t i v e l y , thus i n c r e a s i n g p r o t e i n and c o l l a g e n s y n t h e s i s . The i n c o r p o r a t i o n of p r o l i n e i n t o p r o t e i n i s maximal at 20% (v/v) f o e t a l c a l f serum; that of l y s i n e , at 5% ( v / v ) . The h y d r o x y l a t i o n of c o l l a g e n has been found to be enhanced by serum i n f i b r o b l a s t s of normal t i s s u e s (Bashey and Jimenez, 1977). Serum al s o a f f e c t s the type of c o l l a g e n synthesized. Using human g i n g i v a l f i b r o b l a s t s , synthesis of type I c o l l a g e n increases t h r e e - f o l d i n 20% (v/v) f o e t a l c a l f serum. In 10% (v/v) serum, type I I I production i s increased but only by 1.5 f o l d . Further increases i n serum concentration reverse the trend so that the r a t i o of type I I I to type I decreased. These observations were made i n comparison to serum-free c u l t u r e s (Narayanan and Page, 1977). Collagen degradation i s i n h i b i t e d by the presence of serum. Mammalian collagenases are i n h i b i t e d completely upon a d d i t i o n of 10% (v/v) f o e t a l c a l f serum (Bienkowski et a l . , 1978a). Serum contains a l - a n t i t r y p s i n and a2-macro-g l o b u l i n , which bind to and i n a c t i v a t e collagenases (Birkedale-Hansen et a l . , 1976). Although human d i p l o i d dermal and lung f i b r o b l a s t s have been found to - 19 -ingest e x t r a c e l l u l a r l y formed o^-macroglobulin-protease complexes, no s i g n i f i c a n c e i s attached to such endocytosis. P o s s i b l y , the proteases are l i b e r a t e d from the complex f o r r e - e x c r e t i o n by the c e l l s (Leuven e_t a l . , 1978). To summarize, serum-mediated m o d i f i c a t i o n s of p r o t e i n metabolism vary w i t h the o r i g i n and type of c e l l but such e f f e c t s have not been e l u c i d a t e d completely i n human dermal and mouse L-929 f i b r o b l a s t s . I I . Ascorbate Ascorbate functions as a c o f a c t o r of l y s y l hydroxylase and as c o f a c t o r and a c t i v a t o r of p r o l y l hydroxylase (Peterkofsky and Udenfriend, 1965). In a d d i t i o n , ascorbate stimulates c o l l a g e n s e c r e t i o n from the c e l l s . The r o l e of ascorbate a s an a c t i v a t i n g agent of p r o l y l hydroxylase i n mouse L-929 f i b r o b l a s t s was examined by Stassen e_t a l (1973). Ascorbate, added to c u l t u r e s i n l a t e l o g a r i t h m i c phase of growth, evoked a f i v e - f o l d increase i n the a c t i v i t y of p r o l y l hydroxylase; no dependence on RNA and p r o t e i n synthesis was noted. The p o s s i b i l i t y that the s t i m u l a t i o n of enzyme a c t i v i t y i n the presence of ascorbate stemmed from the a c t i v a t i o n of a pre-formed precursor received support from immunological experiments i n which a constant amount of p r o l y l hydroxylase precursor p r o t e i n was found. D i t h i o -t h r e i t o l converted q u a n t i t a t i v e l y the a c t i v e enzyme to smaller i n a c t i v e molecules. Treatment with ascorbate r e s t o r e d the a c t i v i t y and p r o p o r t i o n a t e l y decreased the concentrations of precursors. Therefore, i t was suggested t h a t , i n the presence of ascorbate, the e n z y m a t i c a l l y - i n a c t i v e subunits of p r o l y l hydroxylase (Stassen et a l . , 1973) aggregate to form an a c t i v e tetramer (Chen-Kiang et a l . , 1977). - 20 -L a t e r , Berg et^ a_l. (1976), using a d i f f e r e n t assay f o r p r o l y l hydroxy-l a t i o n , showed that ascorbate was incapable of a c t i v a t i n g p r o l y l hydroxylase i n mouse L-929 f i b r o b l a s t s . However, the lack of ascorbate d i d not appear to prevent completely the h y d r o x y l a t i o n of p r o l i n e (Nolan et a l . , 1978) nor a f f e c t c e l l u l a r p r o l i f e r a t i o n and rate of c o l l a g e n synthesis (Peterkofsky, 1972). The absence of gulonolactone oxidase i n L-929 c e l l s confirmed the i n a b i l i t y of these c e l l s to synthesize ascorbate (Nolan et a l . , 1978). Although c e r t a i n embryonic t i s s u e s , such as e a r l y chick blastoderm, embryonic mesenchyme and developing c a r t i l a g e , do synthesize ascorbate endogenously (Meier and Solursh, 1978), c e l l s derived from most adult t i s s u e s lose t h e i r c a p a c i t y to produce ascorbate; adult chicken kidney and l i v e r are exceptions. The a d d i t i o n of 1.78 Ug of ascorbate to 10^ c e l l s had the e f f e c t of hy d r o x y l a t i n g c o l l a g e n to 67%; t h i s was reduced to 19% i f only 50 ng ascorbate were added. Hence, although the absence of ascorbate d i d not i n h i b i t h y d r o x y l a t i o n completely, i t s presence d i d enhance the process (Nolan et a l . , 1978). I l l . Glutamine Glutamine i s present i n high concentration i n most t i s s u e c u l t u r e media, the r a t i o n a l e being i t i s the sole precursor of purine bases. Hence, i t pro-motes c e l l growth. Judged by i n c o r p o r a t i o n of t r i t i a t e d p r o l i n e i n t o hydroxy-p r o l i n e , the presence o f 2 to 4 mM glutamine, the concentration u s u a l l y found i n t i s s u e c u l t u r e media, reduced c o l l a g e n production by 80%. In c o n t r a s t , decreasing the concentration of glutamine to 40 ]JM increased c o l l a g e n synthesis by 40% (Ronnemaa et a l . , 1977). Corroborative data (Lehtinen et a l . , 1978) showed increases of c o l l a g e n synthesis of 72.3% i n c e l l s and 20.8% i n the - 21 -medium with 10 UM glutamine; 2 mM glutamine suppressed c o l l a g e n s y n t h e s i s by 62% i n c e l l s and 60% i n the medium. In high c o n c e n t r a t i o n , glutamine may compete with the uptake of p r o l i n e i n t o the c e l l s or d i l u t e the p r o l i n e pool by m e t a b o l i z i n g to glutamic a c i d to glutamic semialdehyde to p r o l i n e . However, i n low concentrations, glutamine provides n i t r o g e n and ammonia, thus enhancing the i n t r a c e l l u l a r production of amino acids and consequently, p r o t e i n synthesis (Ronnemaa e_t a l . , 1977). J . COLLAGEN METABOLISM OF CELLS IN VITRO I . Mouse L-929 F i b r o b l a s t s Cultured mouse L-929 c e l l s , a w e l l - e s t a b l i s h e d l i n e of chemically t r a n s -formed embryonic f i b r o b l a s t s , synthesize and secrete c o l l a g e n ( G r i b b l e et a l . , 1969). Indeed, collagen forms a higher p r o p o r t i o n of the t o t a l p r o t e i n than that found i n human dermal f i b r o b l a s t s (Kerwar ejt a_l., 1973). Matsubayashi e_t a l . , (1977) found that c o l l a g e n accounted for 16% of p r o t e i n synthesized during a 48 hour i n c u b a t i o n p e r i o d . During the l o g a r i t h m i c growth stage, 25 to 30% of the p r o t e i n i n the medium i s r e p r e s e n t a t i v e of c o l l a g e n . The a c t i v i t y of p r o l y l hydroxylase i s low i n the e a r l y l o g a r i t h m i c phase of growth ( K i v i r i k k o and R i s t e l i , 1976). Since immunological methods have demonstrated the presence of the enzyme, i t , most l i k e l y , i s present i n an i n a c t i v e form. Hydroxyproline, i n d i c a t i v e of increased enzymatic a c t i v i t y , does not appear u n t i l the l a t e l o g a r i t h m i c phase when m i t o t i c a c t i v i t y i s low ( G r i b b l e et a l . , 1969). The c o l l a g e n of the medium and the c e l l l a y e r i s hydroxylated to about 20% of maximum i n the e a r l y l o g a r i t h m i c phase, i n c r e a s i n g to 60% i n the s t a t i o n a r y phase. A c t i v a t i o n of p r o l y l hydroxylase occurs through subunit aggregation (McGee and Udenfriend, 1972). F o l l o w i n g h y d r o x y l a t i o n , the - 22 -a f f i n i t y of the c o l l a g e n f o r p r o l y l hydroxylase diminishes w i t h the subsequent release of the enzyme (Kuttan et_ a l . , 1975). According to Kerwar et a l . (1973), the predominant forms of the c o l l a g e n i n the medium are pro a l and pro a2 chains; the low a c t i v i t y of p r o c o l l a g e n peptidase precludes the presence of a l l but a few a chains. Only p r o c o l l a g e n chains are found i n the c e l l u l a r c o l l a g e n ; procollagen peptidase a c t i v i t y i s undetectable i n the c e l l u l a r compartment (Kerwar et a l . , 1973). In c o n t r a s t , peptidase a c t i v i t y i s noted i n the study by Matsubayashi et aj.. (1977) who found tro p o c o l l a g e n molecules comprising a l and a2 chains as w e l l as two species of p e p s i n - s e n s i t i v e c o l l a g e n precursors. U n l i k e normal human dermal f i b r o b l a s t s , mouse L-929 f i b r o b l a s t s synthesize only one type of c o l l a g e n , type I . Type I I I c o l l a g e n i s undetectable (Matsubayashi et a l . , 1977). I I . Human Dermal F i b r o b l a s t s The human dermal f i b r o b l a s t i s t h i n , f u s i f o r m or oval-shaped with long cytoplasmic extensions. A small amount of cytoplasm and a l a r g e nucleus w i t h prominent n u c l e o l i c o n s t i t u t e the main body of the c e l l . Prominent d i l a t e d rough endoplasmic r e t i c u l a are present i n the a c t i v e c o l l a g e n - s y n t h e s i z i n g f i b r o b l a s t s (Hashimoto, 1978). The conversion of c o l l a g e n precursors i n c u l t u r e r e q u i r e s many hours (Taubman et a l . , 1976; Goldberg, 1977). Consequently, most of the molecules i n the e x t r a c e l l u l a r medium are procollagen which d i s s o c i a t e upon red u c t i o n w i t h 2-mercaptoethanol to form free s i n g l e a l and a2 chains ( L i c h t e n s t e i n et a l . , 1975; Goldberg et^ a l . , 1972). Associated with the smaller volume of the c e l l l a y e r are the intermediate form of c o l l a g e n , the p-collagen chains, and a small q u a n t i t y of the n a t i v e tropocollagen f i b r i l s (Goldberg, 1977) which are - 23 -derived mainly from the conversion of the pr o c o l l a g e n i n the e x t r a c e l l u l a r space of the c e l l l a y e r s (Taubman et a l . , 1976). Collagen represents 5-10% of the t o t a l p r o t e i n i n the human dermal f i b r o b l a s t s (Goldberg et a l . , 1964; Matsubayashi et a l . , 1977). F o l l o w i n g a 24 hour i n c u b a t i o n p e r i o d w i t h t r i t i a t e d p r o l i n e , 60 to 80% of the l a b e l l e d c o l l a g e n i s present i n the e x t r a c e l l u l a r medium (Goldberg et a l . , 1972). Types I and I I I collagens are synthesized by human dermal f i b r o b l a s t s , the former predominating i n normal healthy adult c e l l s ( L i c h t e n s t e i n et a l . , 1975). Each c e l l i s capable of s y n t h e s i z i n g both types of c o l l a g e n simultaneously (Gay et al., 1976). The conversion of type I pr o c o l l a g e n to native tropocollagen molecules d i f f e r s from that of type I I I collagenous p r o t e i n s . In f i b r o b l a s t c u l t u r e s of murine and human dermal f i b r o b l a s t s , n a t i v e tropocollagen i s generated from type I pr o c o l l a g e n but such conversion i s not observed with type I I I procollagen i n the same c u l t u r e s . These observations suggest that d i f f e r e n t enzyme systems are resp o n s i b l e f o r the processing of the two types of c o l l a g e n . The enzymes converting type I I I p r o c o l l a g e n appear to be l a c k i n g or l e s s e f f i c i e n t than those f o r p r o c o l l a g e n type I , leading to the d i s p r o p o r t i o n a t e accumulation of type I I I p r o c o l l a g e n i n the medium (Goldberg, 1977). K. GLUCOCORTICOID EFFECTS ON CELLULAR PROLIFERATION AND COLLAGEN METABOLISM I. In V i t r o Studies (a) C e l l Culture Examination of the e f f e c t s of g l u c o c o r t i c o i d s on col l a g e n metabolism i n v i t r o has not been confined to human dermal f i b r o b l a s t s ; at l e a s t as many studies have been d i r e c t e d towards f i b r o b l a s t s from other t i s s u e s and - 24 -species. For i n s t a n c e , chick embryo tendon f i b r o b l a s t s are popular, p r i m a r i l y because of t h e i r high metabolic a c t i v i t y and the f a c t that c o l l a g e n represents 70 to 80% of the t o t a l p r o t e i n ( S a a r n i , 1977). ( i ) chick embryo tendon f i b r o b l a s t s By measuring the i n c o r p o r a t i o n of p r o l i n e i n t o hydroxyproline i n these c e l l s , Saarni et a l . (1976), compared the e f f e c t s on c o l l a g e n synthesis of se v e r a l g l u c o c o r t i c o i d s , i n c l u d i n g hydrocortisone butyrate. Kligman and Frosch (1979) b e l i e v e d the l a t t e r to be atrophogenic; Saarni et a l . (1976) disagreed. Of the g l u c o c o r t i c o i d s studied, only hydrocortisone succinate at -4 10 M had no d i s c e r n i b l e e f f e c t on c o l l a g e n synthesis (Saarni et a l . , 1976). Manipulation of the dosage of t h i s g l u c o c o r t i c o i d produced i n t e r e s t i n g and contrary responses. A s l i g h t increase i n c o l l a g e n synthesis was e l i c i t e d -5 -3 at 10 M but i n c r e a s i n g the concentration to 10 M produced a profound i n h i b i t i o n of 90% ( S a a r n i , 1977). These observations c l e a r l y demonstrate the b i p h a s i c e f f e c t of g l u c o c o r t i c o i d s whereby enhancement of c o l l a g e n synthesis i s produced at low concentrations and i n h i b i t i o n i s r e g i s t e r e d w i t h e l e v a t e d q u a n t i t i e s of g l u c o c o r t i c o i d s . The free a l c o h o l form of hydrocortisone was -4 capable of suppressing hydroxyproline formation by 46% and 20% at 10 M and 10 "'M, r e s p e c t i v e l y ( S a a r n i , 1977). S i m i l a r r e s u l t s had been obtained e a r l i e r with the phosphate and butyrate d e r i v a t i v e s of hydrocortisone as w e l l as prednisolone (Saarni et a l . , 1976). Collagen synthesis was reduced 24 and -4 -3 35% at 10 M hydrocortisone butyrate or prednisolone and 56% at 10 M hydrocortisone phosphate. Lower concentrations had no e f f e c t . With betamethasone-17-valerate, the depression was 90% at 10~^M and 20% at 10 "*M. Dexamethasone, i t s phosphate d e r i v a t i v e , and the a l c o h o l and phosphate d e r i v a t i v e s of betamethasone-17-valerate e l i c i t e d no a l t e r a t i o n s i n - 25 -c o l l a g e n production perhaps due to the lack of conversion to some a c t i v e forms of the s t e r o i d . With a l l dosages of g l u c o c o r t i c o i d s that were e f f e c t i v e , n e i t h e r an accumulation of unhydroxylated c o l l a g e n i n the c e l l s nor an i n h i b i t i o n of the s e c r e t i o n of c o l l a g e n was reported. The observed p a r a l l e l decrease i n q u a n t i t y of c o l l a g e n and t o t a l p r o t e i n suggested a general d e c l i n e of p r o t e i n s y n t h e s i s . These studies demonstrated the d i f f e r e n t potencies of various d e r i v a t i v e s of hydrocortisone i n the depression of c o l l a g e n s y n t h e s i s . Both monosodium phosphate and sodium succinate d e r i v a t i v e s e x h i b i t e d m i l d e r potencies than the f r e e a l c o h o l and butyrate forms of hydrocortisone and prednisolone. The most potent was betamethasone-17-valerate. I n t e r e s t i n g l y enough, betamethasone-17-valerate generates atrophy i n greater s e v e r i t y than hydrocortisone, hydrocortisone butyrate or prednisolone. In c l i n i c a l exper-ience, hydrocortisone butyrate and betamethasone-17-valerate are comparable i n anti-inflammatory potencies. No c o r r e l a t i o n e x i s t s between atrophogenic property and anti-inflammatory potency ( S a a r n i , 1977). Oikarinen (1977a), using the same c e l l type, a l s o observed reduced syn-t h e s i s of c o l l a g e n i n the presence of betamethasone-17-valerate. A s p e c i f i c a l t e r a t i o n i n c o l l a g e n synthesis was suggested by depression of p r o l i n e i n c o r -p o r a t i o n and by the marked reduction i n hydroxyproline b i o s y n t h e s i s . The d e c l i n e i n c o l l a g e n synthesis was not associated with any a b e r r a t i o n i n p r o l y l hydroxylase a c t i v i t y . However, i f exposed to the g l u c o c o r t i c o i d i n v i v o , p r o l y l hydroxylase a c t i v i t y decreased s i g n i f i c a n t l y suggesting e i t h e r i n a c t i v a t i o n or i n h i b i t i o n i n the synthesis of the enzyme (Oikari n e n , 1977a). In a l a t e r i n v i t r o study using the same c e l l type, Oikarinen (1977b) observed no a l t e r a t i o n i n the a c t i v i t i e s of p r o l y l hydroxylase and the other enzymes, l y s y l hydroxylase, g a l a c t o s y l and g l u c o s y l t r a n s f e r a s e s i n the presence of - 26 -C o r t i s o l acetate despite a depression i n collagen synthesis. Prolonged or repeated exposure to the g l u c o c o r t i c o i d did r e s u l t in reduction o f the enzymatic a c t i v i t i e s , e s p e c i a l l y of the hydroxylases. Thus, i t would appear from the foregoing data (Saarni, 1977; Oikarinen, 1977a) that c e r t a i n glucocorticoids i n h i b i t collagen synthesis by a f f e c t i n g the synthesis of the polypeptide chains d i r e c t l y and possibly by i n h i b i t i n g the a c t i v i t i e s of those enzymes responsible for the p o s t - t r a n s l a t i o n a l changes in collagen, ( i i ) f o e t a l human dermal f i b r o b l a s t s Kirk and Mittwoch (1977) compared the e f f e c t s of the g l u c o c o r t i c o i d , C o r t i s o l , and i t s more potent synthetic fluorinated d e r i v a t i v e , fluocinolone acetonide, on the c e l l u l a r p r o l i f e r a t i o n of human d i p l o i d dermal f i b r o b l a s t s from aborted foetuses. Treatment with these glucocorticoids for 24 hours in doses ranging from a p h y s i o l o g i c a l l e v e l of 0.05 yg per ml to a pharmaco-l o g i c a l l e v e l of 150 yg per ml of growth medium produced higher m i t o t i c indices (percentage of c e l l s i n m i t o t i c phase) than untreated control cultures. The increase in the m i t o t i c index could have arisen from an i n h i b i t i o n of a cer-tain m i t o t i c phase or could r e f l e c t a stimulation of p r o l i f e r a t i o n . C o r t i s o l , at concentrations up to 50 yg per ml produced no s i g n i f i c a n t increase in the p r o l i f e r a t i v e rate. However, at s i m i l a r concentrations, fluocinolone acetonide did produce a s l i g h t but s i g n i f i c a n t increase in the rate of p r o l i f e r a t i o n . Furthermore, the c e l l s were induced to enter e a r l i e r into the period of DNA synthesis which was increased s u b s t a n t i a l l y following g l u c o c o r t i c o i d treatment (Kirk and Mittwoch, 1977). Harvey et a l . (1974) found that 10 yg prednisolone or C o r t i s o l per ml of medium i n h i b i t e d the p r o l i f e r a t i o n of human embryonic skin f i b r o b l a s t s a f t e r a 9 day incubation period. When the g l u c o c o r t i c o i d concentration was reduced to - 27 -0.1 ug C o r t i s o l or 0.01 Ug prednisolone per ml of medium, the number of c e l l s f l o a t i n g i n the c u l t u r e medium was decreased, p o s s i b l y i n d i c a t i n g enhanced c e l l u l a r adhesion. The rate of t r i t i a t e d thymidine i n c o r p o r a t i o n was elevat e d by 0.01 Ug of e i t h e r g l u c o c o r t i c o i d but decreased by 20% i n the presence of 1.0 Ug per ml and 50% at 10 ug per ml. The depression of thymidine i n c o r p o r -a t i o n preceded the re d u c t i o n of c e l l numbers. The observed s t i m u l a t i o n and suppression of thymidine i n c o r p o r a t i o n at the lower and higher g l u c o c o r t i c o i d c o n c e n t r a t i o n s , r e s p e c t i v e l y , r e f l e c t s the b i p h a s i c e f f e c t of g l u c o c o r t i c o i d s (Ueki et a l . , 1976). Collagen s y n t h e s i s was enhanced by C o r t i s o l and pred-nisolone at concentrations ranging from 0.01 to 1.0 Ug per ml medium; maximal s t i m u l a t i o n occurred at 0.1 Ug per ml of each s t e r o i d . These concentrations extend over the range of p h y s i o l o g i c a l and pharmacological doses (Harvey et a l . , 1974). The concentration of 0.1 Ug per ml of C o r t i s o l i s w i t h i n the range of 0.04 to 0.2 Ug per ml found i n plasma ( A s f e l d t , 1971). A l a t e r study of Doherty e_t a_l. (1976) a l s o examined g l u c o c o r t i c o i d e f f e c t s on col l a g e n synthesis by confluent human f o e t a l s k i n f i b r o b l a s t s of passage 5 to 10. At concentration of 10 ^ M hydrocortisone sodium succinate d i d not i n h i b i t the synth e s i s of c o l l a g e n or non-collagen p r o t e i n a f t e r a 5 hour i n c u b a t i o n . But, at 10 M, c o l l a g e n synthesis was stimulated markedly, as determined by i n c o r p o r a t i o n of r a d i o a c t i v e p r o l i n e i n t o hydroxyproline and t o t a l p r o t e i n . Non-collagen p r o t e i n synthesis was stimulated but not to a s t a t i s t i c a l l y s i g n i f i c a n t degree. The s t i m u l a t i o n of non-collagenous p r o t e i n and co l l a g e n syntheses both i n the medium and i n the c e l l l a y e r was enhanced by prolonging the i n c u b a t i o n to 11 h i n the presence of 10 ^ M hydrocortisone sodium succinate (Doherty et al., 1976). - 28 -More r e c e n t l y , studies using normal human f o r e s k i n c e l l s before the twentieth passage confirmed the concentration-dependent a l t e r a t i o n of c e l l u l a r p r o l i f e r a t i o n . At concentrations between 0.001 yg per ml to 1.0 yg per ml of medium, c e l l u l a r p r o l i f e r a t i o n was enhanced by hydrocortisone, betamethasone v a l e r a t e and c l o b e t a s o l propionate. S i g n i f i c a n t depression of p r o l i f e r a t i o n was evident w i t h 10 yg of the foregoing g l u c o c o r t i c o i d s per ml medium ( P r i e s t l e y , 1978). Again, the b i p h a s i c e f f e c t of g l u c o c o r t i c o i d s i s evident. These f i n d i n g s are i n contrast to those of B e r l i n e r and Ruhmann (1967) and Brotherton (1971) who observed a progressive depression of c e l l u l a r p r o l i f e r -a t i o n of mouse L-929 f i b r o b l a s t s with increased g l u c o c o r t i c o i d dosage. Clo b e t a s o l propionate at 10 yg per ml i n h i b i t e d c o l l a g e n s y n t h e s i s by 42%, as measured by s u s c e p t i b i l i t y of the c o l l a g e n to d i g e s t i o n by protease-free collagenase. At 25 yg per ml of medium, c o l l a g e n production was depressed more than 50% by betamethasone v a l e r a t e and c l o b e t a s o l propionate but t o t a l p r o t e i n synthesis was reduced to a l e s s e r extent, thus adding support to the view that g l u c o c o r t i c o i d s a f f e c t the metabolism of c o l l a g e n s p e c i f i c a l l y ( P r i e s t l e y , 1978). A dose- and time-dependent e f f e c t of g l u c o c o r t i c o i d s on c o l l a g e n synthesis was demonstrated by Saarni and Tammi (1978). Incubation of confluent c u l t u r e s of human f o e t a l dermal f i b r o b l a s t s of passage 9 to 11 w i t h 10 VjM C o r t i s o l f o r 12 to 36 hours produced a s i g n i f i c a n t increase i n c o l l a g e n s y n t h e s i s . S t i m u l -a t i o n was a l s o reported with 1 ]JM C o r t i s o l at 12 hours, and with 10 nM C o r t i s o l at 96 hours. However, dosages i n excess of 10 nM were i n h i b i t o r y when incub-ated f or 96 hours. The synthesis was followed by measurement of the in c o r p o r -a t i o n of p r o l i n e i n t o hydroxyproline. Saarni and Tammi (1978) claimed that c o l l a g e n synthesis was a l t e r e d more than that of other p r o t e i n s , suggesting - 29 -that C o r t i s o l , l i k e betamethasone v a l e r a t e and c l o b e t a s o l propionate ( P r i e s t l e y , 1978) a f f e c t s c o l l a g e n synthesis s p e c i f i c a l l y . Thymidine uptake was unaffected over the f i r s t 12 hours but dropped s i g n i f i c a n t l y a f t e r 36 hours with 10 VJM C o r t i s o l . At 10 * JJM, DNA synthesis was i n h i b i t e d a f t e r 96 hours. Saarni and Tammi (1978) suggest that C o r t i s o l a f f e c t s the e a r l i e r part of G^ phase of the c e l l c y c l e supporting an e a r l i e r report that showed the r e t a r d a t i o n of the c e l l s to the phase. In c o n t r a s t , K i r k and Mittwoch (1977) found that C o r t i s o l and f l u o c i n o l o n e acetonide shorten the G^  phase, thereby promoting an e a r l y entry i n t o the S phase. Thus, the e f f e c t of gluco-c o r t i c o i d s on the c e l l c y c l e remains unclear i n l i g h t of such c o n t r a d i c t o r y f i n d i n g s . The previous studies have demonstrated the dose- and time-dependent a l t e r -ations of c e l l u l a r p r o l i f e r a t i o n and co l l a g e n synthesis by g l u c o c o r t i c o i d s i n c u l t u r e s of human f o e t a l f i b r o b l a s t s . D i f f e r e n t techniques have been employed to determine c o l l a g e n s y n t h e s i s and c e l l u l a r p r o l i f e r a t i o n , producing v a r y i n g r e s u l t s . The great number of v a r i a b l e parameters, for instance, dosage, duration of g l u c o c o r t i c o i d treatment and stage of c e l l growth, u n f o r t u n a t e l y , hinder the e l u c i d a t i o n of g l u c o c o r t i c o i d a c t i o n s i n v i t r o . ( i i i ) adult human dermal f i b r o b l a s t s Human f o e t a l c e l l s have been used w i d e l y , as has been evident from the papers discussed so f a r . Less data i s a v a i l a b l e on c e l l s i s o l a t e d from ad u l t man. Ponec e_t a l . (1977a) subjected l o g a r i t h m i c c u l t u r e s of dermal f i b r o -b l a s t s from a young adult and of passage 17 to 21 to g l u c o c o r t i c o i d s at concentrations compatible with those found i n s k i n a f t e r t o p i c a l treatment w i t h c l i n i c a l l y employed dosages. At 5 yg per ml of medium f o r 5 days, triamcinolone acetonide, betamethasone-17-valerate or hydrocortisone 17-- 30 -butyrate caused 30 to 50% i n h i b i t i o n of c e l l u l a r p r o l i f e r a t i o n . Hydro-c o r t i s o n e at 5 Ug per ml had no d i s c e r n i b l e e f f e c t but the same concentration of clobetasol-17-propionate suppressed c e l l u l a r d i v i s i o n almost completely. E i t h e r 16 Ug hydrocortisone or 1 Ug clobetasol-17-propionate per ml produced 30 to 50% i n h i b i t i o n . D i f f e r e n t r e s u l t s were generated i n confluent c u l t u r e s of adult human dermal f i b r o b l a s t s . Neither depression of c e l l u l a r p r o l i f e r a t i o n nor increase i n c e l l turnover was observed. Instead, w i t h triamcinolone acetonide, hydrocortisone 17-butyrate and betamethasone-17-valerate, the c e l l d e nsity at confluency was 20% greater than that of c o n t r o l c u l t u r e s (Ponec et a l . , 1977b). These observations lend support to those of Cunningham et a l . (1974) who a l s o found that triamcinolone acetonide, dexamethasone, C o r t i s o l and c o r t i c o s t e r o n e increased c e l l d e nsity of confluent c u l t u r e s of 3T3 f i b r o b l a s t s . The synthesis of c o l l a g e n was followed by measurement of the formation of hydroxyproline. T o t a l p r o t e i n synthesis was a f f e c t e d to a l e s s e r extent than that of c o l l a g e n f o l l o w i n g a 6 day inc u b a t i o n w i t h the g l u c o c o r t i c o i d s . The depression, though s l i g h t at 5 Ug hydrocortisone per ml of medium, was pronounced at 16 Ug/ml. However, only 5 Ug triamcinolone acetonide, betamethasone-17-valerate or hydrocortisone 17-butyrate per ml was necessary to cause a 30 to 50% re d u c t i o n i n hydroxyproline content. Clobetasol-17-propionate was even more a c t i v e , 5 Ug per ml l i m i t e d t o t a l p r o t e i n synthesis markedly; 1 Ug per ml depressed hydroxyproline synthesis by 60%. The degree of i n h i b i t i o n of hydroxyproline s y n t h e s i s was unr e l a t e d to f l u o r i n a t i o n , c e l l u l a r p r o l i f e r a t i o n , c e l l u l a r turnover or p r o t e i n synthesis (Ponec et a l . , 1977a,b). - 31 -( i v ) mouse L-929 f i b r o b l a s t s Mouse L-929 f i b r o b l a s t s are also s u s c e p t i b l e to g l u c o c o r t i c o i d s . U n l i k e primary human s k i n f i b r o b l a s t s , the p r o l i f e r a t i o n of these c e l l s was i n h i b i t e d . . . 3 4 . by g l u c o c o r t i c o i d s at concentrations 10 to 10 times lower than those encountered i n the t o p i c a l a p p l i c a t i o n of g l u c o c o r t i c o i d s . The i n h i b i t i o n of c e l l u l a r p r o l i f e r a t i o n of the L-929 c e l l s extended over a range of concen-4 5 . t r a t i o n s , 10 to 10 times broader than that of the human f i b r o b l a s t s . The e f f e c t i v e minimal concentrations were 0.1 pg clobetasol-17-propionate per ml, 1 pg triamcinolone acetonide per ml, 10 pg betamethasone-17-valerate or hydrocortisone 17-butyrate per ml, 100 pg hydrocortisone-21-acetate per ml and 1 ng hydrocortisone per ml. I n h i b i t i o n occurred even at 10 ug per ml, the highest c o n c e n t r a t i o n studied. Thus, only a l t e r a t i o n s i n degree, not k i n d , of response were produced by the g l u c o c o r t i c o i d s i n t h i s e s t a b l i s h e d mouse l i n e (Ponec et a l . , 1977a). The i n h i b i t o r y e f f e c t s of hydrocortisone and triamcinolone acetonide on c e l l u l a r p r o l i f e r a t i o n of the mouse L-929 f i b r o b l a s t were confirmed by R u n i k i s et a l . , 1978. The e f f e c t i v e range was 10 ng to to 1 ug hydrocortisone per ml; f o r triamcinolone acetonide and desonide, 100 pg to 1 ug/ml. Desonide was the most, hydrocortisone the l e a s t , potent i n h i b i t o r of c e l l p r o l i f e r a t i o n . Dermal d i p l o i d f i b r o b l a s t s from adult humans, i n c o n t r a s t to the mouse c e l l s , were stimulated c o n s i s t e n t l y by comparable concentrations of the foregoing gluco-c o r t i c o i d s (Runikis e t ^ a l . , 1978). Although the l a t t e r r e s u l t s are i n c o n t r a s t to those of Ponec et a l . (1977a), i t must be noted that the g l u c o c o r t i c o i d concentrations used by Ponec were much higher, ranging from 1 to 16 ug per ml. Indeed, the data from both studies are not n e c e s s a r i l y c o n f l i c t i n g but - 32 -may merely r e f l e c t the v a r i a b l e dose-response e f f e c t of g l u c o c o r t i c o i d s on c e l l u l a r p r o l i f e r a t i o n , (b) Organ c u l t u r e Organ c u l t u r e s , although not as widely employed as c e l l c u l t u r e systems, have a l s o been used to i n v e s t i g a t e the e f f e c t s of g l u c o c o r t i c o i d s on c o l l a g e n metabolism. Although concentrations of 10 Ug hydrocortisone acetate, f l u o c i n -olone acetonide, f l u c l o r o l o n e acetonide, betamethasone-17-valerate or fluprednyliden-21-acetate per ml had no d i s c e r n i b l e e f f e c t on c o l l a g e n synthesis i n human s k i n s l i c e s , i n c r e a s i n g l y severe i n h i b i t i o n of hydroxy-hydroxyproline formation was observed from 30 to 300 Ug of the g l u c o c o r t i c o i d per ml. Betamethasone-17-valerate e x h i b i t e d the most pronounced i n h i b i t i o n , hydrocortisone acetate the l e a s t , at a l l concentrations used. The amount of p r o l i n e i n c o r p o r a t i o n i n t o t o t a l p r o t e i n p a r a l l e l e d that of hydroxyproline, an index of c o l l a g e n synthesis ( U i t t o et aJL, 1972). A study by Koob £ a l . (1974) showed that human s k i n i n organ c u l t u r e was capable of s y n t h e s i z i n g collagenases. Contrary to Houck's (1968) f i n d i n g s of increased c o l l a g e n degradation f o l l o w i n g g l u c o c o r t i c o i d treatment, Koob et a l . (1974) found that 10 nM dexamethasone and 100 nM hydrocortisone were incapable of e l i c i t i n g increased c o l l a g e n o l y s i s . However, collagenase a c t i v i t y was reduced by 80 to 90%, the degree of i n h i b i t i o n depending upon the s t e r o i d c o n c e n t r a t i o n , the lower l i m i t s being 1 j>M dexamethasone and 10 nM hydro-c o r t i s o n e . P r o t e i n synthesis remained unaffected o v e r a l l . The i n h i b i t i o n of collagenase expression was s p e c i f i c ; t h i s was corroborated by a s i m i l a r decrease i n the l e v e l of d i a l y z a b l e h y d r o x y p r o l i n e - c o n t a i n i n g peptides. From these data, Koob et a l . (1974) concluded that i n human s k i n explants gluco-c o r t i c o i d s prevent endogenous c o l l a g e n degradation. - 33 -The e f f e c t s of g l u c o c o r t i c o i d s on c o l l a g e n metabolism have a l s o been examined i n organ c u l t u r e s of t i s s u e s other than s k i n or of humans. Based on a collagenase assay, treatment of chick embryo c a l v a r i a e w i t h large doses of triamcinolone acetonide had no e f f e c t on c o l l a g e n synthesis (Cohen et^ al. , 1977). However, col l a g e n formation was depressed i n t i b i a e exposed to C o r t i s o l . High doses of C o r t i s o l reduced the l e v e l s of h y d r o x y p r o l i n e , g l y c o s y l a t e d and t o t a l hydroxylysine as w e l l as the uptake of p r o l i n e and l y s i n e r e s i d u e s . A l s o , high doses of hydrocortisone acetate, hydrocortisone phosphoric a c i d complex or hydrocortisone sodium succinate i n h i b i t e d p r o l y l h y d r o x y l a t i o n , r e s u l t i n g i n reduction of c o l l a g e n s e c r e t i o n i n t o the medium (Bluraenkrantz and Asboe-Hansen, 1976). Thus, the lower l e v e l of c o l l a g e n found i n the medium of organ c u l t u r e s could be the consequence of two processes: (1) a defect i n peptide synthesis and (2) a suppression of the p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s of c o l l a g e n . ( I I ) In Vivo Studies C o n f l i c t i n g , v a r y i n g r e s u l t s and o c c a s i o n a l m i s i n t e r p r e t a t i o n s have prevented the e l u c i d a t i o n of g l u c o c o r t i c o i d e f f e c t s i n i n v i t r o systems. Unfo r t u n a t e l y , the in v i v o studies shed l i t t l e l i g h t on the a c t i o n of g l u c o c o r t i c o i d s on c o l l a g e n metabolism. Admittedly, biochemical data from human subjects i s sparse. Nonetheless, the h i s t o p a t h o l o g i c a l f i n d i n g s of s e v e r a l studies of human subjects do present some common fe a t u r e s . Treatment of normal human s k i n with f l u o c i n o l o n e acetonide induced e a r l y onset of s k i n atropy. S i g n i f i c a n t decreases i n the diameter of c o l l a g e n f i b r i l s , t h i n n i n g , d i s o r g a n i z a t i o n and fragmentation of c o l l a g e n bundles and reduced numbers of f i b r o b l a s t s were observed f o l l o w i n g f l u o c i n o l o n e acetonide - 34 -and flumethasone p i v a l a t e treatment. C h a r a c t e r i s t i c s of a c t i v e p r o t e i n s y n t h e s i s , d i l a t e d rough endoplasmic r e t i c u l u m , elaborate g o l g i apparatus and numerous mitochondria were not present. A prominent feature was the endo-c y t o s i s of c o l l a g e n f i b r i l s . In close v i c i n i t y were dense lysosomal bodies, an i n d i c a t i o n of the formation of phagolysosomes (Jablonska et a l _ . , 1979; Groniowska et a_l., 1976). That dermal atrophy could be a consequence of c o l l a g e n degradation i s f u r t h e r supported by the biochemical f i n d i n g s of Cohen et a l . (1977) who observed r e g r e s s i o n of human k e l o i d s f o l l o w i n g i n t r a l e s i o n a l i n j e c t i o n s w i t h triamcinolone. Regression was not due to decreased c o l l a g e n s y n t h e s i s . Indeed, c o l l a g e n s y n t h e t i c r a t e was comparable with that of untreated b i l a t e r a l earlobe k e l o i d s . Although c o l l a g e n o l y s i s was not i n v e s t i g a t e d , Cohen et al. (1977) hypothesized that triamcinolone enhanced co l l a g e n degradation. More studies have been performed on rodents. Dexamethasone was found to i n h i b i t c o l l a g e n synthesis i n skins of two s t r a i n s of mice. One s t r a i n had a higher c o l l a g e n s y n t h e t i c r a t e . Following g l u c o c o r t i c o i d treatment, c o l l a g e n synthesis i n t h i s s t r a i n was depressed to a greater extent than that of the other s t r a i n . The former s t r a i n also encountered greater loss of non-collagenous p r o t e i n s a f t e r dexamethasone a d m i n i s t r a t i o n . Hence, the proportion of c o l l a g e n was a l t e r e d . Two important conclusions emerged from t h i s study. S t r a i n d i f f e r e n c e s determined the e f f e c t of g l u c o c o r t i c o i d s on dermal c o l l a g e n . Both synthesis and degradation of p r o t e i n s were a f f e c t e d by dexamethasone (Robey, 1979). Extensive studies of g l u c o c o r t i c o i d m o d i f i c a t i o n s of c o l l a g e n metabolism have been reported f o r organs other than s k i n as w e l l as s k i n from other species. Prednisolone, a popular g l u c o c o r t i c o i d , was examined f o r i t s e f f e c t s - 35 -on the a c t i v i t i e s of the hydroxylases and g l y c o s y l t r a n s f e r a s e s i n r a t s k i n and l i v e r . The i n h i b i t i o n of a c t i v i t i e s of p r o l y l and l y s y l hydroxylases, g a l a c t o s y l - and g l u c o s y l - t r a n s f e r a s e s was dose-dependent; a l l were depressed to a s i m i l a r degree at each pharmacological dose l e v e l . Suppression of enzyme a c t i v i t i e s could p o s s i b l y a r i s e from a general r e d u c t i o n i n enzyme p r o t e i n s y n t h e s i s . Indeed, p r o l y l hydroxylase a c t i v i t y diminished to the same degree as the amount of the enzyme. Q u a n t i t a t i o n of the other enzyme p r o t e i n s has yet to be done. Any r e d u c t i o n i n the a c t i v i t y of enzymes r e s p o n s i b l e for p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s of c o l l a g e n can be manifested i n a suppression of c o l l a g e n synthesis ( R i s t e l i , 1977). These f i n d i n g s corroborated the e a r l i e r work on r a t l i v e r by Cutroneo et al_. (1971). P r o l y l hydroxylase was i n h i b i t e d i n granuloma t i s s u e by i n c r e a s i n g concentrations of the g l u c o c o r t i -c o i d s , methylprednisolone, triamcinolone d i a c e t a t e , hydrocortisone acetate; triamcinolone d i a c e t a t e had the most i n h i b i t o r y e f f e c t . Dexamethasone al s o caused a 50 to 70% i n h i b i t i o n of c o l l a g e n synthesis i n mouse granulomas; t o t a l p r o t e i n synthesis was depressed by only 25%. Kruse et a l . (1978) reported a concomitant r e d u c t i o n of f i b r o b l a s t number. The degree of depression of p r o l y l hydroxylase a c t i v i t y and of t o t a l p r o t e i n synthesis was comparable. Hence, i n the l a t t e r study n e i t h e r h y d r o x y l a t i o n of p r o l y l residues nor c o l l a g e n synthesis was s p e c i f i c a l l y a f f e c t e d by dexamethasone. U n l i k e Kruse et a l . (1978), a more marked depression of the enzymatic a c t i v i t y r e l a t i v e to p r o t e i n synthesis was observed by Cutroneo et a l . (1971), i n d i c a t i n g a s p e c i f i c e f f e c t of g l u c o c o r t i c o i d s on c o l l a g e n s y n t h e s i s . U n l i k e the g l u c o c o r t i c o i d s discussed above, betamethasone disodium phos-phate had no d i s c e r n i b l e e f f e c t on the a c t i v i t y of p r o l y l hydroxylase. The i n c o r p o r a t i o n of l a b e l l e d p r o l i n e i n t o hydroxyproline was i n h i b i t e d to a greater extent than that i n t o non-collagen p r o t e i n , i n d i c a t i n g a s p e c i f i c e f f e c t of betamethasone on col l a g e n synthesis (Nakagawa ejt a l . , 1971). A s i g n i f i c a n t s p e c i f i c dose- and time-dependent suppression of c o l l a g e n polypeptide synthesis was observed i n s k i n of newborn r a t s i n j e c t e d i n t r a -p e r i t o n e a l l y w i t h triamcinolone d i a c e t a t e (Cutroneo and Counts, 1975; Newman and Cutroneo, 1978). Although s i m i l a r reductions i n the a c t i v i t i e s of p r o l y l and l y s y l hydroxylases and a concomitant d e c l i n e i n hydroxyproline formation were observed, no synthesis or accumulation of underhydroxylated c o l l a g e n occurred. Hydroxyproline formation p a r a l l e l e d the synthesis of the nascent c o l l a g e n chains. DNA synthesis was suppressed only a f t e r m u l t i p l e i n t r a -p e r i t o n e a l i n j e c t i o n s of triamcinolone d i a c e t a t e . In a l a t e r study (Slaga et a l . , 1978), DNA synthesis was shown to be stimulated 48 hours a f t e r t o p i c a l a p p l i c a t i o n of f l u o c i n o l o n e acetonide to newborn mice. These c o n t r a s t i n g observations were due not only to the g l u c o c o r t i c o i d s used but als o to d i f f e r e n c e s i n the animal s p e c i e s , route of g l u c o c o r t i c o i d a d m i n i s t r a t i o n , dosage and duration of treatment and animal species. Data c o r r o b o r a t i n g those of Cutroneo and Counts (1975) and Newman and Cutroneo (1978) were obtained using modified techniques (Counts et al., 1979). Rats were i n j e c t e d i n t r a -p e r i t o n e a l l y w i t h 15 mg triamcinolone d i a c e t a t e per kg body weight, and f i b r o b l a s t s were derived from the dermis for a n a l y s i s for c o l l a g e n . These c e l l s were incubated f o r 0, 1.0, 3.0 and 6.5 hours p r i o r to the estimations of p r o l y l hydroxylase a c t i v i t y , t o t a l p r o l i n e incorporated using a collagenase assay, hydroxyproline formation and l a s t l y , the r a t i o of hydroxyproline to p r o l i n e i n the i n t r a c e l l u l a r c o l l a g e n peptides. Suppression was observed i n the t o t a l i n c o r p o r a t i o n of p r o l i n e , the hydroxyproline formation and i n the content of c e l l u l a r c o l l a g e n . Kruse a l . (1978) a l s o grew f i b r o b l a s t s from granuloma t i s s u e of mice i n j e c t e d i n t r a m u s c u l a r l y w i t h 0.35 mg dexamethasone per kg body weight. The r e s u l t s obtained c o n t r a d i c t e d those of Counts et a l . (1979), f o r the r a t e of c e l l u l a r growth and syntheses of t o t a l p r o t e i n and c o l l a g e n were unaffected. The d i f f e r e n c e s i n type, dosage and route of g l u c o c o r t i c o i d a d m i n i s t r a t i o n and the animal species, most l i k e l y , account for the discrepancy. Only with the a d d i t i o n of 0.3 UM hydrocortisone to the c u l t u r e medium of these f i b r o b l a s t s were t o t a l p r o t e i n and c o l l a g e n syntheses suppressed. The a c t i v i t y of p r o l y l hydroxylase was a l s o reduced. Again, the degree of i n h i b i t i o n v a r i e d w i t h dose and duration of incubation with the s t e r o i d (Kruse et a l . , 1978). A d i f f e r e n t i n v i v o system from the foregoing experiments was employed by U i t t o and M u s t a k a l l i o (1971) to t e s t the e f f e c t s of hydrocortisone acetate, f l u o c i n o l o n e acetonide, f l u c l o r o l o n e acetonide, betamethasone-17-valerate and fluprednyliden-21-acetate on c o l l a g e n b i o s y n t h e s i s determined as the amount of hydroxyproline formed. A l l the g l u c o c o r t i c o i d s i n h i b i t e d the formation of hydroxyproline i n chick embryo t i b i a e when applied onto c h o r i o a l l a n t o i c membranes of chick embryos. The degree of suppression was dependent on dose and duration of treatment but was comparable with a l l the g l u c o c o r t i c o i d s . A d i s t i n c t i v e observation that evolved from t h i s study was the requirement of higher concentrations to i n h i b i t c o l l a g e n formation in v i t r o than v i v o ( U i t t o and M u s t a k a l l i o , 1971). - 38 -SUMMARY The mechanism of g l u c o c o r t i c o i d i n t e r a c t i o n with c o l l a g e n metabolism and i t s relevance to dermal atrophy are s t i l l obscure, l a r g e l y because of the many v a r i a b l e s involved i n the experimental models, the c u l t u r e systems, the assays, and the g l u c o c o r t i c o i d treatment. Confusion i s heightened by la c k of information about g l u c o c o r t i c o i d i n t e r a c t i o n s along the e n t i r e metabolic pathway of co l l a g e n s y n t h e s i s and degradation. Most studies have been devoted to an a n a l y s i s of one or two aspects of co l l a g e n synthesis a f t e r g l u c o c o r t i c o i d treatment. Often no a s s o c i a t i o n e x i s t s between bi o c h e m i c a l , u l t r a s t r u c t u r a l and c l i n i c a l data, thereby making i n t e r p r e t a t i o n of data d i f f i c u l t , i f not impossible. In v i v o experimental models have v a r i e d ; they includ e chick embryos, r a t s , mice, monkeys and man. Within each model are such v a r i a b l e s as age, sex, and species. The type and concentration of g l u c o c o r t i c o i d , d u r a t i o n and frequency of g l u c o c o r t i c o i d treatment, and the route of a d m i n i s t r a t i o n are but a few v a r i a b l e s i n i n v i v o experiments. The solvent or v e h i c l e f or the gluco-c o r t i c o i d , yet another v a r i a b l e , determines the ease and r a p i d i t y of i t s p e n e t r a t i o n . Using c u l t u r e systems, the i n v e s t i g a t o r has at h i s d i s p o s a l the use of c e l l s or organs. Popular c e l l types have been f i b r o b l a s t s derived from chick embryos, human foetuses and neonatal babies. The s i t e s from which the c e l l s have been derived are, l a r g e l y , from tendon and t i b i a e i n chick embryos, dermis and, of greater p o p u l a r i t y , f o r e s k i n of humans. S u r p r i s i n g l y few studies centre on adul t human i n d i v i d u a l s . Indeed, a survey to date has produced one study that employed f i b r o b l a s t s derived from ad u l t human models. - 39 -Human s k i n and l i v e r have been the predominant t i s s u e s i n organ c u l t u r e s . Regardless of the choice of c u l t u r e systems, the studies discussed thus f a r are fraught with a la r g e number of v a r i a b l e s . Where c e l l u l a r aspects are concerned, the growth phase of f i b r o b l a s t s and the passage number of the c e l l c u l t u r e s are important c o n s i d e r a t i o n s . The lack of optimal c o n d i t i o n s f o r c e l l u l a r growth can prevent the e l u c i d a t i o n of biochemical e f f e c t s of gluco-c o r t i c o i d s on c e l l u l a r metabolic a c t i v i t i e s . More v a r i a b l e s are posed i n the form of type, dosage, frequency and duration of g l u c o c o r t i c o i d treatment of c e l l s and organs, as w e l l as the ki n d of solvent or v e h i c l e which determines the ease and r a p i d i t y of penetrance i n t o the f i b r o b l a s t s or t i s s u e s . Some workers (Kruse et_ al., 1978; Counts £t al., 1979) have gone so f a r to combine i n v i v o and in v i t r o experiments. For instance, c o l l a g e n metabolism of granuloma f i b r o b l a s t s derived from dexamethasone-treated mice was examined i n c e l l c u l t u r e ; i n f a c t , some of these f i b r o b l a s t s were l a t e r f u r t h e r exposed to hydrocortisone (Kruse et a l . , 1978). Such experiments can add only f u r t h e r confusion. The most popular c o l l a g e n assays have been the es t i m a t i o n of p r o l y l hydroxylase a c t i v i t y and the i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n estimated e i t h e r w i t h b a c t e r i a l collagenase or by the amount of hydroxyproline formed. The l a t t e r procedure should not be regarded as an index of c o l l a g e n s y n t h e s i s , although i t has been used as such by seve r a l i n v e s t i g a t o r s (Nakagawa et a l . , 1971; Ponec et a l . , 1977a,b; Saarni and Tammi, 1978), since the content of hydroxyproline r e f l e c t s the h y d r o x y l a t i o n process as w e l l . Information about the e f f e c t s of g l u c o c o r t i c o i d s on other aspects of co l l a g e n metabolism i s scant because few studies have been devoted to the i n t e r a c t i o n of g l u c o c o r t i c o i d s w i t h c e l l u l a r uptake of precursor amino a c i d s , - 40 -s p e c i f i c a c t i v i t y a n d s i z e o f i n t r a c e l l u l a r p r o l i n e p o o l , i n t r a c e l l u l a r c o l l a g e n d e g r a d a t i o n , g l y c o s y l a t i o n , s e c r e t i o n o f p e p t i d e c h a i n s , a c t i v i t y t h e p r o c o l l a g e n p e p t i d a s e , t h e e x t e n t o f c r o s s - l i n k i n g o f t h e c o l l a g e n o u s f i b r i l s a n d f i n a l l y , t h e t y p e s o f c o l l a g e n f o r m e d . - 41 -APPROACH Despite the f a c t that g l u c o c o r t i c o i d s have been i n use c l i n i c a l l y f or many years, v i r t u a l l y nothing i s known about t h e i r a c t i o n on c e r t a i n metabolic a c t i v i t i e s i n many types of c e l l s . G l u c o c o r t i c o i d s have been found to c o n s i s t e n t l y i n h i b i t the c e l l u l a r p r o l i f e r a t i o n of murine L-929 f i b r o b l a s t s . However, no study to date has been performed on g l u c o c o r t i c o i d e f f e c t s on col l a g e n metabolism i n these c e l l s which were of great i n t e r e s t because they had been used to p r e d i c t anti-inflammatory potencies of g l u c o c o r t i c o i d s based on growth i n h i b i t i o n ( B e r l i n e r and Ruhmann, 1967; Brotherton, 1971; R u n i k i s et a l . , 1978). Since c o l l a g e n metabolism i n these c e l l s i s not u n l i k e that of human dermal f i b r o b l a s t s ( G r i b b l e et a\_., 1969), an attempt was made to examine co l l a g e n synthesis of mouse L-929 f i b r o b l a s t s i n the presence of a s t e r o i d . C e l l u l a r p r o l i f e r a t i o n i n g l u c o c o r t i c o i d - t r e a t e d f i b r o b l a s t s of human o r i g i n are, r e p o r t e d l y , not a l t e r e d c o n s i s t e n t l y i n kind and degree. Very few biochemical analyses have been performed on c o l l a g e n s y n t h e s i s i n g l u c o c o r t i c o i d - t r e a t e d a d u l t human dermal f i b r o b l a s t s . Thus, t h i s work was al s o undertaken to attempt to c l a r i f y the actions of a g l u c o c o r t i c o i d on growth and co l l a g e n production i n adult human dermal f i b r o b l a s t s . The c e l l c u l t u r e system, as opposed to organ c u l t u r e system, was chosen to study the e f f e c t s of a g l u c o c o r t i c o i d . The former system permits the poten-t i a l l y obscuring homeostatic mechanisms and humoral f a c t o r s to be bypassed. Furthermore, only one c e l l type i s exposed to a given c o n c e n t r a t i o n of gluco-c o r t i c o i d , thus p e r m i t t i n g q u a n t i t a t i v e measurements to be made. L a s t l y , the c e l l c u l t u r e system i s amenable to m o d i f i c a t i o n s of the environment and so may - 42 -lead to the f u r t h e r understanding of the r o l e of exogenous f a c t o r s on c e l l u l a r p r o l i f e r a t i o n and c o l l a g e n s y n t h e s i s . To study the s y n t h e t i c processes of c o l l a g e n metabolism without i n t e r f e r e n c e from the degradative aspects, serum must be present. Aside from being e s s e n t i a l for maintenance of c e l l growth, attachment and other metabolic a c t i v i t i e s , i t has the added advantage of suppressing collagenase a c t i v i t y due to the presence of ot2-macroglobulin and a l - a n t i t r y p s i n which complex w i t h the enzyme (Birkedale-Hansen et a l . , 1976). The g l u c o c o r t i c o i d of choice was triamcinolone acetonide which has been known to cause dermal atrophy i n man (Kligman and Frosch, 1979) and animal models (Young et al., 1977). The degree of atrophy has been reported to be of great s e v e r i t y (Kligman and Frosch, 1979). As dermal atrophy has been postu-l a t e d to a r i s e from an a b e r r a t i o n of c o l l a g e n metabolism (Jablonska, 1979), t h i s g l u c o c o r t i c o i d was s e l e c t e d to t e s t i t s actions on c o l l a g e n b i o s y n t h e s i s i n v i t r o . An added reason for the choice of t h i s g l u c o c o r t i c o i d i s that triamcinolone acetonide does not bind to t r a n s c o r t i n or c o r t i c o s t e r o i d - b i n d i n g g l o b u l i n i n serum (Runikis et_ al., 1978; Ganong, 1975). To o b t a i n optimal c o l l a g e n s y n t h e s i s , confluent c u l t u r e s were chosen because the a c t i v i t i e s of enzymes res p o n s i b l e for p o s t - t r a n s l a t i o n a l m o d i f i -c a t i o n s are maximal during t h i s phase of growth ( G r i b b l e et_ al^., 1969). However, i f triamcinolone acetonide was not e f f e c t i v e during t h i s growth phase, the l o g a r i t h m i c stage of growth would be s e l e c t e d f o r the adminis-t r a t i o n of the g l u c o c o r t i c o i d . U n l i k e many of the studies mentioned (Ponec et a l . , 1977a,b; Saarni and Tammi, 1978) where i n c o r p o r a t i o n of p r o l i n e i n t o hydroxyproline was used as an index of c o l l a g e n s y n t h e s i s , t h i s present study employed a d i f f e r e n t means of q u a n t i t a t i n g c o l l a g e n production, namely, the use of b a c t e r i a l collagenase. - 43 -This enzyme s e l e c t i v e l y degrades c o l l a g e n . The i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n s was examined i n medium and c e l l u l a r f r a c t i o n s , independently of the h y d r o x y l a t i o n process. To seek co n f i r m a t i o n of r e s u l t s obtained, r a d i o a c t i v e p r o t e i n s c o n t a i n i n g c o l l a g e n were subjected to pepsin treatment and electrophoresed i n polyacrylamide gels which were s l i c e d and counted for r a d i o a c t i v i t y . - 44 -MATERIALS AND METHODS A. MATERIALS Triamcinolone acetonide, a g i f t from E.R. Squibb and Sons, Montreal, P.Q., was d i s s o l v e d i n propylene g l y c o l at a concentration of 116 or 98 yg per ml and stored i n the dark at 4°C. The concentration was confirmed during the course of experimentation by g a s - l i q u i d chromatography (Au, R u n i k i s , Abbott, unpublished procedure). 3 G-( H) - L - p r o l i n e of s p e c i f i c a c t i v i t y 677 C i per mol i n 2% (v/v) ethanol 14 was purchased from Amersham-Searle Corp., O a k v i l l e , Ont.; U-( C ) - L - p r o l i n e of s p e c i f i c a c t i v i t y 225 C i per mol i n 0.01 N-HC1 was purchased from New England Nuclear (Canada) L t d . , Lachine, P.Q.; a c e t y l - l a b e l l e d c o l l a g e n of s p e c i f i c a c t i v i t y 0.3 C i per kg was a g i f t from M. Gisslow and B.C. McBride, Dept. of Micr o b i o l o g y (1975). Collagenous p r o t e i n s were estimated using c h r o m a t o g r a p h i c a l l y - p u r i f i e d C l o s t r i d i u m h i s t o l y t i c u m collagenase (Form I I I ) (Advanced B i o f a c t o r s Corp., Lynbrook, NY, USA). T w i c e - r e c r y s t a l l i z e d pepsin containing approximately 2,500 u n i t s per mg was purchased from Worthington Biochemical Corporation, Freehold, NJ, USA. A l l other chemicals were the purest grade commercially a v a i l a b l e . B. CELL LINES Stock L-929 mouse dermal f i b r o b l a s t s were obtained from Flow L a b o r a t o r i e s , Inglewood, CA, USA. Human dermal f i b r o b l a s t s , derived from explants of a biopsy from a healthy male i n h i s l a t e twenties, were used i n passages 5 to 8. C e l l s not required immediately were t r a n s f e r r e d to p l a s t i c v i a l s c o n t a i n i n g medium plus 20% (v/v) f o e t a l bovine serum and 10% (v/v) dimethyl sulphoxide and stored i n l i q u i d n i t r o g e n . - 45 -C. CELL CULTURE The c e l l s were maintained i n Dulbecco's Modified Eagle's Medium c o n t a i n i n g L-glutamine but no p r o l i n e (Grand I s l a n d B i o l o g i c a l Co., Berkeley, CA, USA), supplemented with 1.8 g NaHCOg per l i t r e , 100 u n i t s of p e n i c i l l i n per ml, 100 Ug of streptomycin per ml (Grand I s l a n d B i o l o g i c a l Co., Berkeley, CA, USA) and 15% (v/v) f o e t a l bovine serum (Flow L a b o r a t o r i e s , Inglewood, CA, USA). Stock c e l l s were maintained and grown to confluency at 37°C i n 5% (v/v) C02~95% (v/v) a i r i n 100 mm p l a s t i c c u l t u r e dishes (Falcon P l a s t i c s , F i s h e r S c i e n t i f i c Co., L t d . , Montreal, P.Q.). The medium was changed twice a week. Confluent c e l l s were tre a t e d for 10 min at 37°C with 0.25% (w/v) t r y p s i n i n Hank's buffered s a l i n e s o l u t i o n (Grand I s l a n d B i o l o g i c a l Co., Berkeley, CA, USA), detached by gentle scraping w i t h a policeman, then disaggregated by drawing up and e x p e l l i n g the suspension repeatedly w i t h a pasteur p i p e t t e . C e l l s from s e v e r a l p l a t e s were pooled for each experiment. Mouse (10^) or human (1.5 x 10 ) c e l l s were seeded i n t o r o l l e r b o t t l e s 2 of surface area 780 cm c o n t a i n i n g 150 ml medium supplemented with 15% (v/v) o f o e t a l bovine serum and incubated at 37 C. C e l l u l a r morphology was examined d a i l y ; those b o t t l e s showing i n f e c t i o n , c e l l u l a r degeneration or abnormally retarded growth were discarded. The medium was changed once every three or four days. To expose the c e l l s to triamcinolone acetonide, the medium was replaced w i t h f r e s h medium con t a i n i n g the supplements, 50 Ug a s c o r b i c a c i d (J.T. Baker Chemical Co., P h i l l i p s b e r g , NJ, USA) per ml and triamcinolone acetonide i n propylene g l y c o l to a f i n a l c oncentration of 0.1 Ug per ml; simultaneous preparations of c o n t r o l b o t t l e s were i d e n t i c a l but contained no g l u c o c o r t i c o i d . - 46 -F o r t y - e i g h t hours f o l l o w i n g the i n i t i a l a d m i n i s t r a t i o n of triam c i n o l o n e acetonide to the c e l l s , the medium was replaced w i t h 50 ml of medium con t a i n i n g the g l u c o c o r t i c o i d or propylene g l y c o l , a s c o r b i c a c i d , serum, 10 3 14 yC i of H- or C-L-proline and 50 yg of the lathyrogen, beta-amino-p r o p i o n i t r i l e (Sigma Co., St. L o u i s , MO, USA), per ml to prevent the formation of c r o s s - l i n k s i n the c o l l a g e n . The c e l l s were incubated f o r a f u r t h e r twenty-four hours. D. CELL HARVESTING, COUNTING AND SIZING A f t e r i n c u b a t i o n w i t h r a d i o a c t i v e - l a b e l l e d p r o l i n e , the medium was de-canted, the c e l l l a y e r s washed twice w i t h 10 ml serum-free medium and the washes pooled w i t h the decanted medium. The c e l l l a y e r was t r y p s i n i z e d , disaggregated and suspended i n 100 ml serum-free medium. W i t h i n an hour of detachment, a l i q u o t s of suspended c e l l s were counted w i t h a haemacytometer and an impedance-triggered e l e c t r o n i c counter. One hundred to four hundred c e l l s were counted i n each of four charges of the counting chamber of a haemacyto-meter; 1500 to 3000 c e l l s were counted i n each of f i v e a l i q u o t s i n an e l e c t r o n i c counter (Celloscope Model 112 CLTH/RWP, P a r t i c l e Data Inc., Cordis L a b o r a t o r i e s , Miami, FL, USA). The s i z e d i s t r i b u t i o n of both human and mouse f i b r o b l a s t s was determined by a f i l a r micrometer. Harvested c e l l s were spotted on a haemacytometer and measured under high dry m a g n i f i c a t i o n using an eyepiece micrometer c a l i b r a t e d w i t h a stage micrometer. A t o t a l of 125 to 200 c e l l s were measured. S i z e d i s t r i b u t i o n s were also determined using an e l e c t r o n i c counter i n which the impedance was a l t e r e d to provide a range of thresholds corresponding to d i f f e r e n t c e l l diameters. - 47 -E. ISOLATION OF RADIOACTIVE-LABELLED COLLAGEN AND TREATMENT WITH COLLAGENASE A l l manipulations were c a r r i e d out at 4°C. Collagen was i s o l a t e d using a m o d i f i c a t i o n of the procedure of U i t t o et <al. (1976). 100 Ug of c y c l o -heximide (Sigma Co., St. L o u i s , MO, USA) per ml and 1 mM a , a ' - d i p y r i d y l ( F i s h e r S c i e n t i f i c Co., V a n e , B.C.) were added to the pooled medium to i n h i b i t p r o t e i n synthesis and h y d r o x y l a t i o n , r e s p e c t i v e l y . P r o t e o l y s i s was i n h i b i t e d by the presence of 10 mM N-ethylmaleimide ( P i e r c e Chemical Co., Rockford, IL, USA), 0.3 mM a - t o l u e n e s u l p h o n y l f l u o r i d e (Sigma Chemical Co., St. L o u i s , MO, USA) and 20 mM disodium ethylenediaminetetraacetate ( F i s h e r S c i e n t i f i c Co., Vane., B.C.). A f t e r removal of c e l l u l a r debris by c e n t r i f u g a t i o n at 9,000 g f o r 30 min, the collagenous p r o t e i n s were p r e c i p i t a t e d by the a d d i t i o n of ammonium sulphate as a saturated s o l u t i o n to give a concentration of 20% (w/v) (Church et a l , 1973). The p r e c i p i t a t e was c o l l e c t e d a f t e r 16. h by c e n t r i f u g a t i o n at 18,000 g_ for 1 hour, washed twice with 20% (w/v) ammonium sulphate, d i s s o l v e d i n 2.0 ml 0.15 M NaCl co n t a i n i n g 0.01 M C a C l 2 and 0.05 M T r i s . H C l , pH 7.6, then d i a l y z e d against the foregoing buffered s a l i n e . A 1.0 ml p o r t i o n of the o re t e n t a t e was removed, weighed, and digested f or 15 hours at 37 C with 100 Ug of b a c t e r i a l collagenase i n the presence of 2.5 mM N-ethylmaleimide. The d i g e s t i o n was h a l t e d by the a d d i t i o n of 0.1 ml 0.25 M EDTA and the small collagenous peptides were e l i m i n a t e d by d i a l y s i s versus 0.15 M NaCl c o n t a i n i n g 0.05 M T r i s . H C l , pH 7.6. Co n t r o l samples i n which the b a c t e r i a l collagenase was absent were run simultaneously. The d i f f e r e n c e between the counts observed i n the presence and absence of collagenase d i g e s t i o n was used as an index of the amount of r a d i o a c t i v e l y - l a b e l l e d c o l l a g e n synthesized. Treatment of the c e l l u l a r f r a c t i o n consisted of c e n t r i f u g a t i o n at 9,000 g for 20 min to sediment the c e l l s . The p e l l e t from one b o t t l e was suspended i n - 48 -10 ml 0.5 M a c e t i c a c i d and placed on a shaker overnight. The e x t r a c t , c o l l e c t e d by c e n t r i f l i g a t i o n , was d i a l y z e d against 0.02 M disodium phosphate. The i n s o l u b l e collagenous p r o t e i n s were c o l l e c t e d by c e n t r i f u g a t i o n at 9,000 g for 30 min, d i s s o l v e d i n 2.0 ml 0.15 M NaCl c o n t a i n i n g 0.01 M C a C l 2 and 0.05 M T r i s . H C l , pH 7.6, then d i a l y z e d against the same b u f f e r . Enzymatic d i g e s t i o n of 1.0 ml portions of the r e t e n t a t e was done as before. F. PEPSINIZATION OF NATIVE COLLAGEN Collagenous p r o t e i n s i s o l a t e d from both c e l l s and medium, by the foregoing procedure, were d i s s o l v e d i n 0.5 M a c e t i c a c i d . To ensure maximal cleavage of the t e l o p e p t i d e s and complete d i g e s t i o n of the noncollagenous p r o t e i n s , pepsin was added to each sample to a f i n a l concentration of 100 ug per ml (Burke et a l . , 1977). A f t e r d i g e s t i o n for 5 hours at 15°C, the r e a c t i o n was h a l t e d by n e u t r a l i z a t i o n with 0.5 N NaOH. The t e l o p e p t i d e d i g e s t s were then removed by d i a l y s i s against 0.4 M NaCl c o n t a i n i n g 0.1 M T r i s . H C l , pH 7.6. 14 G. IDENTIFICATION AND QUANTITATION OF PEPSINIZED C-LABELLED COLLAGEN IN POLYACRYLAMIDE GELS The procedure used was a m o d i f i c a t i o n of that of Furthmayr and Timpl (1971), Byers et a l . (1974) and Sykes et a l . (1976). Reagent A: To 10 g acrylamide (Matheson, Coleman and B e l l Manufacturing Chemists, Norwood, OH, USA) and 0.26 g N,N 1-methylenebisacrylamide (Eastman Kodak Co., Rochester, NY, USA) d i s s o l v e d i n 50 ml 0.2 M phosphate b u f f e r , pH 7.2, were added 10 ml 2% (w/v) sodium dodecyl sulphate (Matheson, Coleman and B e l l Manufacturing Chemists, Norwood, OH, USA) and 0.1 ml N,N,N',N'-tetramethylethylenediamine (Eastman Kodak Co., Rochester, NY, USA) followed by d i l u t i o n to 100 ml w i t h d i s t i l l e d water. Reagent B: 150 mg ammonium persulphate ( F i s h e r S c i e n t i f i c Co., Vane., BC) were d i s s o l v e d i n 50 ml 0.1 M phosphate b u f f e r , pH 7.2. Both - 49 -reagents A and B were stored at 4°C i n the dark. When equal volumes of so l u t i o n s A and B were mixed, p o l y m e r i z a t i o n occurred w i t h i n two hours. To the c o l l a g e n e x t r a c t s was added one-tenth volume of a t r a c k i n g dye comprising of 50% (w/v) sucrose i n 0.1% (w/v) bromophenol blue. The c o l l a g e n was denatured f or 30 minutes at 50°C before a p p l i c a t i o n of 50 y l to the ge l . Reference type I and type I I I collagens derived from r a t t a i l tendon and human placenta (prepared by Tan, E.M.L. and Tan, S.Y.L.) were tre a t e d s i m i l a r l y and included i n each e l e c t r o p h o r e t i c run. The gels were electrophoresed at 8 mA per g e l i n 0.1 M phosphate b u f f e r , pH 7.2 c o n t a i n i n g 0.1% (w/v) sodium dodecyl sulphate. When the t r a c k i n g dye had migrated to the end of each g e l , the e l e c t r o p h o r e s i s was terminated. The reference gels were removed and stained overnight i n 0.2% (w/v) Coomassie B r i l l i a n t Blue R-250 (Bio-Rad Co., Richmond, CA, USA) i n 50% (v/v) methanol/5% (v/v) a c e t i c a c i d . The unstained r a d i o a c t i v e gels were s l i c e d i n t o 2 mm s l i c e s . Each s l i c e was s o l u b i l i z e d w i t h 0.2 ml 60% p e r c h l o r i c a c i d ( F i s h e r S c i e n t i f i c Co., V a n e , BC) and 0.4 ml 30% (v/v) hydrogen peroxide ( F i s h e r S c i e n t i f i c Co., V a n e , BC) f o r 3 to 5 hours at 60°C (Kobayashi and H a r r i s , 1974) i n separate 22 mm l i n e a r p o l y -ethylene s c i n t i l l a t i o n v i a l s (New England Nuclear (Canada) L t d . , Lachine, PQ),. H. ASSAY OF RADIOACTIVITY OF COLLAGEN For the i n i t i a l experiment with L-929 c e l l s , 1.0 ml of the non-d i a l y z a b l e c o l l a g e n a s e - r e s i s t a n t p r o t e i n was placed i n a p l a s t i c m i n i v i a l (New England Nuclear (Canada) L t d . , Lachine, PQ). An equal volume of 0.15 M NaCl containing 0.05 M T r i s . H C l , pH 7.6 and 4 ml of Aquasol-2 (New England Nuclear (Canada) L t d . , Lachine, PQ) were added and the mixture shaken. These samples were loaded i n a U n i l u x I I l i q u i d s c i n t i l l a t i o n spectrometer (Searle A n a l y t i c Inc., Des P l a i n e s , I L , USA) and counted by a channels r a t i o method at 4°C. - 50 -For the experiment w i t h the human c e l l s , the collagenous p r o t e i n s were counted i n 22 mm l i n e a r polyethylene s c i n t i l l a t i o n v i a l s w i t h 9.0 ml Aquasol-2 i n a Mark I I I l i q u i d s c i n t i l l a t i o n system (Searle A n a l y t i c Inc., Des P l a i n e s , IL, USA) using a channels r a t i o method. 14 The reference standard was 7-( C) benzoic a c i d (New England Nuclear (Canada) L t d . , Lachine, PQ) d i s s o l v e d i n a small excess of saturated NaOH, n e u t r a l i z e d to pH 7.0 and d i l u t e d g r a v i m e t r i c a l l y w i t h d i s t i l l e d water.. A weighed a l i q u o t of benzoic a c i d was adjusted to 0.15 M NaCl buffered to pH 7.6 with 0.05 M T r i s . H C l . 9.0 ml Aquasol-2 was added to a 1.0 ml weighed a l i q u o t of the benzoic a c i d . Volumes of 5 to 50 Ul nitrobenzene were added to produce v a r i a b l e quenching. The absolute counts of the col l a g e n samples were c a l -c ulated from a p l o t of channels r a t i o versus counting e f f i c i e n c y . For the g e l e l e c t r o p h o r e t i c experiment w i t h the L-929 f i b r o b l a s t s , the . . o s l i c e s were s o l u b i l i z e d at 60 C, cooled and 10 ml Aquasol-2 added to each v i a l w i t h thorough mixing. These samples and a set of commercial 14 C-standards (New England Nuclear (Canada) L t d . , Lachine, PQ) were counted as before i n a Mark I I I l i q u i d s c i n t i l l a t i o n system. I . STATISTICAL ANALYSIS Student's t - t e s t was used to compare two groups of r e p l i c a t e s w i t h c e l l s derived from a common pool. A two-way a n a l y s i s of variance was used to analyze experiments where r e p l i c a t e p a i r s of b o t t l e s u t i l i z i n g d i f f e r e n t pools of c e l l s were i n v o l v e d . The l a t t e r technique permitted the separation of the e f f e c t of the g l u c o c o r t i c o i d from that of batch-to-batch v a r i a b i l i t y of the c e l l s . In t h i s a n a l y s i s , the r e s i d u a l i s used as the e r r o r and included both the variance of r e p l i c a t e s and the batch-to-batch d i f f e r e n c e s i n response to the drug. - 51 -EXPERIMENTAL AND RESULTS A. CELL SIZE AND DISTRIBUTION F i b r o b l a s t s were i n i t i a l l y counted i n haemacytometers but were l a t e r counted i n an e l e c t r o n i c counter. The haemacytometer a f f o r d s the advantage of v i s u a l e l i m i n a t i o n of c e l l u l a r debris and nonviable c e l l s but lends i t s e l f to greater v a r i a b i l i t y i n counting. Therefore, the e l e c t r o n i c counter was s u b s t i t u t e d because the counts tended to be more re p r o d u c i b l e and accurate. However, the e l e c t r o n i c counter does not d i s c r i m i n a t e between v i a b l e and nonviable c e l l s . To use the e l e c t r o n i c counter, one had to screen out e l e c t r i c noise to avoid f a l s e elevated counts. The lower and upper thresholds had to be set a c c o r d i n g l y . To do t h i s r e q uired information about c e l l s i z e s . The c e l l s used f o r s i z i n g were mouse L-929 and human dermal f i b r o b l a s t s . A p l a t e of murine L-929 f i b r o b l a s t s was t r y p s i n i z e d and disaggregated. An a l i q u o t of the c e l l suspension was a p p l i e d to the haemacytometer. An eyepiece micrometer was attached to a l i g h t microscope and c a l i b r a t e d w i t h a stage micrometer. Under lOx m a g n i f i c a t i o n 120 mouse L-929 f i b r o b l a s t s were examined. Th e i r diameters ranged from 12 to 25 Um. Approximately 23% of the c e l l s , representing the m a j o r i t y of the c e l l p o p u l a t i o n , measured 16 Um as evident by the prominent peak i n f i g u r e 5. 34% and 43% of the c e l l s f e l l below and above t h i s peak, r e s p e c t i v e l y ( F i g . 5 ) . To confirm these f i n d i n g s , c e l l d i s t r i b u t i o n according to diameter s i z e s was examined i n a c a l i b r a t e d impedence-triggered e l e c t r o n i c counter. A s e r i e s of thresholds was set and the d i f f e r e n c e i n c e l l number between thresholds generated a s i m i l a r d i s t r i -b ution curve of diameter s i z e s as that obtained with micrometer measurements. The c e l l s examined i n the e l e c t r o n i c counter were derived from the same pool - 52 -32 28 h 24 h 20 h 16 h CQ z 8 _ L JL AO 12 14 16 18 20 DIAMETER (/zm) 22 24 26 Figure 5. The d i s t r i b u t i o n of diameters of mouse f i b r o b l a s t s measured by micrometer. C e l l s were harvested, suspended i n medium and the diameters measured i n a wet mount under 10 x m a g n i f i c a t i o n u s i n g a micrometer eyepiece c a l i b r a t e d w i t h a stage micrometer. 120 c e l l s were measured. - 52 -32 r 28 24 C O =! 20 UJ U U _ 0 16 U J C D 1 12 z 8 10 _ l I I : 1 1— 12 14 16 18 20 DIAMETER (pm) 22 24 26 Figure 5. The d i s t r i b u t i o n of diameters of mouse L-929 f i b r o b l a s t s measured  by micrometer. C e l l s were harvested, suspended i n medium and the diameters measured i n a wet mount under 10 x m a g n i f i c a t i o n u s i n g a micrometer eyepiece c a l i b r a t e d w i t h a stage micrometer. 120 c e l l s were measured. - 53 -o f c e l l s o b s e r v e d u n d e r l i g h t m i c r o s c o p e a t l O x m a g n i f i c a t i o n . M e a s u r e m e n t s b y b o t h m e a n s w e r e p e r f o r m e d w i t h i n t h e h o u r o f o n e a n o t h e r . T o a v o i d e r r o r a r i s i n g f r o m c e l l u l a r a g g r e g a t i o n , c o u n t s w e r e c o m p l e t e d n o t l a t e r t h a n a n h o u r f o l l o w i n g i s o l a t i o n a n d d i s a g g r e g a t i o n p r o c e d u r e s . S i m i l a r p r o c e d u r e s w e r e a p p l i e d t o a d u l t h u m a n d e r m a l f i b r o b l a s t s . A p l a t e o f c e l l s was t r y p s i n i z e d a n d d i s a g g r e g a t e d . A n a l i q u o t w a s a p p l i e d t o a h a e m a c y t o m e t e r . U n d e r l O x m a g n i f i c a t i o n , m i c r o m e t e r r e a d i n g s i n d i c a t e d d i a m e t e r s i z e s o f 10 t o 30 Um. O f 126 c e l l s , 46% h a d d i a m e t e r s i z e s o f 18 t o 20 Um. A b o u t 23% o f t h e c e l l s w e r e s m a l l e r t h a n 18 Um a n d 31% l a r g e r t h a n 20 Um ( F i g . 6 ) . C e l l u l a r d i s t r i b u t i o n a c c o r d i n g t o d i a m e t e r s i z e s was t h e n e x a m i n e d w i t h i n an h o u r o f t h e m i c r o m e t e r r e a d i n g s i n a n e l e c t r o n i c c o u n t e r . T h e d i f f e r e n c e i n c e l l n u m b e r s b e t w e e n t h r e s h o l d s w a s c a l c u l a t e d a n d g e n e r a t e d a s i m i l a r d i s t r i b u t i o n c u r v e ( F i g . 7 ) . A s was o b s e r v e d p r e v i o u s l y , a p r o m i n e n t p e a k r e p r e s e n t i n g t h e m a j o r i t y o f c e l l s was o b t a i n e d a t 18 t o 20 Um. T h e d i s t r i b u t i o n c u r v e was f a i r l y r e p r o d u c i b l e w i t h i n two h o u r s o f i s o l a t i o n a n d d i s a g g r e g a t i o n o f c e l l s . Two h o u r s a f t e r i s o l a t i o n t h e r e w a s a s l i g h t d e c r e a s e i n c e l l s m e a s u r i n g 25 t o 30 Um. T h e r e was a c o n c o m i t a n t i n c r e a s e i n t h o s e m e a s u r i n g 12 t o 15 Um. A f t e r 2 h o u r s , c e l l s b e g a n t o a g g r e g a t e g i v i n g r i s e t o e r r o n e o u s c o u n t s . C e l l c o u n t s f o r s u b s e q u e n t e x p e r i m e n t s w e r e p e r f o r m e d w i t h i n a n h o u r o f i s o l a t i o n a n d d i s a g g r e g a t i o n . C o m p a r i s o n o f t h e r a n g e o f d i a m e t e r s i z e s o f m u r i n e L - 9 2 9 a n d h u m a n d e r m a l f i b r o b l a s t s b y b o t h m i c r o m e t r i c a n d e l e c t r o n i c m e a n s i n d i c a t e d t h a t t h e y w e r e s i m i l a r i n s i z e . S e t t i n g s f o r t h e e l e c t r o n i c c o u n t e r w e r e , t h e r e f o r e , i d e n t i c a l . P a r t i c l e s l e s s t h a n 10 Um w e r e e l i m i n a t e d i n c o u n t i n g f o r b o t h c e l l l i n e s . - 54 -co Z 10 -•" 20 DIAMETER (jim) > Figure 6. The d i s t r i b u t i o n of human f i b r o b l a s t s measured by micrometer. C e l l s were harvested, suspended i n medium, and the diameters measured i n a wet mount under 10 x m a g n i f i c a t i o n using a micrometer eyepiece c a l i b r a t e d w i t h a stage micrometer; 126 c e l l s were measured. - 55 -10 20 DIAMETER [pm] 30 Figure 7. The d i s t r i b u t i o n o f diameters o f .human f i b r o b l a s t s measured i n an  e l e c t r o n i c p a r t i c l e counter. C e l l s were harvested, suspended i n medium and the number of c e l l s over a s e r i e s of thresholds was measured i n a c a l i b r a t e d p a r t i c l e counter. The number of c e l l s between thresholds was c a l c u l a t e d by d i f f e r e n c e . The s o l i d l i n e represents counts obtained immediately a f t e r i s o l a t i o n ; the dotted l i n e represents counts determined 2 hours a f t e r i s o l a t i o n . - 56 -B. GLUCOCORTICOID EFFECTS ON CELLULAR PROLIFERATION AND PROLINE INCORPORATION: COLLAGENASE ASSAY STUDIES I. Mouse L-929 F i b r o b l a s t s The study of mouse L-929 f i b r o b l a s t s was i n i t i a t e d before that of the human dermal f i b r o b l a s t s because of the greater ease i n m a i n t a i n i n g the former c e l l s i n c u l t u r e . A l s o , a n a l y s i s of co l l a g e n synthesis i n g l u c o c o r t i c o i d -t r e a t e d murine f i b r o b l a s t s had not been done by other i n v e s t i g a t o r s . Since c o l l a g e n synthesis i n c u l t u r e d f i b r o b l a s t s had been reported to be maximal at l a t e l o g a r i t h m i c phase of growth ( G r i b b l e et a l . , 1969), mouse L-929 f i b r o -b l a s t s were seeded and grown to t h i s stage at which time triamcinolone acetonide was administered to the c e l l s f o r 72 hours. In the f i r s t experiment, A, t r i p l i c a t e c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d b o t t l e s were derived from a common pool of c e l l s . Due to the inconvenience of seeding t r i p l i c a t e samples and the high incidence of contamination, l a t e r experiments, B, c o n s i s t e d of b o t t l e s i n which each p a i r was seeded from a d i f f e r e n t pool of c e l l s . The r e s u l t s of experiments A and B are i n Table I , Pa r t s 1 and 2. At l e a s t four separate c e l l counts were obtained from each sample; counts i n excess of 10 c e l l s were corrected for coincidence. At no time d i d the standard d e v i a t i o n of counts exceed \% of the t o t a l number of c e l l s . The number of c e l l s f l o a t i n g i n each b o t t l e i n the medium was counted but deemed n e g l i g i b l e . In every experiment, triamcinolone acetonide suppressed c e l l u l a r p r o l i f e r a t i o n , thus confirming a previous study ( R u n i k i s et a l . , 1978). In experiment A, a n a l y s i s by Students' t - t e s t showed that suppression of c e l l u l a r p r o l i f e r a t i o n by triamcinolone acetonide was c o n s i s t e n t and s i g n i f i c a n t - 57 -Table I , Pa r t s 1 and 2: The e f f e c t of triamcinolone acetonide on c e l l u l a r p r o l i f e r a t i o n and synthesis of c o l l a g e n a s e - s e n s i t i v e p r o t e i n of confluent mouse L-929 f i b r o b l a s t s . R o l l e r b o t t l e s c o n t a i n i n g 150 ml medium were seeded w i t h 10^ c e l l s and maintained u n t i l the c e l l s reached the l a t e l o g a r i t h m i c phase of growth (4 to 5 days). Medium c o n t a i n i n g a s c o r b i c a c i d and e i t h e r 0.116 Ug triamcinolone acetonide per ml i n propylene g l y c o l (present) or propylene g l y c o l alone (absent) was added and the i n c u b a t i o n continued 48 hours at which time the medium was replaced w i t h 50 ml medium c o n t a i n i n g , i n a d d i t i o n to the above, 3 14 b e t a - a m i n o p r o p i o n i t r i l e and e i t h e r G-( H)- or U-( C ) - L - p r o l i n e . A f t e r 24 hours, the medium was removed, the c e l l l a y e r washed, and the c e l l s harvested and counted. Collagen was i s o l a t e d from both medium and c e l l s ; p o r t i o n s of each f r a c t i o n were tre a t e d with collagenase and d i a l y z e d . The d i g e s t and an undigested c o n t r o l were assayed for r a d i o a c t i v i t y . In experiment A, t r i p l i c a t e c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d samples were derived from a common pool of c e l l s ; i n the subsequent experiments (B), each p a i r of b o t t l e s was derived from a d i f f e r e n t pool of c e l l s . Table I , Part 1: Medium f r a c t i o n C e l l s DPM DPM t o t a l Loss w i t h DPM collagenase-Experiment G l u c o c o r t i c o i d per per p r o t e i n collagenase s e n s i t i v e b o t t l e b o t t l e per c e l l (%) p r o t e i n per c e l l ( x l ( T 6 ) ( x l O 6 ) ( x l O 6 ) Present 44.22 37.97 36.23 45350 59710 55168 1025.55 1572.14 1522.30 12*** 26*** 18*** Absent 174.94 185.57 185.17 214698 269976 156168 1227.27 1454.77 843.33 23*** 48*** 7 P* 0.001  Present 41.62 36196 896.68 75 704.44 Absent 52.08 107840 2070.66 88 1780.77 Present 18.79 42372 2255.03 75 1691.27 Absent 64.56 146725 2272.69 88 1999.97 Present 15.03 44304 2947.70 90 2652.93 Absent 60.80 99569 1637.65 88 1441.13 Present 16.12 33202 2060.00 80 1648.00 Absent 69.68 127774 1833.73 79 1448.65 P** 0.01 * P r o b a b i l i t y that the drug had no e f f e c t , by Student's t - t e s t * * P r o b a b i l i t y that the drug had no e f f e c t , by two-way a n a l y s i s of var i a n c e ***Collagenase d i g e s t i o n at 4°C Table I , Part 2: C e l l u l a r f r a c t i o n C e l l s DPM DPM t o t a l Loss with DPM collagenase-Experiment G l u c o c o r t i c o i d per per p r o t e i n collagenase s e n s i t i v e b o t t l e b o t t l e per c e l l (%) p r o t e i n per c e l l ( x l O - 6 ) ( x l O 6 ) (xlO 6) 44.22 11060 250.11 67 167.57 Present 37.97 11482 287.27 63 180.98 36.23 8700 240.13 64 153.68 174.94 21350 122.04 69 84.21 Absent 185.57 16928 91.22 68 62.03 185.17 19614 105.92 70 74.14 P^ 0.001 0.01 P 0.001 0.01 P 0.001 Present 41.62 5078 122.01 - 59.71 Absent 52.08 4022 77.23 16 18.92 Present 18.79 5610 298.56 20 59.71 Absent 64.56 6430 99.60 19 18.92 Present 15.03 12668 842.85 7 59.00 Absent 60.80 1360 22.37 31 6.93 Present 16.12 218 13.52 48 6.49 Absent 69.68 420 6.03 35 2.11 P** 0.01 0.18 0.85 A and B P** 0.08 0.004 * P r o b a b i l i t y that the drug had no e f f e c t , by Student's t - t e s t * * P r o b a b i l i t y that the drug had no e f f e c t , by two-way a n a l y s i s of variance - 60 -(P = 0.001). The c e l l s used i n experiment B were derived from d i f f e r e n t pools; hence a two-way a n a l y s i s of variance was done. Again, c e l l u l a r p r o l i f e r a t i o n was suppressed markedly and s i g n i f i c a n t l y (P = 0.01) (see Table I , Parts 1 and 2). The i n c o r p o r a t i o n of p r o l i n e i n t o t o t a l p r o t e i n and c o l l a g e n a s e - s e n s i t i v e p r o t e i n was studied to assess the e f f e c t of 0.1 Ug triamcinolone acetonide per ml (0.23 UM) medium on b i o l o g i c a l a c t i v i t i e s other than p r o l i f e r a t i o n . In a l l experiments, triamcinolone acetonide was administered to l a t e l o g a r i t h m i c growing c u l t u r e s for 72 hours; r a d i o a c t i v e - l a b e l l e d p r o l i n e was present during the l a s t 24 hours of i n c u b a t i o n . T r i t i u m - l a b e l l e d p r o l i n e was employed i n i t i a l l y but because of some l o s s of r a d i o a c t i v i t y i n the form of water 14 molecules during h y d r o x y l a t i o n , C-proline was s u b s t i t u t e d . F r a c t i o n s c o n t a i n i n g c o l l a g e n i n medium and c e l l s were i d e n t i f i e d by s u s c e p t i b i l i t y to protease-free C l o s t r i d i u m h i s t o l y t i c u m collagenase (Peterkofsky and D i e g e l -mann, 1971). In Table I , Part 1, p r o l i n e i n c o r p o r a t i o n i n t o t o t a l p r o t e i n per b o t t l e of medium was c o n s i s t e n t l y l e s s w i t h g l u c o c o r t i c o i d treatment. Subse-quent experiments (B) confirmed these r e s u l t s . However, the a c t i v i t y of t o t a l p r o t e i n per c e l l was not c o n s i s t e n t l y lower i n the g l u c o c o r t i c o i d - t r e a t e d b o t t l e s e i t h e r i n experiment (A) or i n subsequent experiments (B) (P = 0.88). P r o l i n e i n c o r p o r a t i o n i n t o the c o l l a g e n a s e - s u s c e p t i b l e p r o t e i n of the f i r s t experiment A could not be measured due to the low f r a c t i o n of r a d i o a c t i v i t y o l o s t w i t h collagenase at 4 C. 50 Ug per ml of collagenase was used i n the foregoing experiment (Table I , P a r t 1). The low losses suggested that the concentration or the temperature or both f a c t o r s were not conducive for maximal enzymatic a c t i v i t y . Consequently, the experiments i n S e c t i o n C were undertaken to determine the c o n d i t i o n s necessary for e f f i c i e n t collagenase d i g e s t i o n . These experiments e s t a b l i s h e d an increase i n a c t i v i t y at 37 UC (see Tables IV and V). The concentration of collagenase used i n subsequent experiments was 100 Ug per ml. G l u c o c o r t i c o i d e f f e c t on the c o l l a g e n a s e - s u s c e p t i b l e p r o t e i n per c e l l of the medium was i n c o n s i s t e n t i n the l a s t four experiments (B) (see Table I , Part 1). Collagenase e l i m i n a t e d 79% to 88% and 75% to 90% of the r a d i o a c t i v e counts i n the c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d b o t t l e s , r e s p e c t i v e l y ; the samples were th e r e f o r e r e l a t i v e l y free of r a d i o a c t i v e - l a b e l l e d noncollagenous p r o t e i n s . Examination of the c e l l l a y e r s i n experiments B showed that the amounts of r a d i o a c t i v i t y per b o t t l e were a l t e r e d i n c o n s i s t e n t l y i n c o n t r o l and e x p e r i -mental groups. However, a c o n s i s t e n t increase i n r a d i o a c t i v i t y per c e l l was observed i n t o t a l (P = 0.18) and c o l l a g e n a s e - s u s c e p t i b l e p r o t e i n (P = 0.85) of triamcinolone acetonide-treated b o t t l e s ; the few samples analyzed d i d not al l o w t h i s o b s e r v a t i o n to be s i g n i f i c a n t s t a t i s t i c a l l y . In the experiment A, the g l u c o c o r t i c o i d treatment enhanced p r o l i n e i n c o r p o r a t i o n i n t o t o t a l and c o l l a g e n a s e - s e n s i t i v e p r o t e i n per c e l l s i g n i f i c a n t l y (0.01 P 0.001). The data for experiments A and B were confirmed by p a i r i n g r e p l i c a t e s of experiment A a r b i t r a r i l y . Although the elevated i n c o r p o r a t i o n of p r o l i n e i n t o t o t a l p r o t e i n per c e l l was not s t a t i s t i c a l l y s i g n i f i c a n t (P = 0.08), that of collagenase-s u s c e p t i b l e p r o t e i n per c e l l was h i g h l y s i g n i f i c a n t (P = 0.004) (see Table I , Part 2). Thus, the observation of a c o n s i s t e n t p o s i t i v e e f f e c t of t r i a m -cinolone acetonide on p r o l i n e i n c o r p o r a t i o n i n t o c o l l a g e n a s e - s u s c e p t i b l e p r o t e i n was more s i g n i f i c a n t when a r b i t r a r i l y - p a i r e d b o t t l e s of experiment A were combined s t a t i s t i c a l l y w i t h those of experiments B. - 62 -I I . A dult Human Dermal F i b r o b l a s t s (a) short-term incubation w i t h triamcinolone acetonide The e f f e c t s of 0.1 Ug triamcinolone acetonide per ml on c e l l u l a r p r o l i f e r -a t i o n and col l a g e n synthesis i n a c e l l l i n e derived from adult human were examined under i d e n t i c a l c o n d i t i o n s to those f o r the mouse L-929 f i b r o b l a s t s . Under these c o n d i t i o n s , triamcinolone acetonide had an e f f e c t on c e l l u l a r p r o l i f e r a t i o n and c o l l a g e n s y n t h e s i s i n the c e l l l a y e r s of the mouse f i b r o -b l a s t s . Therefore, i t was of i n t e r e s t to determine i f human dermal f i b r o -b l a s t s responded s i m i l a r l y to the g l u c o c o r t i c o i d . 6 R o l l e r b o t t l e s were seeded w i t h 1.5 x 10 c e l l s per b o t t l e , 1.5 times more c e l l s per b o t t l e than that r e q u i r e d f o r the previous study. U n l i k e the mouse f i b r o b l a s t s , human f i b r o b l a s t s a t t a i n e d confluency an average of 24 hours l a t e r . The c e l l s were grown to l a t e l o g a r i t h m i c phase of growth at which time tri a m c i n o l o n e acetonide was administered. Triamcinolone acetonide, administered f o r 72 hours during l a t e l o g a r i t h m i c phase of growth, increased c e l l numbers by 11 to 27% i n four of the f i v e experiments, confirming the fin d i n g s of R u n i k i s et a l . (1978); the f i f t h decreased by 17% (Table I I , P a r t s A and B). Therefore, t r i a m c i n o l o n e acetonide had no consi s t e n t e f f e c t on c e l l u l a r p r o l i f e r a t i o n (P = 0.15). The e f f e c t of triamcinolone acetonide on c e l l u l a r p r o l i f e r a t i o n was tested using a two-way a n a l y s i s of va r i a n c e . Since, i n every experiment, the paired b o t t l e s were derived from a separate pool of c e l l s , the d i f f e r e n c e s between batches of c e l l s was also t e s t e d . However, t h i s s t a t i s t i c a l a n a l y s i s pools r e p l i c a t e v a r i a b i l i t y with batch-to-batch d i f f e r -ences i n the response to g l u c o c o r t i c o i d . Since the number of c e l l s i n both the c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d b o t t l e s v a r i e d between 27 and 95 m i l l i o n , the d i f f e r e n c e s between experiments was r e l a t i v e l y large (P = 0.0009). - 63 -Table I I , P a r t s A and B: The e f f e c t of triamcinolone acetonide on c e l l u l a r  p r o l i f e r a t i o n and on the i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e  p r o t e i n by adult human dermal f i b r o b l a s t s . R o l l e r b o t t l e s c o n t a i n i n g 150 ml medium were seeded with 1.5 x 10^ c e l l s and maintained u n t i l the f i b r o b l a s t s had reached the l a t e l o g a r i t h m i c phase o f growth (5 to 6 days) when medium c o n t a i n i n g ascorbic a c i d and e i t h e r 0.116 yg triamcinolone acetonide per ml i n propylene g l y c o l (present) or propylene i g l y c o l alone (absent) was added; the incubation was continued f or 48 h. The medium was replaced w i t h 50 ml medium c o n t a i n i n g , i n a d d i t i o n to the above, 14 b e t a - a m i n o p r o p i o n i t r i l e and U-( C ) - L - p r o l i n e . A f t e r 24 hours, the medium was decanted, the c e l l l a y e r harvested and an a l i q u o t counted f o r c e l l number. F r a c t i o n s c o n t a i n i n g c o l l a g e n were i s o l a t e d from medium and c e l l s ; one p o r t i o n of each f r a c t i o n was treated with b a c t e r i a l collagenase and d i a l y z e d . The digest and undigested portions were assayed for r a d i o a c t i v i t y . Each experiment co n s i s t e d of p a i r e d b o t t l e s derived from a d i f f e r e n t pool of c e l l s . - 64 -Table I I , Part A: Medium f r a c t i o n G l u c o c o r t i c o i d C e l l s per b o t t l e ( x l O - 6 ) DPM per b o t t l e DPM t o t a l p r o t e i n per c e l l ( x l O 6 ) Loss with collagenase (%) DPM collagenase-s e n s i t i v e p r o t e i n per c e l l ( x l O 6 ) Present Ab sent 47.47 42.77 10664 23092 224.65 539.91 53 60 119.06 323.95 Present Absent 28.57 34.58 7822 6926 273.79 200.29 63 56 172.49 112.16 Present Absent 106.01 94.81 51126 75784 482.29 799.28 72 67 347.25 535.52 Present Ab sent 78.88 63.95 60236 49782 763.67 778.40 53 70 404.75 544.88 Present Ab sent 34.73 27.42 41454 8138 1193.47 296.82 56 48 668.34 142.47 P* 0.15 0.79 0.93 * P r o b a b i l i t y that the drug had no e f f e c t , by two-way a n a l y s i s of v a r i a n c e . - 65 -Table I I , Part B: C e l l u l a r f r a c t i o n G l u c o c o r t i c o i d C e l l s per b o t t l e ( x l O - 6 ) DPM per b o t t l e DPM t o t a l prote i n per c e l l ( x l O 6 ) Loss with collagenase (%) DPM collagenase-s e n s i t i v e p r o t e i n per c e l l ( x l O 6 ) Present 47 .47 452 9.52 17 1.62 Ab sent 42.77 304 7.11 33 2.35 Present 28.57 658 23.03 59 13.59 Absent 34.58 1384 40.02 85 34.02 Present 106.01 6106 57.60 69 39.74 Absent 94.81 12388 130.65 81 105.83 Present 78.88 746 21.48 75 16.11 Absent 63.95 3588 56.10 70 39.27 Present 34.73 — _ _ Absent 27.42 — — — — p* 0.15 0.09 0.08 * P r o b a b i l i t y that the drug had no e f f e c t , by two-way a n a l y s i s of v a r i a n c e . - 66 -In t h i s experiment, f i v e b o t t l e s of confluent c u l t u r e s of human dermal 14 f i b r o b l a s t s were tre a t e d with triamcinolone acetonide for 72 hours. U-( C)-L - p r o l i n e was added during the 24 hour pe r i o d p r i o r to h a r v e s t i n g . No c o n s i s -tent increase or decrease was noted i n the dpm per b o t t l e of the medium. Nor was there a c o n s i s t e n t d i f f e r e n c e i n dpm per c e l l between the g l u c o c o r t i c o i d -treated and c o n t r o l samples before or a f t e r d i g e s t i o n by collagenase (P = 0.79 and P = 0.93, r e s p e c t i v e l y ) . Three out of the f i v e b o t t l e s r e g i s t e r e d an i n -. 14 * crease i n the amount of U-( C)-L-prolme incorporated i n t o t o t a l p r o t e i n per c e l l . I n c o r p o r a t i o n was decreased i n the other two. I n t e r e s t i n g l y enough, the i n c o r p o r a t i o n i n t o c o l l a g e n a s e - s u s c e p t i b l e p r o t e i n was s i m i l a r l y a f f e c t e d . Where an increase i n i n c o r p o r a t i o n was r e g i s t e r e d i n t o t a l p r o t e i n of the medium, a s i m i l a r e l e v a t i o n was also obtained w i t h the c o l l a g e n a s e - s u s c e p t i b l e f r a c t i o n . Loss of a c t i v i t y f o l l o w i n g b a c t e r i a l collagenase treatment ranged from 48 to 72%. No d i f f e r e n c e s were found between experiments w i t h i n the c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d samples (P = 0.54) (see Table I I , P a r t A). With g l u c o c o r t i c o i d treatment three out of four b o t t l e s showed an i n h i b -i t i o n , the l a s t showed a s t i m u l a t i o n (P = 0.09) of i n c o r p o r a t i o n of p r o l i n e i n t o t o t a l p r o t e i n of the c e l l l a y e r per b o t t l e . A s i m i l a r trend was also observed when c a l c u l a t i o n s were based on a per c e l l b a s i s . The p r o l i n e i n c o r -p o r a t i o n of both c o n t r o l and experimental groups d i f f e r e d from experiment to experiment (P = 0.005). Triamcinolone acetonide caused a c o n s i s t e n t depression i n the i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n per c e l l i n a l l four b o t t l e s (P = 0.08). More experiments may have made t h i s observation s t a t i s t i c a l l y s i g n i f i c a n t . B a c t e r i a l collagenase d i g e s t i o n reduced the r a d i o -a c t i v i t y from 33% to 85% i n the c o n t r o l and 17% to 75% i n the g l u c o c o r t i c o i d -t r e a t e d b o t t l e s (see Table I I , Part B). - 67 -(b) Long-term inc u b a t i o n w i t h triamcinolone acetonide The foregoing experiments were conducted with confluent f i b r o b l a s t s because the enzymes responsible f o r the p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s of c o l l a g e n and the amount of c o l l a g e n synthesized were maximal at that stage of growth ( G r i b b l e et a l . , 1969). Since human f i b r o b l a s t s have been known to respond to g l u c o c o r t i c o i d s only during the l o g a r i t h m i c phase of growth (Kruse £t al., 1978) l a t e r experiments using p a i r e d b o t t l e s of c e l l s , one t r e a t e d w i t h t r i a m -cinolone acetonide and the other a c o n t r o l , were conducted with the a d d i t i o n of the g l u c o c o r t i c o i d at t h i s growth phase to determine i f the c e l l numbers would be a l t e r e d c o n s i s t e n t l y and the p r o l i n e i n c o r p o r a t i o n suppressed more markedly. Saarni and Tammi (1978) reported a minimum i n c u b a t i o n requirement of 90 hours to suppress p r o l i n e i n t o hydroxyproline and p r o t e i n s i n f o e t a l human f i b r o b l a s t s ; hence, i n the f o l l o w i n g experiments, the f i b r o b l a s t s were exposed to triamcinolone acetonide 24 hours a f t e r seeding f o r a prolonged period of 180 hours. Triamcinolone acetonide had no c o n s i s t e n t e f f e c t on c e l l numbers i n four b o t t l e s (P = 0.44). The c e l l numbers, among the c o n t r o l b o t t l e s , ranged from 7 to 23 m i l l i o n ; those of the g l u c o c o r t i c o i d - t r e a t e d b o t t l e s , 6.5 to 25 m i l l i o n . There was a s i g n i f i c a n t v a r i a t i o n from experiment to experiment which was independent of the g l u c o c o r t i c o i d (P = 0.03). Examination of p r o l i n e i n c o r p o r a t i o n i n t o the medium showed that the i n c o r p o r a t i o n of r a d i o a c t i v e - l a b e l l e d p r o l i n e i n t o t o t a l p r o t e i n per b o t t l e was increased i n one of the four b o t t l e s treated w i t h triamcinolone acetonide. The other three r e g i s t e r e d a d e c l i n e . C a l c u l a t e d on a per c e l l b a s i s , no c o n s i s t e n t e f f e c t was observed e i t h e r on p r o l i n e i n c o r p o r a t i o n i n t o t o t a l p r o t e i n (P = 0.25) or on that of c o l l a g e n a s e - s u s c e p t i b l e p r o t e i n (P = 0.40) - 68 -(Table I I I , P a r t A). Two of the four b o t t l e s showed an increase i n t o t a l p r o t e i n and c o l l a g e n a s e - d i g e s t i b l e p r o t e i n per c e l l . Where an increase i n t o t a l p r o t e i n per c e l l was obtained, so was there a s i m i l a r increase i n the c o l l a g e n a s e - d i g e s t i b l e p r o t e i n per c e l l . F o l l o w i n g collagenase d i g e s t i o n 49% to 75% and 47% to 67% of the counts were removed from the c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d samples, r e s p e c t i v e l y . Between experiments, no d i f f e r -ence i n p r o l i n e i n c o r p o r a t i o n i n t o t o t a l p r o t e i n (P = 0.12) and collagenase-s e n s i t i v e p r o t e i n (P = 0.21) were present. U n l i k e the i n i t i a l experiments w i t h short-term i n c u b a t i o n of the glu c o -c o r t i c o i d , no c o n s i s t e n t trend was observed i n the c e l l l a y e r , e i t h e r i n p r o l i n e i n c o r p o r a t i o n per c e l l i n t o t o t a l (P = 0.71) or i n t o collagenase-s u s c e p t i b l e p r o t e i n (P =0.31). When subjected to collagenase d i g e s t i o n , 72% to 90% of the counts were d i a l y z a b l e i n the c o n t r o l , but only 60% to 72% were i n the g l u c o c o r t i c o i d - t r e a t e d samples, i n d i c a t i n g l e s s e r p u r i t y of c o l l a g e n i n the l a t t e r (Table I I I , Part B). Although, no s t a t i s t i c a l a n a l y s i s could be done on p o s s i b l e v a r i a t i o n of g l u c o c o r t i c o i d e f f e c t between experiments, some glucocorticoid-independent v a r i a t i o n was noted i n the amount of r a d i o a c t i v e p r o l i n e incorporated i n t o t o t a l (P = 0.005) and c o l l a g e n a s e - d i g e s t i b l e p r o t e i n s (P = 0.09). C. DETERMINATION OF CONDITIONS FOR EFFICIENT COLLAGENASE DIGESTION In the i n i t i a l experiment w i t h murine L-929 f i b r o b l a s t s , collagenase d i g e s t i o n was conducted at a concentration of 50 yg per ml of c o l l a g e n s o l u t i o n at 4°C. A small p r o p o r t i o n of r a d i o a c t i v i t y was l o s t from the medium f r a c t i o n c o n t a i n i n g c o l l a g e n (Table I , Part A). Hence, the temperature - 69 -Table I I I , P a r t s A and B: The e f f e c t of prolonged exposure to triamcinolone  acetonide on c e l l u l a r p r o l i f e r a t i o n and the synthesis of c o l l a g e n of human  dermal f i b r o b l a s t s . 6 R o l l e r b o t t l e s c o n t a i n i n g 150 ml medium were seeded with 1.5 x 10 c e l l s and maintained f o r 24 hours, when replacement medium was added c o n t a i n i n g e i t h e r 0.098 yg triamcinolone acetonide per ml i n propylene g l y c o l (present) or propylene g l y c o l alone (absent) and the in c u b a t i o n continued f o r 96 hours. The replacement medium contained 100 yg ascorbic a c i d per ml i n a d d i t i o n to the above. A f t e r a f u r t h e r 84 hours, replacement medium was added c o n t a i n i n g 50 yg asc o r b i c a c i d per ml and, i n a d d i t i o n to the above, beta-14 a m i n o p r o p i o n i t r i l e and U-( C ) - L - p r o l i n e . A f t e r 24 h, the medium was removed, the c e l l s washed and harvested and those over 10 ym diameter counted e l e c t r o n i c a l l y . F r a c t i o n s c o n t a i n i n g c o l l a g e n were i s o l a t e d from medium and c e l l s ; one p o r t i o n of each f r a c t i o n was exposed to collagenase and d i a l y z e d . The digest and undigested c o n t r o l were assayed for r a d i o a c t i v i t y . Each experiment co n s i s t e d of p a i r e d b o t t l e s derived from a d i f f e r e n t pool of c e l l s . - 70 -Table I I I , P a r t A: Medium f r a c t i o n G l u c o c o r t i c o i d C e l l s per b o t t l e ( x l O - 6 ) DPM per b o t t l e DPM t o t a l p r o t e i n per ce 11 ( x l O 6 ) Loss with collagenase (%) DPM collagenase-s e n s i t i v e p r o t e i n per c e l l ( x l O 6 ) Present 6.51 446942 68,663.40 67 46004.48 Absent 16.39 677940 41,365.11 49 20268.90 Present 8.72 480544 55,123.18 47 25907.89 Ab sent 7.21 114290 15,859.21 60 9436.23 Present 25.75 149620 5,811.36 52 3021.91 Ab sent 23.42 352136 15,034.79 75 11276.09 Present 11.00 192234 17,475.82 48 8388.39 Absent 13.22 273972 20,721.29 72 14919.33 0.44 0.25 0.40 * P r o b a b i l i t y that the drug had no e f f e c t , by two-way a n a l y s i s of variance - 71 -Table I I I , P a r t B: C e l l u l a r f r a c t i o n C e l l s DPM DPM t o t a l Loss wit h DPM collagenase-G l u c o c o r t i c o i d per per p r o t e i n collagenase s e n s i t i v e b o t t l e b o t t l e per c e l l (%) p r o t e i n per c e l l ( x l O - 6 ) ( x l O 6 ) ( x l O 6 ) Present 6.51 7380 1133.78 72 816.32 Absent 16.39 12242 746.96 72 537.81 Present 8.72 12130 1391.43 70 967.04 Ab sent 7.21 11924 1654.61 90 1489.15 Present 25.75 26606 1033.40 60 620.04 Ab sent 23.42 37486 1600.5 79 1264.40 Present 11.00 5418 492.55 62 305.38 Absent 13.22 4558 344.7 86 296.44 P* 0.44 0.71 0.31 * P r o b a b i l i t y that the drug had no e f f e c t , by two-way a n a l y s i s of variance - 72 -and concentration of enzyme were test e d to determine co n d i t i o n s r e q u i r e d f o r increased collagenase d i g e s t i o n . The a c t i v i t y of b a c t e r i a l collagenase was tested by two methods: (1) formation of d i a l y z a b l e hydroxyproline (Woessner, 1961) from p u r i f i e d p l a c e n t a l c o l l a g e n as the s u b s t r a t e , (2) formation of d i a l y z a b l e r a d i o a c t i v i t y from a c e t y l a t e d c o l l a g e n . In the f i r s t method, t r i p l i c a t e samples were employed. o Zero, 50, 250 and 1250 Ug of b a c t e r i a l collagenase per ml were tested at 4 C and 37°C. In the absence of the enzyme, the amount of d i a l y z a b l e hydroxy-p r o l i n e was n e g l i g i b l e . Therefore, no other proteases appear to be present to dige s t c o l l a g e n . At 50 Ug of enzyme per ml at 4°C, no d i g e s t i o n of c o l l a g e n was apparent, i n d i c a t i n g that the concentration or temperature or both were i n s u f f i c i e n t f o r degradation of c o l l a g e n . Increasing the temperature from 4°C to 37°C r e s u l t e d i n a 75 to 80% l o s s of c o l l a g e n . I n c r e a s i n g the enzyme concentration from 50 to 250 Ug per ml d i d not have any e f f e c t at 4°C. But at 37°C, the l o s s of co l l a g e n by enzymatic d i g e s t i o n ranged from 75 to 83%. S i m i l a r observations were made with 1250 Ug enzyme per ml at 4°C and 37°C. Concentrations exceeding 50 Ug per ml d i d not enhance the o degradation of co l l a g e n at 37 C. Temperature manipulation had a profound e f f e c t on the amount of c o l l a g e n digested (Table I V ) . Several i n v e s t i g a t o r s use b a c t e r i a l collagenase at a concentration of 100 ug per ml (Peterkofsky e_t a l . , 1971; P r i e s t l e y , 1978). Hence a second method was invoked to confirm the foregoing f i n d i n g s . Using a c e t y l a t e d collagen as the s u b s t r a t e , 100 Ug b a c t e r i a l collagenase per ml at 4°C removed n e g l i g i b l e r a d i o a c t i v i t y ; at 37°C, 85% to 87% of the r a d i o a c t i v i t y was e l i m i n a t e d , thus confirming the increased a c t i v i t y of b a c t e r i a l collagenase at t h i s temperature and concentration (see Table V). - 73 -Table IV: The a c t i v i t y of b a c t e r i a l collagenase at d i f f e r e n t concentrations  and temperatures. 0.130 to 0.180 ug of p u r i f i e d p l a c e n t a l c o l l a g e n , a mixture of Types I and I I I , per ml were exposed to 0, 50, 250, or 1250 Ug b a c t e r i a l collagenase ( C l o s t r i d i u m h i s t o l y t i c u m ) per ml at 4 or 37°C for 24 hours. The r e a c t i o n was terminated w i t h EDTA and the degradation products e l i m i n a t e d by d i a l y s i s against 0.15 M NaCl c o n t a i n i n g 0.05 M T r i s HCl, pH 7.6. The r e t e n t a t e was analyzed f o r hydroxyproline. Table IV. The a c t i v i t y of b a c t e r i a l collagenase at d i f f e r e n t concentrations and temperatures. 4°C 37°C Enzyme Expt. A Expt. B Expt. A Expt. B ug/ml ug/ml c o l l a g e n ug/ml c o l l a g e n ug/ml c o l l a g e n ug/ml c o l l a g e n 0.176 0.137 0.151 0.138 0 0.158 0.138 0.166 0.143 0.156 0.134 0.156 0.138 0.166 0.140 0.039 0.033 50 0.159 0.141 0.038 0.035 0.153 0.133 0.040 0.035 0.166 0.140 0.028 0.034 250 0.166 0.137 0.029 0.033 0.166 0.138 0.031 0.033 1250 0.138 0.137 0.026 0.028 0.028 - 75 -Table V: Concentration and temperature-dependent degradation of a c e t y l a t e d  c o l l a g e n by b a c t e r i a l collagenase. S i m i l a r amounts of a c e t y l a t e d c o l l a g e n was exposed to 100 Ug b a c t e r i a l o o . . . . collagenase per ml at 4 or 37 C. The r e a c t i o n was terminated by a d d i t i o n of EDTA. The degraded products were removed by d i a l y s i s versus 0.15 M-NaCl con t a i n i n g 0.05 M T r i s HC1, pH 7.6. The re t e n t a t e s were assayed for t h e i r r a d i o a c t i v i t y . - 76 -Table V. Concentration and temperature-dependent degradation of a c e t y l a t e d  c o l l a g e n by b a c t e r i a l collagenase. Temperature 4°C 37°C cpm/ml cpm/ml Co n t r o l 20992 20938 0 ug/ml 22059 22201 enzyme 100 Ug/ml 20899 2892 enzyme 21496 3119 2779 - 77 -D. POLYACRYLAMIDE GEL ELECTROPHORETIC STUDIES I. R a t i o n a l e Triamcinolone acetonide a f f e c t e d the i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n i n the c e l l l a y e r of mouse L-929 f i b r o b l a s t s . A c o n s i s t e n t , s t a t i s t i c a l l y s i g n i f i c a n t increase i n p r o l i n e i n c o r p o r a t i o n was obtained. To o b t a i n c o n f i r m a t i o n of the i d e n t i t y of t h i s p r o t e i n , p o l y a c r y l -amide g e l e l e c t r o p h o r e s i s was employed and an attempt was made to q u a n t i t a t e the amount of r a d i o a c t i v i t y of the peaks or bands. Reference collagens con-s i s t i n g of p u r i f i e d u n l a b e l l e d type I collagens from human placenta and r a t -t a i l tendon as w e l l as co l l a g e n l a b e l l e d by methylation were subjected to e l e c t r o p h o r e s i s w i t h the experimental samples to permit i d e n t i f i c a t i o n of the p r o t e i n i n the l a t t e r samples. I I . Methodological Tests f or E f f e c t s of S t a i n i n g and D e s t a i n i n g P o l y a c r y l - amide Gels The r e c o v e r i e s of a c e t y l a t e d c o l l a g e n from stained and destained gels were compared with those of unstained gels (see Table V I ) . The recovery of the r a d i o a c t i v i t y added to the gels was 80% to 83% i n the t r i p l i c a t e samples of unstained g e l s , whereas only 8% to 18% of the a c t i v i t y was recovered a f t e r s t a i n i n g w i t h Coomassie Blue and d e s t a i n i n g i n 7% (v/v) a c e t i c a c i d (Table V I ) . Low re c o v e r i e s were a l s o obtained by Kerwar et a l . (1972) who recovered 55% of the added counts from gels stained w i t h Amido-Schwartz s t a i n . P o s s i b l e reasons f o r low rec o v e r i e s are l o s s of r a d i o a c t i v e m a t e r i a l by e l u t i o n during s t a i n i n g and d e s t a i n i n g procedures and quenching due to the Coomassie Blue s t a i n of the co l l a g e n bands. Thus, subsequent gels were not sta i n e d before s l i c i n g and d i g e s t i o n of g e l s l i c e s . - 78 -Table VI. Recovery of r a d i o a c t i v e c o l l a g e n from polyacrylamide g e l s . Known amounts of a c e t y l a t e d c o l l a g e n were applied to d u p l i c a t e p o l y a c r y l -amide g e l s . Upon completion of the e l e c t r o p h o r e t i c run, one gel of each p a i r was stained w i t h 0.2% (w/v) Coomassie Blue R-250 and destained i n 7% (v/v) a c e t i c a c i d . A l l gels were cut i n t o s l i c e s 2mm t h i c k , each s l i c e was digested with 0.2ml 60% (w/v) p e r c h l o r i c a c i d and 0.4ml 30% (v/v) hydrogen peroxide for 3 to 5h at 60°C i n separate s c i n t i l l a t i o n v i a l s . 9.0 ml Aquasol-2 was added to each v i a l . Samples were then assayed f o r r a d i o a c t i v i t y . - 79 -Table VI. Recovery of r a d i o a c t i v e c o l l a g e n from stained and unstained  polyacrylamide g e l s . R a d i o a c t i v i t y (d.p.m.) Ap p l i e d Recovered Recovery (%) Stained 96491.16 8023.20 8.3 12413.72 2263.40 18.2 14281.27 1688.70 11.8 Unstained 96401.16 76511.10 79.6 89785.21 74515.70 82.9 12633.40 9636.70 76.3 - 80 -I I I . Recovery of Radioactive P r o t e i n s F o l l o w i n g Pepsin Treatment of P r o t e i n s  Containing Collagen. F o l l o w i n g pepsin treatment of pr o c o l l a g e n , t e l o p e p t i d e s were removed by d i a l y s i s against 0.4 M NaCl co n t a i n i n g 0.1 M T r i s . HC1, pH 7.6. The d i a l y s i s s o l u t i o n was replaced w i t h 0.1 M a c e t i c a c i d and t h e r e a f t e r the proteins were l y o p h i l i z e d . An unsuccessful attempt was made to d i s s o l v e the l y o p h i l i z e d product i n 0.5 M a c e t i c a c i d before a p p l i c a t i o n to polyacrylamide g e l s . There was incomplete s o l u b i l i z a t i o n of the m a t e r i a l i n 0.5 M a c e t i c a c i d . M o d i f i -cations i n the procedure were i n s t i g a t e d to ensure complete s o l u b i l i z a t i o n . The e f f i c a c y of the procedures adopted was assessed by monitoring the r a d i o -a c t i v i t y at each step (see F i g . 8). T r i p l i c a t e c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d samples were t r e a t e d as f o l l o w s : p r o t e i n s c o n t a i n i n g c o l l a g e n i s o l a t e d from the medium were t r e a t e d w i t h pepsin; the digest was n e u t r a l i z e d and d i a l y z e d . The cloudy r e t e n t a t e was c e n t r i f u g e d and the supernate A removed. To the p r e c i p i t a t e C was added 2 ml of 0.5 M a c e t i c a c i d . The r e s u l t a n t cloudy suspension was c e n t r i f u g e d , the supernate B decanted and the p r e c i p i t a t e D was d i s s o l v e d i n 100 y l of 0.01 M phosphate b u f f e r , pH 7.2, c o n t a i n i n g 0.2 (w/v) sodium dodecyl sulphate and 2 M o urea, and heated at 60 for 30 minutes. The p r e c i p i t a t e d i s s o l v e d . Both supernatant f r a c t i o n s (A and B) and the d i s s o l v e d p r e c i p i t a t e were pooled and l y o p h i l i z e d ; the product was d i s s o l v e d i n 100 y l of the foregoing phosphate budfer and a p p l i e d to polyacrylamide gels (see F i g . 8 and Table V I I ) . C e l l u l a r p r oteins c o n t a i n i n g c o l l a g e n were also t r e a t e d by t h i s procedure. C e l l s from a common pool were seeded i n s i x r o l l e r b o t t l e s of which three were c o n t r o l and three were t r e a t e d w i t h t r i a m c i n o l o n e acetonide -The c e l l s from each group were pooled (see Table V I I ) . - 81 -Figure 8. Treatment of c o l l a g e n - r i c h f r a c t i o n s of c e l l u l a r l a y e r s f o r  polyacrylamide g e l e l e c t r o p h o r e s i s . Procedure 1. PROTEINS CONTAINING COLLAGEN Pepsin d i g e s t i o n D i a l y s i s C e n t r i f u g a t i o n SUSPENSION SUPERNATE A PRECIPITATE C 0.5 M HOAc C e n t r i f u g a t i o n SUPERNATE B PRECIPITATE D D i s s o l v e L y o p h i l i z a t i o n Disso l v e SOLUTION E l e c t r o p h o r e s i s GEL SLICES Count s l i c e s - 82 -Table V I I . Recovery of the r a d i o a c t i v i t y of p r o t e i n s c o n t a i n i n g c o l l a g e n from  polyacrylamide g e l s . P r o t e i n s c o n t a i n i n g c o l l a g e n were treated with pepsin. The digest was n e u t r a l i z e d , d i a l y z e d and c e n t r i f u g e d . Supernate A was removed and 2 ml of 0,5 M a c e t i c a c i d was added to p r e c i p i t a t e C. The l a t t e r s o l u t i o n was c e n t r i -fuged, supernate B decanted and the remaining p r e c i p i t a t e D d i s s o l v e d i n 100 y l of 0.01 M phosphate b u f f e r , pH 7.2, c o n t a i n i n g 0.2% (w/v) sodium dodecyl sulphate and 2 M urea. The p r e c i p i t a t e was d i s s o l v e d by heating f o r 30 minutes o . . . at 60 C. Both supernate A and B and the d i s s o l v e d p r e c i p i t a t e were pooled, l y o p h i l i z e d and d i s s o l v e d i n the foregoing b u f f e r before a p p l i c a t i o n to polyacrylamide g e l s . Table V I I . Recovery of the r a d i o a c t i v i t y of proteins containing c o l l a g e n from polyacrylamide g e l s . C e l l Layer % Medium % Step Control Triamcinolone Acetonide Control Triamcinolone Acetonide (Supernate B + P r e c i p i t a t e D ) / P r e c i p i t a t e C 89.0 117.6 92.9 99.4 95.1 92.2 92.4 107.4 Solution/(supernate A + supernate B + p r e c i p i t a t e D) 81.4 86.3 100.2 90.4 84.3 80.9 98.3 83.9 Gel s l i c e s / s o l u t i o n 97.8 90.5 80.1 89.9 80.8 80.0 . 90.2 86.1 Gel s l i c e s / ( s u p e r n a t e A + p r e c i p i t a t e C) 91.0 89.0 88.8 81.2 82.9 78.8 81.7 89.9 - 84 -I s o l a t i o n of the collagenous p r o t e i n s from the foregoing medium and c e l -l u l a r f r a c t i o n s was modified s l i g h t l y i n an unsuccessful attempt to minimize the excessive manipulative procedures. Again, c e l l s from a common pool were used to seed eight r o l l e r b o t t l e s of which four were c o n t r o l s and four were treated w i t h triamcinolone acetonide. The c e l l s from each group were then pooled for the i s o l a t i o n of collagenous p r o t e i n s (see F i g . 9 and Table V I I I ) . As before, the p r o t e i n f r a c t i o n s were treated w i t h pepsin, and the d i g e s t was n e u t r a l i z e d and d i a l y z e d against 0.4 M NaCl co n t a i n i n g the 0.1 M T r i s . HC1, pH 7.6. The retentates were l y o p h i l i z e d . The products were suspended i n 0.5 M a c e t i c a c i d and the suspensions c e n t r i f u g e d to p r e c i p i t a t e the i n s o l u b l e p r o t e i n s (Residue D). The supernate A was set aside and the residue D re - e x t r a c t e d w i t h 0.5 M a c e t i c a c i d and c e n t r i f u g e d . The residue was set asi d e , the supernate was pooled with supernate A. Saturated (NH^^SO^ was added to the pooled supernate B to a f i n a l c o ncentration of 30% (w/v) and o allowed to stand overnight at 4 C to p r e c i p i t a t e c o l l a g e n . Assay of the supernate C showed n e g l i g i b l e r a d i o a c t i v i t y . The p r e c i p i t a t e and the residue D were d i s s o l v e d i n phosphate b u f f e r as described p r e v i o u s l y . Recovery of the r a d i o a c t i v i t y was followed at each step (Table V I I I ) . A major disadvantage of the foregoing procedures was the excessive manipulation involved. Hence, a s i m p l i f i e d procedure was devised. F r a c t i o n s c o n t a i n i n g c o l l a g e n from the medium and c e l l u l a r l a y e r were treated w i t h pepsin. The enzymatic r e a c t i o n was terminated by n e u t r a l i z a t i o n with 0.5 N NaOH and d i a l y s i s versus 0.4 M NaCl co n t a i n i n g 0.1 M T r i s . HC1, pH 7.6. The ret e n t a t e was l y o p h i l i z e d , d i s s o l v e d i n 0.01 M phosphate b u f f e r , pH 7.2, containing 0.2% (w/v) sodium dodecyl sulphate and 2 M urea and heated at 60°C f o r 30 min. The sample was then a p p l i e d to the g e l ( F i g . 10). - 85 -Figure 9. Treatment of c o l l a g e n - r i c h f r a c t i o n s of medium and pooled c e l l u l a r  f r a c t i o n s for a p p l i c a t i o n to polyacrylamide g e l e l e c t r o p h o r e s i s . Procedure 2. PROTEINS CONTAINING COLLAGEN Pepsin treatment Ly oph i l i z at ion D i s s o l v e i n 0.5M HOAc SUSPENSION RESIDUE D Re-extract 0.5M HOAc Centr i f u g e SOLUTION SUPERNATE A SUPERNATE B (NH4) 2S04 ppt SUPERNATE C PRECIPITATE E l e c t r o p h o r e s i s GEL SLICES Count S l i c e s - 86 -Table V I I I . Recovery of r a d i o a c t i v i t y of pooled c e l l u l a r p r o t e i n s from polyacrylamide g e l e l e c t r o p h o r e s i s . P r o t e i n s c o n t a i n i n g c o l l a g e n were tre a t e d with pepsin. The digest was n e u t r a l i z e d , d i a l y z e d and l y o p h i l i z e d . The l y o p h i l i z e d product was suspended i n 0.5 M a c e t i c a c i d and the suspension c e n t r i f u g e d to p r e c i p i t a t e the i n s o l u b l e p r o t e i n s (Residue D). Supernate A was decanted, residue D r e - e x t r a c t e d w i t h 0.5 M a c e t i c a c i d and c e n t r i f u g e d . This supernate was pooled with supernate A. Saturated (NH^^SO^ was added to t h i s pooled supernate B to p r e c i p i t a t e c o l l a g e n which was combined w i t h residue D and d i s s o l v e d i n phosphate b u f f e r to be applied to polyacrylamide g e l s . Recoveries (%) from c e l l l a y e r Comparison of Steps C o n t r o l % G l u c o c o r t i c o i d % suspension/pepsin-treated c o l l a g e n f r a c t i o n 57.3 39.9 ( p r e c i p i t a t e + residue D)/suspension 94.6 81.1 g e l c o u n t s / ( p r e c i p i t a t e + residue D) 81.6 82.3 - 87 -Figure 10. Treatment of p r o t e i n s c o n t a i n i n g c o l l a g e n i n medium and c e l l u l a r  l a y e r for polyacrylamide g e l e l e c t r o p h o r e s i s . Procedure 3. PROTEINS CONTAINING COLLAGEN Pepsin treatment D i a l y s i s SUSPENSION L y o p h i l i z a t i o n D i s s o l v e phosphate b u f f e r pH 7.2/SDS/urea, 60°C, 30 min SOLUTION E l e c t r o p h o r e s i s GEL SLICES Count s l i c e s - 88 -Recoveries of added r a d i o a c t i v e counts d i d not f a l l below 80% (Table I X ) . In a l l samples, s o l u b i l i z a t i o n of the products derived from pepsin-treatment was more complete i n the phosphate b u f f e r , pH 7.2, c o n t a i n i n g 0.2% (w/v) sodium o dodecyl sulphate and 2 M urea when heated at 60 C for 30 mm, than i n 0.5 M a c e t i c a c i d . IV. G l u c o c o r t i c o i d E f f e c t s on P r o l i n e I n c o r p o r a t i o n Into Collagenous P r o t e i n s  of Medium and C e l l u l a r Layer F r a c t i o n s of Mouse L-929 F i b r o b l a s t s :  Polyacrylamide Gel E l e c t r o p h o r e t i c Studies (a) comparison of peaks of medium f r a c t i o n s of c o n t r o l and triamcinolone acetonide-treated samples A t o t a l o f nine c o n t r o l and ten g l u c o c o r t i c o i d - t r e a t e d samples were examined. A r e p r e s e n t a t i v e from each of the c o n t r o l , g l u c o c o r t i c o i d - t r e a t e d and reference samples can be seen i n f i g u r e 11. Three p r i n c i p a l peaks appeared c o n s i s t e n t l y i n each experimental sample. The reference c o l l a g e n ( ) c o n s i s t e d of three peaks corresponding to y, 3, and a molecules. These r a d i o a c t i v e peaks coincided w i t h those observed v i s u a l l y i n stained p o l y a c r y l -amide gels c o n t a i n i n g u n l a b e l l e d reference c o l l a g e n ( P l a t e 1). The y molecules represent three a chains i n a s s o c i a t i o n w i t h one another, the 8 represent the a s s o c i a t i o n of two a chains and l a s t l y , the a are s i n g l e c o l l a g e n chains. The y bands or peaks have the slowest m o b i l i t y on the polyacrylamide gels because of t h e i r high molecular weight. 8 bands have intermediate m o b i l i t y and the a bands e x h i b i t the most r a p i d m o b i l i t y due to the low molecular weight of s i n g l e a chains. Peaks obtained from c o n t r o l ( ) and triamcinolone acetonide-treated samples corresponded to the y and a peaks of the reference c o l l a g e n . The intermediate peak of the c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d - 89 -Table IX. Recovery of r a d i o a c t i v i t y of p r o t e i n s c o n t a i n i n g c o l l a g e n from  polyacrylamide g e l s . Collagenous p r o t e i n s from the medium and c e l l u l a r l a y e r were tre a t e d w i t h pepsin, n e u t r a l i z e d and d i a l y z e d against 0.4 M NaCl c o n t a i n i n g 0.1 M T r i s . HC1, pH 7.6. The ret e n t a t e was l y o p h i l i z e d and d i s s o l v e d i n 0.01 M phosphate b u f f e r , pH 7.2, co n t a i n i n g 0.2% (w/v) sodium dodecyl sulphate and 2 M urea by heating at 60°C for 30 min. The sample was applied to polyacrylamide g e l s . Medium C e l l u l a r Layer % % Step Con t r o l G l u c o c o r t i c o i d C o n t r o l G l u c o c o r t i c o i d Solution/suspension 89.9 Gel c o u n t s / s o l u t i o n 85.0 Gel counts/suspension 81.9 84.3 90.0 89.9 90.1 97.5 91.1 95.6 86.6 92.8 89.9 82.0 85.0 78.0 94.8 90.5 81.9 - 90 -nyNMJKH BEL ELECTKROEIS OF fEOIlfl FRACTIONS 60 L 2 4 6 8 10 12 U 16 18 20 22 24 26 28 30 32 ft GEL SLICE Figure 11. Polyacrylamide gel electrophoresis of proteins containing collagen  of the medium fr a c t i o n s Pepsinized products i n phosphate buffer containing sodium dodecyl sulphate and urea were applied to polyacrylamide gels and subjected to electrophoresis. The gels were s l i c e s , digested and assayed for r a d i o a c t i v i t y . T y p i c a l p u r i f i e d reference collagen ( ), control ( ), and triamcinolone acetonide-treated (...) samples are shown. - 91 -s a m p l e s w a s l e s s m o b i l e t h a n t h e 8 p e a k o f t h e r e f e r e n c e s a m p l e . N e v e r t h e l e s s , t h e p e a k s o f t h e e x p e r i m e n t a l s a m p l e s s u g g e s t t h e p r e s e n c e o f c o l l a g e n . S m a l l e r e x t r a n e o u s p e a k s o f m o b i l i t y g r e a t e r t h a n t h e a p e a k s w e r e a l s o o b s e r v e d ; n o n e c o r r e s p o n d e d t o a n y k n o w n c o l l a g e n b a n d s . M o s t l i k e l y , t h e p e a k s w e r e d u e t o p e p s i n - r e s i s t a n t n o n - c o l l a g e n o u s p r o t e i n s o r , l e s s l i k e l y , t o d e g r a d a t i o n p r o d u c t s o f c o l l a g e n ( F i g . 11). ( b ) c o m p a r i s o n o f p e a k s o f c e l l u l a r f r a c t i o n s o f c o n t r o l a n d t r i a m c i n o l o n e a c e t o n i d e - t r e a t e d s a m p l e s T h e t h r e e p r i n c i p a l p e a k s o b s e r v e d p r e v i o u s l y ( F i g . 11) w e r e l e s s o r n o t d i s t i n c t i v e i n t h e c e l l u l a r f r a c t i o n s o f f o u r c o n t r o l a n d f o u r t r i a m c i n o l o n e a c e t o n i d e - t r e a t e d s a m p l e s . I n d e e d , o n l y o n e c o n t r o l s a m p l e h a d a d i s t i n c t i v e p e a k c o r r e s p o n d i n g t o t h e a b a n d o f t h e r e f e r e n c e m e t h y l a t e d c o l l a g e n ( • — . ) . N o n e o f t h e g l u c o c o r t i c o i d - t r e a t e d s a m p l e s h a d s u c h a p e a k . A l l s a m p l e s h a d a p e a k c o r r e s p o n d i n g t o t h e y b a n d o f t h e r e f e r e n c e c o l l a g e n . T h e p r e s e n c e o f n u m e r o u s p e a k s o f m o b i l i t y g r e a t e r t h a n t h e a m o l e c u l e s w e r e i n d i c a t i v e o f p e p s i n - r e s i s t a n t p r o t e i n s i n t h e c o n t r o l s a m p l e s ( ) . S u c h p r o t e i n s w e r e , a p p a r e n t l y , a b s e n t i n t h e t r i a m c i n o l o n e a c e t o n i d e - t r e a t e d s a m p l e s ( . . . . ) . I n -d e e d , o t h e r t h a n t h e p e a k c o r r e s p o n d i n g t o t h e y b a n d , n o o t h e r p e a k s w e r e o b -s e r v e d i n t h o s e p e p s i n - t r e a t e d s a m p l e s e x p o s e d t o t h e g l u c o c o r t i c o i d ( F i g . 12). ( c ) c o m p a r i s o n o f p e a k s o f m e d i u m a n d c e l l u l a r f r a c t i o n s T h e t h r e e p e a k s r e s e m b l i n g t h e y , 8, a n d a m o l e c u l e s o f c o l l a g e n w e r e d e f i n i t e l y m o r e p r o m i n e n t i n t h e m e d i u m s u g g e s t i n g t h a t t h e r e a r e m o r e c o l l a g e n o u s p r o t e i n s i n t h e m e d i u m t h a n i n t h e c e l l u l a r l a y e r . T h e r e a p p e a r e d t o b e m o r e p e p s i n - r e s i s t a n t p r o t e i n s i n t h e c e l l u l a r l a y e r t h a n i n t h e m e d i u m a s i n d i c a t e d b y t h e n u m e r o u s p e a k s a s s o c i a t e d w i t h t h e c o n t r o l c e l l u l a r s a m p l e ( F i g . 11 a n d 12). nxttmrnx. sa ELEOKJHRSIS OF CEUJMR FRACTIONS 60 HI 55 50 45 40 35 E Q. 30 25 20 15 10 I A i i i i V 4 6 10 12 14 16 18 20 22 24 26 28 30 32 H GEL SLICE Figure 12. Polyacrylamide gel electrophoresis of proteins containing collagen  of the c e l l u l a r f r a c t i o n s Pepsinized products i n phosphate buffer containing sodium dodecyl sulphate and urea were applied to polyacrylamide gels and subjected to electr o p h o r e s i s . The gels were s l i c e d , digested and assayed for r a d i o a c t i v i t y . T y p i c a l p u r i f i e reference collagen ( ), control ( ) and triamcinolone acetonide-treated (...) samples are shown. DISCUSSION In an e f f o r t to r e l a t e dermal atrophy to a l o s s of collagen, many studies have been performed on g l u c o c o r t i c o i d e f f e c t s on c o l l a g e n metabolism i n a v a r i e t y of c e l l l i n e s , i n c l u d i n g human dermal f i b r o b l a s t s but not mouse L-929 f i b r o b l a s t s , depression of c o l l a g e n s y n t h e s i s . The r e s u l t s obtained thus f a r have been c o n t r a d i c t o r y , i n p a r t , because of the great number of v a r i a b l e f a c t o r s , such as d u r a t i o n , frequency, type and dosage of g l u c o c o r t i c o i d treatment, that bear i n v e s t i g a t i o n . Other v a r i a b l e s include type of c e l l l i n e , the s i t e from which the t i s s u e was b i o p s i e d , age and sex of donor, the passage number and stage of growth of f i b r o b l a s t s . Often, the r e s u l t s have been m i s i n t e r p r e t e d , causing greater confusion. For i n s t a n c e , the measurement of hydroxyproline has often been used as an index of c o l l a g e n synthesis (Saarni and Tammi, 1978; Ponec et a l , 1977b). However, h y d r o x y l a t i o n i s but one of s e v e r a l steps i n the b i o s y n t h e s i s of co l l a g e n and, indeed, hydroxyproline measurements r e f l e c t simultaneously the i n c o r p o r a t i o n and h y d r o x y l a t i o n of p r o l i n e residues i n the peptide chains. Triamcinolone acetonide, a potent anti-inflammatory g l u c o c o r t i c o i d , at a low pharmacological dosage of 0.1 Ug per ml, has been found to a f f e c t c e l l u l a r p r o l i f e r a t i o n i n adult human dermal and mouse L-929 f i b r o b l a s t s ( R u n i k i s e_t a l . , 1978). This g l u c o c o r t i c o i d has also been observed c l i n i c a l l y to cause dermal atrophy (Snyder and Greenberg, 1977, 1974). The o b j e c t i v e s of t h i s study were to determine the e f f e c t of 0.1 Ug per ml of triamcinolone acetonide on the c o l l a g e n synthesis of mouse L-929 f i b r o b l a s t s . These c e l l s respond to g l u c o c o r t i c o i d s . Their c e l l u l a r p r o l i f e r a t i o n i s depressed by a v a r i e t y of g l u c o c o r t i c o i d s at d i f f e r e n t - 94 -concentrations. The degree of i n h i b i t i o n was c o r r e l a t e d w i t h anti-inflammatory potencies i n a p a r a l l e l - l i n e bioassay developed by B e r l i n e r and Ruhmann (1967) and Brotherton (1971). Since c o l l a g e n s y n t h e s i s i n these c e l l s t r e a t e d w i t h g l u c o c o r t i c o i d s had not been examined p r e v i o u s l y , i t was of i n t e r e s t to us to perform t h i s study. Secondly, due to the obvious relevance to the c l i n i c a l f i n d i n g s of dermal atrophy, human dermal f i b r o b l a s t s were i n -cluded i n the study. The c e l l s employed were derived from an adult i n d i v i d u a l . Except f o r one study (Ponec et_ al., 1977b), a l l other human studies have used f i b r o b l a s t s derived e i t h e r from neonatal or f o e t a l humans. Measurement and i d e n t i f i c a t i o n of c o l l a g e n synthesis were accomplished by b a c t e r i a l collagenase assays and polyacrylamide gel e l e c t r o p h o r e s i s i n our study. Only the c e l l u l a r l a y e r s of mouse L-929 f i b r o b l a s t s were a f f e c t e d by the a d d i t i o n of 0.1 yg triamcinolone acetonide per ml of medium. C e l l numbers decreased and the i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n per c e l l i ncreased. The former observation supports that of R u n i k i s et a l . (1978). The accumulation of r a d i o a c t i v i t y w i t h i n the c e l l s might be i n d i c a t i v e of e i t h e r underhydroxylated or abnormally s t r u c t u r e d c o l l a g e n ; e i t h e r would a r r e s t t r i p l e h e l i x formation. Hence, the s e c r e t i o n of c o l l a g e n may be i n h i b i t e d . Indeed, such has been observed i n scurvy ( K l e i n , 1976). Underhydroxylation of c o l l a g e n i n the presence of g l u c o c o r t i c o i d s was observed i n chick embryo t i b i a e t r e a t e d w i t h h i g h doses of h y d r o c o r t i s o n e , hydro-c o r t i s o n e a c e t a t e , hydrocortisone phosphoric a c i d complex or hydrocortisone sodium succinate (Blumenkrantz and Asboe-Hansen, 1976). The o b s e r v a t i o n , suggesting i n h i b i t i o n of the enzyme, p r o l y l hydroxylase, was supported by Counts et a l . (1979). They observed decreases i n c e l l u l a r p r o l y l hydroxylase a c t i v i t y and i n the amount of hydroxyproline i n dermal f i b r o b l a s t s i n c e l l - 95 -c u l t u r e derived from triamcinolone d i a c e t a t e - t r e a t e d r a t s . E a r l i e r work (Cutroneo and Counts, 1975; Newman and Cutroneo, 1978) had shown that the decrease i n p r o l y l hydroxylase a c t i v i t y was achieved only a f t e r m u l t i p l e g l u c o c o r t i c o i d treatments. P r o l y l hydroxylase a c t i v i t y had been shown to be i n h i b i t e d i n r a t s t r e a t e d w i t h t r i a m c i n o l o n e , hydrocortisone or methyl-prednisolone (Cutroneo et_ al., 1971), thus confirming the e f f e c t of gluco-c o r t i c o i d s on t h i s enzyme. Kruse et a l . (1978) found t h a t , although p r o l y l hydroxylase a c t i v i t y was reduced i n sponge granulomas from dexamethasone-trea t e d mice and i n hy d r o c o r t i s o n e - t r e a t e d f i b r o b l a s t c u l t u r e s , p r o t e i n synthesis was i n h i b i t e d to the same degree. Hence, there was no e f f e c t on p r o l y l h y d r o x y l a t i o n per se. S i m i l a r r e s u l t s were obtained from carrageenan granuloma of betamethasone disodium phosphate-treated r a t s (Nakagawa e_t al_. , 1971). Thus, the e f f e c t of g l u c o c o r t i c o i d s on p r o l y l h y d r o x y l a t i o n remains unclear and may be dependent on the type and concentration of the gluco-c o r t i c o i d , p eriod of i n c u b a t i o n w i t h the g l u c o c o r t i c o i d , and the type and o r i g i n of the c e l l s . Accumulation of c o l l a g e n need not a r i s e only from underhydroxylation but could be a consequence of an a b e r r a t i o n i n the molecular s t r u c t u r e which would a r r e s t the t r i p l e h e l i x formation. A z e t i d i n e - 2 - c a r b o x y l i c a c i d , when i n c o r -porated i n t o c o l l a g e n , prevents the formation of the normal h e l i c a l conformation. S e c r e t i o n of col l a g e n i s depressed. Concomitant accumulation of c o l l a g e n occurs (Bienkowski, 1978b). The proteins of the c e l l layers of the mouse L-929 c e l l s were f u r t h e r c h a r a c t e r i z e d i n polyacrylamide gel e l e c t r o p h o r e s i s because of the c o n s i s t e n t increase i n the r a d i o a c t i v i t y w i t h i n the c e l l s f o l l o w i n g g l u c o c o r t i c o i d t r e a t -ment. A l s o , the c o n s i s t e n t low losses of r a d i o a c t i v i t y i n the c o n t r o l and - 96 -experimental groups f o l l o w i n g collagenase d i g e s t i o n suggested the presence of large amounts of c o l l a g e n a s e - r e s i s t a n t proteins which were also c h a r a c t e r i z e d i n the g e l s . Radioactive peaks of pepsin-treated proteins were compared w i t h those of reference methylated c o l l a g e n and the bands of p u r i f i e d c o l l a g e n v i s u a l i z e d i n sta i n e d g e l s . Three p r i n c i p a l peaks or bands of d i f f e r e n t m o b i l i t i e s were observed. The f a s t e s t m i g r a t i n g band or peak represented the s i n g l e a chains which had the lowest molecular weight of the three peaks, the intermediate bands represented the 8 chains and the slowest m i g r a t i n g were the v chains which have the l a r g e s t molecular weight. These peaks were prominent i n the medium from c o n t r o l and triamcinolone acetonide-treated samples. The c o l l a g e n was r e l a t i v e l y free of other p r o t e i n s , supporting the f i n d i n g s of the previous experiment; high l o s s of r a d i o a c t i v e counts was obtained a f t e r collagenase d i g e s t i o n . No d i f f e r e n c e s were apparent i n the c o n t r o l and g l u c o c o r t i c o i d - t r e a t e d samples. Although, i n the c e l l layer f r a c t i o n s of c o n t r o l samples, peaks c o r r e s -ponding to the 8 and 0t bands were present they were l e s s d i s t i n c t than those i n the medium samples. Such peaks were completely absent i n g l u c o c o r t i c o i d -t r e a t e d samples. Peaks corresponding to the y bands appeared i n a l l samples. Other peaks, i n d i c a t i v e of p e p s i n - r e s i s t a n t p r o t e i n s , were prominent i n the c o n t r o l but absent i n the triamcinolone acetonide-treated f r a c t i o n s . The presence of large amounts of non-collagenous p e p s i n - r e s i s t a n t proteins i n the c e l l l a y e r s confirmed the data i n the previous experiments; low removal of r a d i o a c t i v i t y was encountered a f t e r b a c t e r i a l collagenase i n both c o n t r o l and g l u c o c o r t i c o i d t r e a t e d samples of the c e l l l a y e r s . - 97 -I n t e r p r e t a t i o n s of the data obtained from the collagenase assay and e l e c t r o p h o r e t i c studies are as f o l l o w s : I f the collagen which accumulated i n the c e l l l a y e r a f t e r g l u c o c o r t i c o i d treatment was abnormally s t r u c t u r e d , i t s a b e r r a t i o n might have allowed the pepsin to destroy i t completely. The predominant e f f e c t of pepsin on normally s t r u c t u r e d c o l l a g e n i s the cleavage of the t e l o p e p t i d e s at both ends of the amino and carboxy t e r m i n i ( M i l l e r , 1972). Since the e f f e c t of g l u c o c o r t i c o i d s on c o l l a g e n metabolism has not been examined i n polyacrylamide gels before, comparisons w i t h other work cannot be made. Common to a l l c o n t r o l c e l l layer samples were numerous peaks, i n d i c a t i v e of non-coilagenous p r o t e i n , that were absent i n the g l u c o c o r t i c o i d - t r e a t e d samples a f t e r pepsin treatment. These observations f u r t h e r suggest that the i n t r a c e l l u l a r p r o t e i n s of triamcinolone acetonide-treated samples are more s u s c e p t i b l e to pepsin d i g e s t i o n . Whether t h i s i s due to a b e r r a t i o n i n molecular s t r u c t u r e i s yet unconfirmed. The p o s s i b i l i t y that the peak corresponding to the y molecule may have represented most or a l l of the co l l a g e n present was considered; exposure of these molecules to b a c t e r i a l collagenase would have i d e n t i f i e d the p r o t e i n . However, the sample a p p l i e d to the gel could have in c l u d e d other l a r g e macro-molecules incapable of pe n e t r a t i n g the gel and, t h e r e f o r e , would have been included i n the y peak. Other types of experiments would have to be done to f u r t h e r c h a r a c t e r i z e the c o l l a g e n . For i n s t a n c e , the contents of hydroxyproline and p r o l i n e w i t h i n the c e l l s should be examined. U n f o r t u n a t e l y , many workers use hydroxyproline alone as an index of c o l l a g e n s y n t h e s i s . But the synthesis of c o l l a g e n includes biochemical processes other than h y d r o x y l a t i o n , f or in s t a n c e , amino - 98 -acid uptake i n t o the c e l l , the s i z e of the amino a c i d p o o l , i n c o r p o r a t i o n of amino acids i n t o the collagen molecule, p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s such as g l y c o s y l a t i o n , processing of the molecules i n t o p r o c o l l a g e n , and e x t r u s i o n of collagen i n t o the e x t r a c e l l u l a r m i l i e u . Any of these might be a f f e c t e d by the drug w i t h e f f e c t s apparent by one method and not by another. G l u c o c o r t i c o i d s have been reported to enhance c e l l u l a r p r o l i f e r a t i o n i n l o g a r i t h m i c a l l y growing c u l t u r e s of human dermal f i b r o b l a s t s ( R u n i k i s et a l . , 1978; K i r k and Mittwoch, 1977) and i n h i b i t that i n confluent c u l t u r e s (Ponec et a l . , 1977a,b; P r i e s t l e y , 1978). One must question the relevance of employing human f o e t a l f i b r o b l a s t s ( P r i e s t l e y , 1978; K i r k and Mittwoch, 1977) whose metabolic a c t i v i t i e s d i f f e r from those of adult human f i b r o b l a s t s . The use of human f i b r o b l a s t s of l a t e passages (15 to 21st) (Ponec et a\_., 1977a,b) to assess the adverse e f f e c t s of g l u c o c o r t i c o i d s on c o l l a g e n s y n t h e s i s must a l s o be questioned. C e l l u l a r metabolic a c t i v i t i e s g e n e r a l l y decrease w i t h the number of passages. Using ad u l t human dermal f i b r o b l a s t s at confluency, our p r e l i m i n a r y experiments have shown that triamcinolone acetonide does not have a s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t on c e l l u l a r p r o l i f e r a t i o n and i n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n . The i n c o r p o r a t i o n of r a d i o -a c t i v e p r o l i n e i n t o c e l l u l a r c o l l a g e n a s e - s e n s i t i v e p r o t e i n was suppressed a f t e r g l u c o c o r t i c o i d treatment, though not s i g n i f i c a n t l y . A d d i t i o n a l experiments might have c l a r i f i e d the s i g n i f i c a n c e of t h i s observation. The inc u b a t i o n period w i t h triamcinolone acetonide may have been too short to show a c o n s i s t e n t e f f e c t on c e l l u l a r p r o l i f e r a t i o n . Hence, the g l u c o c o r t i c o i d was added f o r a prolonged p e r i o d ; again no s i g n i f i c a n t e f f e c t s on c e l l u l a r p r o l i f e r a t i o n and p r o l i n e i n c o r p o r a t i o n were observed, p o s s i b l y , because t h i s - 99 -p a r t i c u l a r c e l l s t r a i n was not very responsive to triamcinolone acetonide. V e r i f i c a t i o n of triamcinolone acetonide e f f e c t s can be made only i f s e v e r a l s t r a i n s of adult human dermal f i b r o b l a s t s have been examined. The lack of a consi s t e n t e f f e c t of triamcinolone acetonide on p r o l i n e i n c o r p o r a t i o n i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n i n adult human dermal f i b r o b l a s t s may have been due to the dosage, 0.1 ug per ml of medium. This dosage i s exceedingly low when compared to those used by other i n v e s t i g a t o r s i n t h e i r i n v i t r o s t u d i e s . For i n s t a n c e , p r o l i n e i n c o r p o r a t i o n i n t o c o l l a g e n a s e - s e n s i t i v e p r o t e i n was suppressed by 10 Ug c l o b e t a s o l propionate or betamethasone v a l e r a t e per ml i n human f o e t a l f i b r o b l a s t s ; the concentration was 100 times greater than that we s e l e c t e d . P r i e s t l e y (1978) claimed that although the concentrations employed in v i t r o do not n e c e s s a r i l y r e f l e c t that found i n the s k i n , the foregoing concentration was r e l a t e d to c l i n i c a l use because about 2.2 Ug hydrocortisone per ml was found i n the dermis and was increased almost n i n e t y - f o l d w i t h the removal of stratum corneum before t o p i c a l a p p l i c a t i o n of 1% g l u c o c o r t i c o i d ; indeed the concentration of the g l u c o c o r t i c o i d i n the dermis was expected to be increased f u r t h e r i n diseased s k i n . Hence, 10 Ug per ml was not u n r e a l i s t i c . Most other workers have also employed concentrations exceeding that used i n our experiments (Ponec et a l . , 1977a,b). P r o l i n e i n c o r p o r a t i o n i n t o hydroxyproline was also depressed by 1 or 5 Ug hydrocortisone, hydrocortisone-17-butyrate, triamcinolone acetonide, betamethasone-17-valerate or clobetasol-17-propionate (Ponec et a l . , 1977a,b). Hence, 0.1 Ug triamcinolone acetonide per ml was probably not s u f f i c i e n t l y high to induce changes i n p r o l i n e i n c o r p o r a t i o n i n t o c o l l a g e n . A survey of studies reported to date does not permit g e n e r a l i z a t i o n of the e f f e c t s of g l u c o c o r t i c o i d on f i b r o b l a s t s i n t i s s u e c u l t u r e since these e f f e c t s - 100 -were many, v a r i e d and o f t e n i n c o n s i s t e n t . Too many v a r i a b l e s , f o r i n s t a n c e , the type, passage number, o r i g i n , and growth phase of the c e l l , the age and sex of the donor, the type and concentration of g l u c o c o r t i c o i d , the length of the incubation p e r i o d , the frequency of g l u c o c o r t i c o i d treatment and system used - i n v i v o , i n v i t r o , organ or c e l l c u l t u r e s - r e q u i r e systematic e v a l u a t i o n to e l u c i d a t e the adverse e f f e c t s of g l u c o c o r t i c o i d s . Recent personal communication with Booth and U i t t o (1979) has f a i l e d to shed any l i g h t on the g l u c o c o r t i c o i d e f f e c t s on collagen synthesis i n adult human dermal f i b r o b l a s t s i n c e l l c u l t u r e . Indeed, there were no co n s i s t e n t e f f e c t s despite numerous experiments which were designed and employed to examine many of the foregoing v a r i a b l e s encountered i n c e l l c u l t u r e systems. One cannot discount the p o s s i b i l i t y that g l u c o c o r t i c o i d s may not a f f e c t c o l l a g e n s y n t h e s i s but may perhaps a l t e r the degradative aspects of c o l l a g e n metabolism. There-f o r e , a l l biochemical steps i n co l l a g e n metabolism and other c e l l u l a r metabolic a c t i v i t i e s as w e l l as the o v e r a l l p h y s i o l o g i c a l responses to g l u c o c o r t i c o i d s have to be examined s e p a r a t e l y and i n concert i f the adverse e f f e c t s of gluco-c o r t i c o i d s are to be understood completely. - 1 0 1 -SUMMARY AND CONCLUSION Triamcinolone acetonide, at a concentration of 0.1 Ug per ml had the f o l l o w i n g e f f e c t s on mouse L-929 f i b r o b l a s t s and a s t r a i n of adult human dermal f i b r o b l a s t s : 1. C e l l u l a r p r o l i f e r a t i o n of confluent mouse L-929 f i b r o b l a s t s was c o n s i s t e n t l y depressed. 2. Inc o r p o r a t i o n of p r o l i n e i n t o t o t a l p r o t e i n and c o l l a g e n a s e - s e n s i t i v e m a t e r i a l i n the medium remained unal t e r e d . 3. However, the i n c o r p o r a t i o n of p r o l i n e i n t o c e l l u l a r p r o t e i n and c o l l a g e n a s e - s e n s i t i v e m a t e r i a l was enhanced c o n s i s t e n t l y and s i g n i f i c a n t l y (0.01 P 0.001 and P = 0.004 r e s p e c t i v e l y ) . 4. C h a r a c t e r i z a t i o n of the c o l l a g e n a s e - s e n s i t i v e m a t e r i a l was accomplished by polyacrylamide g e l e l e c t r o p h o r e s i s . Such m a t e r i a l of the medium f r a c t i o n had peaks whose m o b i l i t i e s c o i n c i d e d w i t h those of p u r i f i e d c o l l a g e n , t h e r e f o r e i d e n t i f y i n g the c o l l a g e n a s e - s e n s i t i v e p r o t e i n as c o l l a g e n . C o l l a g e n a s e - s e n s i t i v e c e l l u l a r m a t e r i a l t r e a t e d with pepsin d i s p l a y e d only one peak i n polyacrylamide g e l s . Indeed, other proteins were not apparent i n gels f o l l o w i n g pepsin treatment. 5. At the l o g a r i t h m i c and confluent phases of growth, c e l l u l a r p r o l i f e r a t i o n of adult human dermal f i b r o b l a s t s exposed to the g l u c o c o r t i c o i d was not a l t e r e d c o n s i s t e n t l y . 6. I n c o r p o r a t i o n of p r o l i n e i n t o c o l l a g e n a s e - s e n s i t i v e m a t e r i a l i n the medium and c e l l u l a r f r a c t i o n s of human f i b r o b l a s t s was a f f e c t e d i n c o n s i s t e n t l y . 7. To conclude, 0.1 Ug per ml triamcinolone acetonide does a f f e c t the metabolic a c t i v i t i e s of mouse L-929 f i b r o b l a s t s . Both c e l l u l a r - 102 -p r o l i f e r a t i o n and c o l l a g e n s y n t h e s i s i n the c e l l l a y e r are a l t e r e d by t h i s c o n c e n t r a t i o n . T o t a l p r o t e i n and c o l l a g e n a s e - s e n s i t i v e p r o t e i n appear to be degraded by pepsin according to polyacrylamide g e l s , i n d i c a t i n g perhaps that the c o l l a g e n a s e - s e n s i t i v e p r o t e i n may be underhydroxylated or abnormal i n s t r u c t u r e . The l i n e of ad u l t human dermal f i b r o b l a s t s used i n t h i s study does not appear to respond to the s i n g l e dose of triamcinolone acetonide. This could be due to the low concentration of the g l u c o c o r t i c o i d or perhaps t h i s p a r t i c u l a r l i n e i s not responsive to g l u c o c o r t i c o i d s . - 103 -BIBLIOGRAPHY A s f e l d t , V.H.: Plasma c o r t i c o s t e r o i d s i n normal i n d i v i d u a l s w i t h reference to the c i r c a d i a n rhythm. Scand. J . 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