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Intracellular signaling pathways regulating integrin-mediated keratinocyte spreading and lamellipodia… Alavian, Keyhan 2000

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I N T R A C E L L U L A R SIGNALING P A T H W A Y S R E G U L A T I N G INTEGRIN-MEDIATED K E R A T I N O C Y T E SPREADING A N D L A M E L L I P O D I A F O R M A T I O N  by KEYHAN ALAVIAN B.Sc, The University of British Columbia, 1997 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T O F REQUIRMENT FOR T H E D E G R E E OF M A S T E R OF SCIENCE In F A C U L T Y OF G R A D U A T E STUDIES (Department of Oral Biological and Medical Sciences, Faculty of Dentistry) We accept this thesis as conforming to the r e q u i r e d standard  T H E UNIVERSITY OF BRITISH C O L U M B I A A U G U S T 2000 © Keyhan Al avian, 2000  In  presenting  degree freely  this  thesis  in  partial  fulfilment  at the University  of  British  Columbia, I agree  available for reference  copying  of  department publication  this or  thesis by  of this  and study.  for scholarly  his thesis  or  her  of  requirements that  I further agree that  purposes  gain  It  is  of  Q#AL  &Joi.oGicAi-.  The University of British C o l u m b i a Vancouver, Canada  DE-6  (2/88)  shall make  by the head  understood  shall not be allowed  (OSbicAL  an advanced it  permission for extensive  permission.  Department  for  the Library  may be granted  representatives.  for financial  the  &c/enA^£^,  that without  of my  copying  or  my written  ABSTRACT The process of wound healing involves formation of long cytoplasmic extensions by keratinocytes followed by the their migration into the wound. This process is regulated through intracellular pathways triggered by integrin-extracellular matrix interactions. Still, little is known about the intracellular pathways involved in formation of these "extended lamellipodia" in keratinocytes and their effects on cell migration. In this study, we used a number of kinase and phosphatase modulators to target various signaling pathways inside keratinocytes spreading on the wound provisional matrix, fibronectin. Our results showed that staurosporine (STP), a broad kinase inhibitor, leads to formation lamellipodia in keratinocytes at 10 nM and extended lamellipodia (E-Lam) at 50 nM. Cell spreading however was not concentration dependent since both STP concentrations increased cell spreading and migration by same amount (200%). Neither cell spreading or E-Lam formation involves modulation of phosphatidylinositol-3 kinase (PI-3K) or extended tyrosine phosphorylation since inhibitors against these proteins had no effect on STP-induced E-Lam  formation  and cell  spreading.  Actin  and microtubule  polymerization, however, are required for the formation of E-Lams. Western blotting experiments showed that keratinocytes treated with 50 nM STP exhibit twice the level of mitogen-activated protein kinase (MAPK) activation as keratinocytes treated with 10 n M  ii  STP. Immunostaining experiments with anti-tubulin antibodies demonstrated that microtubules (MTs) are localized at the tip of the extended lamellipodia when treated with 50 n M STP, suggesting that MTs may play an active role in E-Lam formation. Further experiments using inhibitory ocv and p i antibodies confirmed previous results that cell spreading and E-Lam formation is mediated through av(3l, cx5(3l, and av(36 integrins. Based on these findings, we speculate that differential activation of M A P K may play an active role in regulation of E-Lam formation involved in cell spreading and cell migration, by modulation of integrins binding-affinity, and organization of the cytoskeletal actin and microtubules.  iii  CONTENTS ABSTRACT  ii  CONTENTS  iv  LIST O F T A B L E S  viii  LIST O F F I G U R E S  ix  ABREVIATIONS  xi  AWKNOWLEDGEMENT  xiii  INTRODUCTION  1  LITERATURE REVIEW  3  1.  3  KERATINOCYTES 1.1.  2.  3.  4.  Function  3  FIBRONECTIN  4  2.1.  Expression and Function  4  2.2.  Structure  5  INTEGRINS  6  3.1.  Structure of Integrins  3.2.  Expression of Integrins by Normal Keratinocytes  8  3.3.  Keratinocyte Integrins in Wound Healing  9  3.4.  Function and Regulation of Integrins  12  4.2.  6  3.4.1.  Cells adhesion  12  3.4.2.  Outside-in signal transduction  13  3.4.3.  Inside-out signal transduction  14  MAPK PATHWAY 4.1.  '.  Function of M A P K  17 17  4.1.1.  Transcriptional regulation  17  4.1.2.  Cytoplasmic proteins  17  4.1.3.  Cell adhesion/migration  18  Mechanism of M A P K Activation  19  4.2.1.  Growth factor-mediated activation  21  4.2.2.  Integrin-mediated activation  22  Ras/FAK Dependent activation  22  FAK-independent activation  25  Ras-independent activation  25  Activation through PI-3K pathway  26  iv  5.  6.  KINASE A N D PHOSPHATASE M O D U L A T O R S  29  5.1.  Staurosporine  29  5.2.  Okadaic acid  31  5.3.  Genistein  31  5.4.  Sodium metavanadate  32  5.5.  Bombesin  32  5.6.  Herbimycin A  33  THE CYTOSKELETON  34  6.1.  Actin Cytoskeleton  34  6.2.  Microtubules  37  AIM O F T H E STUDY  40  M A T E R I A L AND METHODS  41  1.  Cell line and cell culture  41  2.  Antibodies  42  3.  Toxicity tests  43  4.  Cell spreading assay  44  5.  Immunochemical staining  46  6.  Western blotting  48  7.  Cell migration assay  49  RESULTS 1.  50  Effects of Protein Kinases and Phosphatases on Keratinocyte Spreading on Fibronectin 1.1.  Staurosporine, wortmannin, and bombesin induce keratinocyte spreading on fibronectin  51  1.2.  Staurosporine induced formation of extended lamellipodia  53  1.3.  The morphology of staurosporine-induced formation of extended lamellipodia is different on different extracellular proteins  1.4. 1.5.  58  Promotion of keratinocyte extended lamellipodia by staurosporine is concentration-dependent  1.6.  55  Staurosporine stimulated keratinocyte spreading is mediated through ccv and (31 integrins  60  Formation of extended lamellipodia is not blocked by other kinase/phoaphatase inhibitors  2.  50  62  The Role of Actin and Microtubule Cytoskeleton in Staurosporine-induced Keratinocyte Cell Spreading  65  2.1.  Actin polymerization is required for cell spreading and lamellipodia formation  2.2.  65  Microtubule polymerization is required for the formation of extended lamellipodia  2.3.  65  Microtubules accumulate to the tip of the extended lamellipodia in STPstimulated keratinocytes  3.  67  Role of Microtubule Dynamics on STP-stimulated Keratinocyte Spreading on Fibronectin 3.1.  70  Taxol and nocodazole have no effect on the fomation of extended lamellipodia  4.  M A P K Activation in Response to Staurosporine  5.  Effects of Staurosporine on Cell Migration 5.1.  70 72 :  74  Staurosporine stimulates cell migration on fibronectin  DISCUSSION 1.  74 76  STP Stimulates Cell Spreading and Formation of Extended Lamellipodia on Fibronectin Through av and (31 Integrins  76  2.  M A P K Activation precedes STP-induced cell spreading and E-Lam formation.. 81  3.  Actin and Microtubule Cytoskeleton are Required For the Formation of  4.  Extended Lamellipodia  82  Summary  86  CONCLUSIONS & SUGGESTIONS F O R F U T U R E R E S E A R C H  89  REFERENCES  91  APPENDICES  108  APPENDIX A - Drug Dilutions  108  APPENDIX B- Toxicity Tests  '.  APPENDIX C- Raw Data for the Graphs  109 113  APPENDIX C I - Effect of protein kinase and phosphatase modulators on HaCaT keratinocyte spreading (Figure 3)  113  APPENDIX C2- Staurosporine-induced cell spreading on different matrices (Figure 5)  113  APPENDIX C3- Effect of inhibitory anti-integrin antibodies on HaCaT cells on fibronectin (Figure 7)  114  APPENDIX C4- Effect of different concentration of staurosporine on cell spreading on fibronectin (Figure 8)  114 vi  APPENDIX C5- Effect of STP10 and STP50 on HaCaT cell spreading on fibronectin in combination with protein kinase and phosphatase modulators (Figure 12)  114  APPENDIX C6- Effect of cytochalasin-D on HaCaT cell spreading on fibronectin (Figure 12)  115  APPENDIX C7- Effect of colchicine on HaCaT cell spreading on fibronectin (Figure 13)  115  APPENDIX C8- Effect of microtubule modulators on HaCaT cell spreading on fibronectin (Figure 16)  115  APPENDIX C9A- Activation of M A P K (ERK1/2) in STP-treated HaCaT cells. (Figure 17B)-Band intensity  116  APPENDIX C9B- Activation of M A P K (ERK1/2) in STP-treated HaCaT cells. (Figure 17C)- % cells spread APPENDIX CIO- effect of STP on HaCaT cell migration (Figure 18)  116 116  vn  LIST OF TABLES Table 1:  Keratinocyte integrins and their ligands  Table 2:  Protein kinase and protein phosphatase modulators and their respective targets used in spreading assay experiments  9  50  viii  LIST OF FIGURES Figure 1:  Structure of the integrin heterodimer  Figure 2:  Hypothetical model for collaboration between integrinmediated signaling pathway leading to M A P K activation  Figure 3:  23  Effect of protein kinase and phosphatase modulators on HaCaT keratinocyte spreading  Figure 4:  7  52  Morphology of HaCaT keratinocytes seeded on fibronectin in presence of protein kinase and phosphatase modulators  54  Figure 5:  Staurosporine-induced cell spreading on different matrixes  56  Figure 6:  Morphology of HaCaT cell spreading on fibronectin, collagen type I and IV, and laminin-5  Figure 7:  Effects of inhibitory anti-integrin antibodies on HaCaT cells on fibronectin  Figure 8:  59  Effect of different concentrations of staurosporine on cell spreading on fibronectin  Figure 9:  57  60  Morphology of HaCaT keratinocyte spreading in presence of different concentrations of staurosporine on fibronectin as viewed by scanning electron microscopy  Figure 10:  61  Morphology of HaCaT cells spreading on fibronectin in presence of STP 50, in combination with various protein kinase and phosphatase modulators  Figure 11:  63  Effect of STP 10 and STP 50 on HaCaT cell spreading on Fibronectin in combination with protein kinase and phosphatase modulators  64  ix  Figure 12:  Effect of cytochalasin-D on HaCaT cell spreading on fibronectin  66  Figure 13:  Effect of colchicine on HaCaT cell spreading on fibronectin  68  Figure 14:  Morphology of HaCaT cells treated with luM colchicine and 50 n M staurosporine on fibronectin  Figure 15:  Immunolocalization of tubulin in HaCaT cells in response to staurosporine  Figure 16:  68  69  Effect of microtubule modulators on HaCaT cell spreading on fibronectin  71  Figure 17:  Activation of M A P K (ERK1/2) in STP-treated HaCaT cells  Figure 18:  Effect of STP on HaCaT cell migration  Figure 19:  Morphology of migrating HaCaT cells treated with  ....73 74  .different concentrations of staurosporine on F N viewed by scanning electron microscopy Figure 20:  A hypothetical  75  model illustrating STP-induced cell  spreading and formation of extended lamellipodia  88  x  ABBREVIATIONS Ape  Adenomatous polyposis coli  ATPase  adenine triphosphatase  BDM  butanedione monoxide  BSA  bovine serum albumin  CHO  Chinese hamster ovary  CSB  cytoskeletal stabilizing buffer  DMEM  dulbecco's modified eagle's medium  DMSO  dimethyl sulfoxide  ECM  extracellular matrix  EDA  extra domain A  EDB  extra domain B  EGF  epidermal growth factor  E-Lam  extended lamellipodia  ERK1  extracellular-regulated signal-regulated kinase 1  ERK2  extracellular-regulated signal-regulated kinase 2  FA  focal adhesion  FAK  focal adhesion kinase  FAT  focal adheion targeting sequence  FBS  fetal bovine serum  FN  fibronectin  FRNK  focal adhesion kinase-related non-kinase  GNEF  guanine nucleotide exchange factor  GTP  guanine triphosphate  kD  kilodalton  KGM  keratinocyte growth medium  mAB  monoclonal antibody  MAPI  microtubule associated protein 1  MAP2  microtubule associated protein 2  MAPK  mitogen activated protein kinase  MEK  M A P K / E R K kinase  MLC  myosin light chain  MLCK  myosin light chain kinase  mRNA  messenger ribonucleic acid  xi  MT  microtubule  OkA  okadaic acid  PBS  phosphate buffer saline  PDGF  platelet derived growth factor  PI  phosphoinositide  PI-3K  phosphoinositol 3-kinase  PIP2  phosphatidyl-4,5-bis-phosphate  PKC  protein kinase C  PM  plasma membrane  PP1  protein phosphatase 1  PP2  protein phosphatase 2  PTP  protein tyrosine kinase  PtyrK  phosphotyrosine kinase  RGD  arginine-glycine-aspartate  RTK  receptor tyrosine kinase  SEM  scanning electron microscope  SH2  Src homology 2  STP  staurosporine  STP10  10 nM staurosporine  STP50  50 nM staurosporine  TGFpl  transforming growth factor beta 1  ACKNOWLEDGEMENTS I would like to extend my thanks and appreciation to the following people who have made the completion of this project possible. To my supervisor, Dr. Hannu Larjava, Head, Division of Periodontics, U B C , for his guidance and support throughout my program. I thank you for trusting me with this project and allowing me the freedom to complete it. To Dr. Lari Hakkinen, assistant professor, Dentistry, UBC. Thank you for your expertise and continuous academic support, as well as your insight as a member of my committee. To Dr. Colin Wiebe, assistant professor, Dentistry, U B C , and Dr. Jukka Uitto, professor, Dentistry, U B C . I also thank you for your valuable suggestions as members of my research committee. To Cristian Sperantia, for your technical and laboratory expertise, as well as your great sense of humor. To Leeni Koivisto, Liangxuan Zhang, Jim Firth, and Ali Reza Sanaie, whose advise has been instrumental for the completion of my project. To my parents. I thank you for all the sacrifices you have made throughout my life. I thank you for being there for me when I needed you the most. Your undying support and valuable advice has brought me to where I am today. To my two brothers, for their charm, friendship and love. To Virginia Kwan, and the many friends whom I had the pleasure of meeting in these last few years at U B C . I thank you for your support and your true friendship.  xiii  INTRODUCTION The process of wound healing involves transition of keratinocytes from a stationary to migratory phenotype (Grinnell, 1992). This change includes formation of long cytoplasmic extensions called lamellipodia which are extended into the wound (Odland and Ross, 1968; Woodley, 1996; Larjava et al., 1996) upon migration. Keratinocytes migrate through the fibronectin-rich provisional matrix, which covers the entire wound once the basement membrane is disrupted (Clark and Brugge, 1995). Migration of keratinocytes on the provisional matrix is mediated through transmembrane receptors called integrins (Grinnell, 1992; Hughes and Pfaff, 1998). Integrins are heterodimeric glycoproteins made up the two subunits, a and (3, whose various combinations give rise to over 20 different integrins (reviewed in Larjava et al,  1996;  Longhurst and Jennings, 1998; Graber et al, 1999). Integrins are in the center of a twoway transfer of information between the extracellular matrix and the proteins inside the cell. Signals generated as a result of integrin and extracellular matrix interactions are conducted through various cytoplasmic protein kinases and phosphatases (e.g. mitogen activated proteins kinase- MAPK), which regulate many changes within the cells such as actin and microtubule organization, lamellipodia formation, protein phosphorylation and integrin activation (reviewed in Yamada, 1997 and Alpin et al,  1998). Although a  tremendous amount of research has been conducted in regards to integrin-mediated cell  1  spreading and lamellipodia formation, many questions have remained to be answered in regards to the protein kinases and phosphatases involved in these processes in keratinocytes. The purpose of the present study was to investigate the role of various protein kinases and phosphatases in keratinocytes including M A P K in the formation of extended lamellipodia, and cell spreading. We also investigated the role of actin and microtubule filaments in cell spreading and formation of extended lamellipodia.  2  LITERATURE REVIEW 1.  KERATINOCYTES  1.1.  Function Integrity of the surface and mucosal epidermis is very important to the vitality of  the organism. In general, keratinocytes maintain epidermal and mucosal surfaces, which serve the organism as a protective barrier against physical, chemical, or microbial agents of the outside world. Also, adhesion between epidermis and the basement membrane is specialized for mechanical stability. In case of an injury, the basal cells serve as the major source of keratinocyte migrating into the wound area (Hakkinen et al, 2000). This process begins at the edges of the wound within 24 hours of the injury, where keratinocytes form long veil-like extensions from the cell periphery into the wound, called lamellipodia (Larjava et al., 1996). Once the entire wound is covered this thin layer of epithilial tissue differentiates  and remodels into a stratified squamous  epithelium (Schaffer and Nanney, 1996). Human keratinocytes can be grown in culture under conditions in which they can attach, spread and migrate on fibronectin surfaces (Takashima and Grinnell, 1984). These can be used to study many aspects of keratinocyte behavior such as formation of lamellipodia involved in cell spreading and migration (Watt and Jones, 1993).  2.  FIBRONECTIN  2.1.  Expression and Function Fibronectins (FN) are a family of closely related adhesive glycoproteins, and one  of the key components of the extracellular (ECM) and provisional wound matrix. Fibronectins contain multiple binding sites, which allow them to link various components of the E C M to each other as well as to receptors on the cell surface. Furthermore, fibronectin molecules can bind to each other, further enhancing their capacity to form a stable network of E C M components and their anchorage to the cell surface (Alberts et al, 1993). A soluble form of fibronectin is present in the blood and other body fluids. This form, called plasma fibronectin, has several binding domains that recognize the bloodclotting protein, fibrin. The ability to bind fibrin makes F N an important component of the plasma clot formed in wounds. F N is also an integral E C M component of the provisional matrix, which serves as the substratum during times of cell migration in wound healing (Alberts et al,  1993). Fibronectin also acts as a chemotactic factor  attracting peripheral blood monocytes to the wound site (Richards and Clark, 1990), and as a general opsonin enhancer during the inflammatory phase of wound repair (Takashima and Grinnell, 1985).  4  2.2.  Structure: A fibronectin molecule consists of two large polypeptides linked near their  carboxyl ends by a pair of disulfide bonds. The two polypeptides are similar but nonidentical. Each has about 2500 amino acids with a mass of approximately 250 kDa and is folded into a series of globular domains connected by short, flexible segments of the polypeptide chain. Some of the domains recognize and bind specific components of the E C M , including several types of collagen (I, II, IV) and specific proteoglycans. Other domains recognize and bind cell surface receptors via a specific tetrapeptide sequence, RGDS (arginine-glycine-aspartate-serine). This RGDS sequence is a common motif recognized by integrins such as a5pT, the "fibronectin receptor" (Alberts et al, 1993). There is only one gene for fibronectin, but there are more than 20 different isoforms. The capacity for the large number of variants exists based on differential splicing of the mRNA. For example, wound healing in adult rat-skin is accompanied by a re-expression of cellular fibronectin which contain both EIIIA (EDA; extra domain A), and EIIIB (EDB; extra domain B) (Buck and Horwitz, 1987). The expression of these domains are absent in plasma FN. This suggests that distinctive splicing patterns leading to presence or absence of the extra domain influences physical and/or functional properties of F N (French-Constant and Hynes, 1988).  5  3.  INTEGRINS Integrins are a family of heterodimeric, transmembrane glycoproteins that act as  cell surface receptors for E C M molecules such as collagen, and fibronectin (Gumbiner 1996). Integrins are in the midst of the two-way signaling between the cell and its external environment, which regulates gene expression and cellular functions including changes in cell morphology associated with actin cytoskeleton organization, cell spreading and migration (Watt and Jones, 1993; Hynes, 1994; Giancotti and Mainiero, 1994; Yamada K M , 1997; Alpin et al. 1998; Lunghurst, 1998 ). Integrins are expressed practically by all cell types, except red blood cells. Some integrins' are cell specific while many are expressed in multiple cell types. For example, a L 3 2 is only expressed by leukocytes, while many (31 integrins by many cell types (Watt and Jones, 1993; Graber et al, 1999, Hakkinen et al, 2000). Also a single cell can express multiple integrins with some that may have similar binding specificity. For example, there are three fibronectin-binding integrins in human keratinocytes: namely ot5pi, avf31, and avP6 (Koivisto et al, 1996).  3.1.  Structure of Integrins Integrins are composed of a pair of non-covalantly linked a and (3 subunits  (Figure 1). Currently there are 18 known a subunits, and 8 p subunits, whose combination gives rise to more than 24 different type of integrins (Larjava et al, 1996;  Figure 1. Structure of the integrin heterodimer.  Alpin et al., 1998; Hakkinen et al., 2000). The combination of particular a and (3 subunits determines the ligand specificity of the integrin complex. Some integrins bind to a single E C M protein (e.g. a5(31), while others recognize several distinct proteins such as collagens, laminins and fibronectins (Larjava et al, 1996). Integrins are asymmetrical and have a large mushroom-like extracellular region and two flexible tails. The extracellular domain is at the N-terminal regions of the a and P subunits and it expands through the membrane ending at the C-terminal of the short cytoplasmic domains of both subunits (with the exception of P4 which has a long extended cytoplasmic domain) (Kieffer and Phillips, 1990).  3.2.  Expression of Integrins By Normal Keratinocytes Human keratinocytes can express at least eight integrin heterodimers that  mediate cellular response to various extracellular matrix proteins (Table 1). These integrins are found in basal epithelial cells of skin and oral mucosa including the gingiva (Larjava et al,  1996), where they participate in mediating adhesion and signal  transduction (Watts and Jones, 1993). The integrins most expressed by normal epithelial cells, namely a2pi, oc3pi, and a6p4, are present both in vivo and vitro. Integrin a2pi is described as a receptor for collagen, while integrin a3pi binds to laminin-5 (reviewed in Hakkinen et al, 2000). Integrin a6p4 is also primarily a receptor for laminin-5. cx6p4 serves as a major  constituent of the hemidesmosomes that attach the basal epithelial cells to basement membrane (Jones et al, 1986; Niessen et al, 1994).  Table 1. Keratinocyte integrins and their ligands INTEGRIN  LlGAND  a2pi  Collagen  cc3pi  Laminin-5  CX6P4  Laminin-5  a5pi, avpl  Serum Fibronectin, Fibronectin E I I I - A  avp5  Vitronectin  avP6  Fibronectin E I I I - A , Fibronectin E I I I - B  a9pi  Tenascin  The level of integrin expression in keratinocytes is not constant. As epithelial cells differentiate, they migration from the basal cell layer to the suprabasal layers, during which their integrin expression gradually drops (Adams and Watts, 1991). On the other hand when keratinocytes migrate laterally across a wound, they express new integrins, namely a5pi and ocvP6, which are not expressed in healthy tissue (Larjava et al, 1993). The role of these integrins will be discussed shortly.  3.3  Keratinocyte Integrins in Wound Healing The process of wound healing involves a predictable but complex series of  events including migration, proliferation, differentiation and apoptosis of cells. This  9  process is divided into four phases: acute inflammation, epithelialization, granulation tissue formation, and tissue remodeling. Due to their particular relevance to this research clotting and epithelialization related to cell spreading and migration will be discussed in more debt than other phases of wound healing. Clotting and epithelialization of the wound are important in providing strength to the site of injury, and in providing an initial barrier against the external environment including bacteria. Clotting is the first event to take place following the wounding. During clotting, a fibrin clot and platelets provide the initial barrier to stop the bleeding. The clot also contains F N which is the main constituent of the provisional matrix needed for cell migration. Epithelialization starts within hours after injury where keratinocytes form and extend long lamellipodia into the wound (Larjava et al,  1996). This is  followed by migration of cells from the edges of the wound to the center of the provisional matrix (Stenn and Malhotra, 1992). To initiate wound healing, keratinocytes require a change from a stationary to a migratory phenotype. They detach from the basement membrane by dissolving their hemidesmosomal connections. This involves expression of new integrins. Migrating wound keratinocytes express a5(31 and ccv(36 integrins, which are not expressed in stationary keratinocytes (Larjava et al,  1993; Haapasalmi et al,  1996). They also  express enhanced levels of (31 subunit associated with a2 and oc3 (Larjava et al, 1993).  10  Integrins cc5pi and avp6 are considered to be specialized receptors for F N (Larjava et al, 1993), with the latter expressed only in epithelial cells (Breuss et al, 1993). Their function includes, cell adhesion, migration, and FN-matrix assembly. Although both a5pi and avP6 integrins have specificity for the same E C M protein, they are expressed at different times during cell migration. a5pi is expressed during early migration while avp6 is expressed during reorganization of the basement membrane. This suggests that they may have different roles in wound healing (Hakkinen et al, 2000). Both integrins however, disappear once the epithelialization is complete. (Larjava et al, 1993). Integrin avP5 is the receptor for vitronectin and it appears to be present during epidermal cell migration in human wounds (Clark et al, 1996) Aside from changing their integrin expression, migrating keratinocytes are also capable of making E C M matrix that they can use to support their migration (Hakkinen et al, 2000). Migrating keratinocytes do not deposit collagen type IV, laminin-1, or collagen type VII (Larjava et al,  1993) but they instead produce laminin-5 and  fibronectin EIIIA (but not EIIIB) (Hakkinen et al, 2000; Larjava et al, 1993). Once the two epithelial sheets confront each other in a wound, laminin-1, collagen type IV and collagen type VII are formed at the basement membrane zone (Larjava et al, 1993). At this point the migrating keratinocytes return to their stationary state.  11  3.4  Function and Regulation of Integrins  3.4.1. Cell adhesion Both the a and (3 subunits are needed for overall integrin function, including cell adhesion (Carter et al, 1990; Weinel RJ et al, 1992; Takada and Puzon, 1993). The extracellular domain of the a subunit has a disulfide cleavage site near the transmembrane region (Tuckwell et al, 1992), and seven repeated sequences of 60-70 residues (Fig. 1). Repeats 2-4 are involved in ligand binding, while repeats 4-7 are the putative binding site for divalent cations (Irie et al  1997). Cations are thought to  influence ligand-binding activity of the integrins by changing the conformation of the receptors. Several a subunits including a l , a2, a L , a M , and a E have a region of about 200 residues called I-domain. I-domain is inserted between repeat 2 and 3, which are thought to be critically involved in ligand binding, a subunits which do not contain Idomain, contain other ligand binding sites for adhesion. For example av subunit binds RGD peptides of fibronectin through its binding sites located at residues 139-349 (Smith and Cheresh, 1990). (3 subunits have a homology of approximately 40%. They also have an extracellular I-like-domain structure, which has been shown to contain conserved residues critical for ligand binding (Puzon-McLaughlin and Takada, 1996; Goodman and Bajt, 1996; Tozer et al, 1996). Similar to a subunit, the ligand-binding affinity (activation) of f3 integrins can also be non-physiologically altered by interactions of  12  certain agents with its extracellular domain. For example, activating anti-(31 mAbs (e.g. 8A2), or inhibiting anti-pi mAb (e.g. mAbl3) can induce high or low affinity states, respectively, by binding to the non-ligand binding site of the p i subunit, thereby changing its conformation (Takada and Puzon-McLaughlin, 1993).  3.4.2  Outside-in signal transduction In addition to cell adhesion, integrins convey signals from the outside  environment to the cell interior though a process called "outside-in" signaling. Outsidein signaling involves adhesion of E C M ligands with the ligand-binding region of the integrin. This interaction will lead to a conformational change in the integrin structure resulting in signal transduction. The ability of integrins to change their conformation (affinity modulation) allows modulation of cell adhesive properties without changes in integrin gene expression (reviewed in Longhurst and Jennings, 1998). Upon adhesion to extracellular molecules, the cytoplasmic subunits of a and p swing apart, allowing the P cytoplasmic domain to directly bind cytoskeletal proteins such as talin and a-actinin. Talin and a-actinin in turn bind other structural proteins such as vinculin, paxillin, and tensin, and actin filaments, which ultimately leads to the formation of focal adhesions (FA) (Yamada and Geiger 1997; Alpin et al, 1998). Focal adhesions are highly complex structures at the start of a series of signaling cascades that lead to various cytoplasmic events such as cytoskeletal rearrangement involved in  13  lamellipodia formation during cell' spreading and migration. As they mature, the actin filaments extend and bundle to form prominent structures termed stress fibers. In addition to extracellular ligand binding, integrin clustering (avidity) is also known to regulate signal transduction. Experiments have shown (Miyomoto et al, 1995) that integrin clustering alone leads to colocalization of tensin (a cytoskeletal protein) and focal adhesion kinase (FAK). This process however does not lead to recruitment of important cytoskeletal proteins such as talin, vinculin, a-actinin, or actin needed for focal adhesion formation. It is thought therefore, that a combination of both ligandbinding and integrin clustering may be needed for complete integrin signaling. It is difficult to determine the effect of ligand binding alone on cytoplasmic protein recruitment, since ligand-binding and clustering often occur simultaneously.  3.4.3  "Inside-out" Signal Transduction "Inside-out" signaling, involves the modulation of integrins ligand-binding  affinity via the interaction of intracellular proteins and the cytoplasmic region of integrin subunits. Integrins (31, f32, |33, and (37 have all been shown to modulate their ligandbinding affinity in response to cytoplasmic signals (Faull et al,  1993; Crowe and  Hunter, 1994). Coordinated activation and de-activation of integrins play a crucial role in cell spreading and migration, since these processes are regulated by rapid, controlled changes in integrin-dependent cell adhesion (Lauffenburger and Horwitz, 1996).  14  The importance of the a and P cytoplasmic subunit in integrin affinity regulation has been shown by the observation that locking integrins in a constitutively high-affinity state can inhibit cell migration (Hottenlocher et al, 1996). Both cytoplasmic domains of the cc and P subunits contain conserved sequences at sites near the cell membrane, and the deletion of these sites leads to high integrin affinity (Crowe et al, 1994, O'Tool et al, 1994). The high affinity of these mutant integrins is independent of cell type and physiological pathway, suggesting that these sequences serve as a membrane-proximal "hinge" (Fig. 1), that lock integrins in their default, low affinity conformation (Hughes et al, 1996). In addition to the conserved membrane-proximal sequences, sequences Cterminal to the a and P conserved motifs are also crucial in integrin activation, and signal transduction. Deletion of these sequences leads to inhibition of cell adhesion, (Williams et al, 1994). Presence of highly conserved tyrosine, threonine and serine residues in integrin's cytoplasmic domain suggests that integrin affinity may be regulated through phosphorylation of these residues. Experiments with inhibitors of tyrosine kinases and phosphatases support this hypothesis (Hughes and Pfaff, 1996). Also, in an experiment involving cotransfection of P3 cytoplasmic domain, it was found that integrin-affinity of alip3 integrins were dose-dependently reduced due to the competition for cytoplasmic factors required for integrin activation. Together, these observations suggest that  15  interactions of a and P cytoplasmic domains with specific regulatory proteins could modulate integrin activation. Of the proteins that regulate integrin affinity, the ras family of small GTPbinding proteins and their effectors are found to be very important. Hughes et al. (1997) demonstrated, activated H-ras and its kinase effector raf-1 inhibit integrin activation in Chinese hamster ovary cells (CHO). This suppression correlates with the activation of M A P K pathway, and is independent of protein synthesis and mRNA transcription. Rras, which is homologous to H-ras has an opposite effect by stimulating integrin ligandbinding affinity. It was found that in leukocytes, R-ras stimulates pi and P2 integrins affinity through the activation of phosphoinositide 3-kinase (PI-3K) (Shimizu and Hunt, 1996). Modulation of integrin affinity by PI-3K has also been demonstrated with wortmannin where its inhibition of PI-3K partially blocks the activation of allb3 integrins in platelets (Zhang et al, 1996). Also, protein kinase C (PKC), a downstream effector of PI-3K, was found to directly interact with vinculin, which is one of the most abundant and important structural proteins in F A . Vinculin contains a potential P K C phosphorylation site in its tail, which becomes available upon adhesion to the PI 3-K product, phosphatidylinositol 3,4-bisphosphate (PI(3,4)P-2) (Weekes et al, 1996). All this evidence suggest that regulation H-ras and R-ras along with their downstream effectors may be a key factor in the regulation of integrin affinity.  16  4.  MAPK PATHWAY. Activation of the mitogen activated protein kinase (MAPK) cascade is a  common event in the response to many diverse extracellular stimuli (Alpin et al, 1998) (Fig. 2). These stimuli could be soluble factors (e.g. peptide growth factor, okadaic acid) or E C M molecules since both can independently lead to the activation of M A P K cascade. M A P kinase also has diverse targets, and its activation leads to different cellular responses, such as lamellipodia formation and cell migration (reviewed in Seger and Krebs, 1995).  4.1  Function of M A P K  4.1.1  Transcriptional Regulation M A P K translocates to the nucleus after integrin-mediated cell adhesion. It can  regulate various transcriptional factors, including c-Myc, Elk-1, and AP-1, (Treisman, 1996) leading to various changes in cell function. Although many M A P K transcriptional factors have been identified, the mechanism and their physiological importance has not yet been fully understood.  4.1.2  Cytoplasmic Proteins In addition to transcriptional factors, M A P K also interacts with targets in the  cytoplasm. One of these targets is the cytoarchitectural system, the microtubule network.  17  M A P K can associate directly with microtubule components (Reszka et al., 1995). Reszka et al. (1997) has been suggested that the activity of microtubules and microtubule-associated M A P K regulate integrin-mediated signaling events and the formation of focal adhesions and stress fibers.  4.1.3  Cell adhesion/migration M A P K plays an important role in regulating the actin-myosin cytoskeleton  which is critical for lamellipodia formation during cell spreading and migration. Myosins are actin-activated ATPases capable of promoting cell contraction needed for migration of cells. (Jay et al., 1995) In nonmuscle cells, myosin II is the one best known. Myosin II function is regulated by phosphorylation of the regulatory light chains, through activation of M A P K . Klemke et al. (1997) demonstrated that expression of mutationally active M E K directly activates myosin light chain kinase (MLCK). This in turn leads to an increase in phosphorylation of the myosin light chain (MLC) and migration of cells on collagen and F N . Inhibition of M A P K activity suppresses phosphorylation of M L C K and in turn M L C , leading to a rapid loss of stress fibers, FAs and contractility needed for cell mobility. However this inhibition was found to have no effect on cell adhesion and in situ cell spreading of fibroblasts, suggesting that integrinmediated M A P K activity is not required for these initial events in this system. M A P K inhibition in macrophages (Ogra and Kitamura, 1998) and hepatocytes (Tanimura et a/.,  18  1998) on the other hand leads to reduced cell spreading which suggests that the effect of M A P K on cell spreading is cell dependent. Either way it is certain that M A P K plays an important role in regulation of cytoskeleton needed cell adhesion and locomotion. It has been proposed that M A P K activation may also contribute to the regulation of cell adhesion and motility through a "feedback mechanism". Hughes et al. (1997) has demonstrated that constitutively active mutants of M A P K pathway components (i.e. Raf, and ras) can suppress the activation of integrins, as well as inhibit fibronectin matrix assembly, which in turn induces cell rounding. Since cells constantly make and break focal adhesions with the E C M during migration, it is possible that M A P K contributing to the dynamic modulation of ligand binding affinity (and cell motility) through a negative feedback mechanism (Fig. 2).  4.2.  Mechanism of M A P K Activation Activation of M A P K by soluble factors as well as insoluble factors has been  under intense research. Integrin-mediated M A P K activation has been demonstrated in a variety of cell types such as fibroblasts, keratinocytes, and endothelial cells. In NIH3T3 fibroblast cells, M A P K activity peaks 10 to 15 minutes after plating on fibronectin, whereas lower, but significant activity persists to 40 to 60 minutes (Clark and Hydes, 1996). Similar results have been observed with other rodent fibroblast cells such as Swiss 3T3 fibroblasts (Chen et al, 1994) as well as human dermal (Miyamoto et al,  19  1996) and lung fibroblasts (Chen et al., 1994). In primary human keratinocytes plated on laminin, peak M A P K activation occurs 30 minutes after plating (Mainiero et al., 1997). Conversely, endothelial cells (HUVECs) plated on fibronectin show peak levels of M A P K activity within 10 minutes of plating (Takahashi and Berk, 1996). Integrinmediated M A P K activation may persist between 60 to 100% of maximum levels for periods ranging from 15 minutes to 2 hrs or more. It is apparent that integrin-mediated M A P K activation varies based on the types of cell, integrin, and matrix involved. The prolonged activation of M A P K by integrins is in sharp contrast to that seen in response to many soluble mitogens such as platelet-derived growth factors (PDGF), which typically elicit a response within 2-5 minutes. It has been suggested that the duration of M A P K activity can affect the nature of the cellular response (Marshal, 1995). In PC12 cells, for example, transient or prolonged activity of M A P K induces either cell division or differentiation. How the duration of integrin-mediated M A P K activity may affect downstream biological events is not yet understood. It is important to note that the combinatorial repertoire of extracellular ligands, integrins, and intracellular signaling molecules differs from one cell to another and even within a given cell at various times. While it contributes to the diversity and specificity of the adhesion and signaling responses of integrins, it also complicates the task of proposing unifying principles. For example, there is substantial evidence supporting that integrin-mediated M A P K activation can be through focal adhesion kinase (FAK) and  20  ras-dependent, or F A K - and ras-independent pathways (Shattil and Ginsberg, 1997). Accordingly, caution must be taken when applying results from one cell type to another and from experiments in vitro to in vivo.  4.2.1. Growth Factor-mediated Activation Growth factor receptors have intrinsic enzymatic activity and their role in activation of M A P K is different from that of integrin-mediated activation (Alpin et al., 1998). Growth factors can elicit M A P K activation in suspension which suggests that they do not require E G F receptors or integrins (hence cell adhesion and the formation of focal adhesions) for M A P K activation. Upon cell treatment with soluble peptide mitogens (e.g. epidermal growth factor; EGF), the respective receptor tyrosine kinase (RTK) homodimerizes and undergoes autophosphorylation on various tyrosine residues. This modification creates binding sites for proteins containing Src homology 2 (SH2) domains. Some of these proteins are enzymes such as kinases (e.g. p85 " ), phosphatases (e.g. SHP), and GTPase activator Pi  3K  proteins (e.g.l20ras ), whereas others are adapter proteins (e.g. She, Grb2) that serve GAP  to relocate specific target proteins to sites of tyrosine phosphorylation. One of these target proteins, Sos, is a ras guanine nucleotide exchange factor (GNEF) and is recruited  21  to activate R T K through its interaction with Grb2 (Schlessinger, 1994). Once at the membrane, Sos promotes conversion of ras to an activated, GTP-bound form, which will in turn form, a stable complex by binding to the N-terminal of raf-1 protein. Once activated, raf-1 phosphorylates and activates M A P K / E R K kinase (MEK), which is the kinase directly responsible for activation of M A P K (i.e. extracellular signal-regulated kinases 1, ERK-1; extracellular signal-regulated kinases 2, ERK-2) (Seger and Krebs, 1995).  4.2.2. Integrin-Mediated Activation Similar to growth factor activation, integrin-dependent M A P K activation is carried through M E K and raf family of protein kinases (Fig. 2). In NIH3T3 cells, raf-1 was activated by adhesion to fibronectin at a time course parallel those for M E K and M A P K . Also, pharmacological inhibition of M E K activation ablates integrin-mediated M A P K activation in fibroblast (Chen et al,  1996) as well as human monocytes  (McGilvray et al, 1997). In this system integrin ligand-binding is required for M A P K activation.  ras/FAK Dependent Activation  raf-1 activation is very complex and it is unclear exactly how it occurs. One accepted mechanism by which integrins can activate raf-1 closely parallels growth 22  Figure 2. Hypothetical model for collaborator! between integrin-mediated signaling pathways leading to MAPK activation. Integrin engagement by ligands such as F N , stimulates multiple signaling pathways involving F A K phosphorylation, which leads to activation of Ras and MAPK. F A K can also activate MAPK through a Ras- independent pathways involving PI-3K. PI-3K can also be activated via a FAKindependent pathways involving direct interaction between PI-3K and integrin cytoplasmic domain. Aside from integrins, G proteins-coupled receptors can also activate MAPK, by direct interaction with PI-3K Ras. Once activated, MAPK leads to modulation of many cell functions such as activation of MLCK. M L C K increases M L C phosphorylation which can lead to contractility, cell spreading and migration. M A P K is also involved in the inside-out modulation of integrins. Other mechanisms leading to inside-out affinity of integrins are through the H-Ras/R-Ras GTPases, as well as PI-3K. It is important to note that not all these pathways apply to keratinocytes, since different cell lines may vary in their intracellular signaling cascades in response to extracellular or intracellular stimuli.  FN  23  factor stimulation; it uses ras activation through the same signaling intermediates used by receptor tyrosine kinase (RTK), with the exception that F A K acts as the tyrosine kinase domain. Overexpression of dominant negative ras (ras ) can block integrinNI7  mediated M A P K activation in several systems including keratinocytes (Mainiero et al, 1997), whereas overexpression of wildtype F A K in transformed human epithelial cells enhances integrin-mediated M A P K activation four-fold. F A K is a highly conserved protein, which can be detected in the majority of cells. In fibroblasts and keratinocytes (Gates et al,  1994), adhesion to the E C M is  required for efficient tyrosine phosphorylation of F A K (Burridge et al, 1992). In this mechanism, engagement of integrins by fibronectin induces F A K autophosphorylation at the F A K  T y r 3 9 7  , which in turn leads to F A K association with c-Src. The interaction of c-  Src with F A K causes further phosphorylation of F A K  T y r 9 2 5  , leading to its maximal  activation. Once fully activated F A K will bind the Grb2 adapter protein, which leads to recruitment of Sos, and subsequent activation of ras. F A K  P h e 3 9 v  , the c-Src binding site  mutant (which Src bind to) inhibits fibronectin-stimulated signaling to ERK2 in human 293T cells (Kaplan et al, 1995). Grb2 binding to F A K on the other hand is not essential to promote integrin signaling to ERK2 since overexpression of wild-type nor the Grb2 binding site mutant of F A K ( F A K  P h e 9 2 5  ) changes integrin-stimulation of ERK2  (Schlaepfer and Hunter, 1997)  24  FAK-independent Activation  In contrast to the discussion above, there is also substantial evidence suggesting that other FAK-independent mechanisms for integrin-mediated M A P K activation exist. Reports have shown that in fibroblasts,, a (31 subunit mutation with a deletion at the putative F A K binding domain can support integrin-mediated M A P K activation even when it lacks F A K tyrosine phosphorylation (Lin et al, 1997). In addition, experiments involving recombinant F N fragments have demonstrated M A P K activation even when there is no detectable tyrosine phosphorylation of F A K (Lin et al, 1997). Another way to prevent integrin-mediated phosphorylation of F A K is through the use of FAK-related non-kinase (FRNK). FRNK is the product of an alternative fak gene splicing, which lacks the central region the whole F A K structure. It possesses no catalytic activity or focal adhesion targeting sequence (FAT) needed for localization of focal adhesion sites (Schaller et al, 1993). Expression of FRNK, although able to block integrin-mediated phosphorylation of F A K , has no effect on integrin-mediated M A P K activation (Lin et al, 1997). ras-independent Activation Integrin-mediated M A P K activation can occur independently of ras function. For example, dominant negative inhibitors of ras-dependent signaling (pZIP-ras ) failed to N19  25  block integrin-mediated activation of M E K in NIH3T3 fibroblasts seeded on F N (Chen et al,  1996). In addition, while treatment with the peptide mitogen E G F clearly  increased GTP-loading of ras, little effect was observed in response to integrindependent cell adhesion. Also raf defective in the ras binding site can still be activated by integrin-mediated adhesion (Alpin et al,  1998). This suggests that raf may be  activated through its interaction with a protein other than ras. One such protein could be the enzyme phosphatidylinositol-3 kinase (PI-3K) which has been suggested to activate raf in a ras independent manner (Alpin et al, 1998).  Activation Through PI-3K pathway  PI-3K enzymes are present in all cell types, and their activity has been shown to be necessary for many different cell regulatory pathways (Duronio et al, 1997) such as cell mitogenesis and differentiation, as well as cytoskeletal organization. The enzyme PI-3K is characterized as a heterodimer containing a 110 kDa catalytic subunit and an 85 kDa regulatory subunit (Katada et al, 1999). Adhesionmediated activation of PI-3K involves association of the two SH2 (Src homology 2) domains of p85 subunit with tyrosine phosphorylated proteins such as F A K and/or ras  26  (Chen et al, 1994; Kapeller and Cantley, 1994). Wortmannin , a specific inhibitor of PI1  3K, was reported to block the activation of M A P K up to 80% in fibroblasts (King et al, 1997), suggesting that PI-3K may be involved in activation of M A P K . The ability of PI3K to become activated by either ras or F A K provides a possible pathway for ras- or a FAK-independent M A P K activation. Once activated, PI-3K can phosphorylate the 3-OH position of the inositol ring of several different phosphoinositides (PI). PI-3K lipid products act as second messengers, and are able to activate certain PKC isoforms; Co-immunoprecipitation experiments with antibodies against PI-3K lipid products (e.g. PI (3,4)P2) and anti-PKC isoforms (e.g. PKC8) confirm direct interactions of PI-3K products with P K C isoforms (Ettinger et al,  1996; Toker et al,  1994). Upon activation, P K C can catalyze  phosphorylation of serine and threonine, where they can directly activate raf (thus MAPK) (King et al, 1997). PKC can in fact activate M A P K in COS-7 cells expressing dominant negative ras (Wennstrom and Downward, 1999). An isoenzyme of PI-3K (plOO/pllO-g), lacking the p85 subunit, was shown to be activated by the Py subunits of G-proteins (Stoyanov et al, 1995), through interaction with its catalytic subunit. This suggests that in addition to integrins and the tyrosine  1  The drug Wortmannin, is a fungal metabolite that inhibits PI-3K activity by irreversibly modifying its  catalytic domain.  27  phosphorylated receptors, G proteins-coupled receptors can also stimulate PI-3K, which in turn may be sufficient to trigger M A P K by itself. A PI-3K isoenzyme, namely p85/plOO-P was found to be activated by both FAK/ras pathway and the G-protein pathway (Stephens et al, 1998). In this system an increase in PI-3K activity was observed when protein tyrosine kinase was activated upon stimulation of G protein-coupled receptors. Furthermore, M A P K activation was attenuated upon inhibition of p85 subunit (Stephens et al, 1993). This leads to the hypothesis that the two pathways may work in synergy. Aside from P K C activation, lipid products of PI-3K are also involved with other intracellular proteins such as Akt/PKB (Andjelkovic et al, 1997), Rac, and Cdc42 (Chou and Blenis, 1996). These targets are beyond the scope of this paper and will not be discussed further.  28  5.  K I N A S E and P H O S P H A T A S E M O D U L A T O R S Phosphorylation of protein kinases and phosphatases are thought to be a crucial  event in the regulation of signaling pathways, as is affinity modulation of integrins leading to changes in cell morphology and lamellipodia formation in association with cell spreading and migration (Tamaoki et al, 1990). Protein kinases are generally classified into serine/threonine and tyrosine kinases. Protein kinase and phosphatase modulators can be used to target specific pathways to elucidate the physiological roles of the various protein kinases in formation of lamellipodia and cell migration.  5.1  Staurosporine Staurosporine (STP) is a protein kinase inhibitor, targeted towards the A T P  binding site of the kinases catalytic domain. However, since the A T P binding site is conserved in several protein kinases, inhibitors (e.g. STP) interacting with such domains provide non-specific selectivity. In fact, staurosporine is found to inhibit the activity of several kinases such as the PKC, PKA (Tamaoki et al, 1990) and M L C K (Watson et al, 1988). Because of its broad specificity, STP has different morphological effects depending on its concentration and the type of cell. For example, in keratinocytes, treatment with 60 nM STP leads to the development of dendritic shape cells resembling  29  that induces by phorbol esters (activator of PKC) (Sako et al,  1998). In rat  pheochromocytoma PC12 cells, neural outgrowth is observed (rasouly et al, 1994). In megakaryoblastic leukemia cells (MEG-01) STP-stimulated cell elongation along with reorganization and/or activation of both integrin and the actin-based cytoskeleton has also been observed. Changes in cytoskeletal proteins with STP treatment has been reported by several groups (Yoshimura et al, 1995; Maroney et al, 1995), but little is known about its relation with cytoplasmic elongation. It is possible that STP modulates protein phosphorylation in adhesion complexes to induce the elongation in Meg-01 cells. Furthermore, STP has a dose dependent effect on the integrin-mediated cell elongation, spreading and migration of Meg-01 cells on FN. This effect however, was not observed in the CMK-7 human megakaryoblastic cell line (Yamazaki et al, 1999). In human colon cancer cell line (Col 201) seeded on F N , STP-induced spreading was observed, but via a non-integrin pathway (Yoshimura et al,  1995.) It is not clear  whether these phenomena are due to effects on isoenzymes of P K C , another protein kinase or some completely different molecule target (Ruegg and Burgess, 1989). The diversity of these results suggest that signaling pathways leading to cell morphology, spreading and migration differ depending on the cell line and the adhesive proteins (Yoshimura et al, 1995).  30  5.2  Okadaic Acid Okadaic acid (OkA) is a potent and specific inhibitor of protein phosphatase type  1 and 2A. Gotoh Y (1990) proposes that M A P kinase is negatively regulated by protein phosphatases 1 and/or 2A (PP1 and PP2A, respectively) in quiescent fibroblast cells and therefore can be activated by inhibiting these protein phosphatases (Shelden and Wadsworth, 1996). Also, induction of PP1 in fibroblasts leads to a decrease in M L C phosphorylation, which coincides with loss of stress fibers, focal adhesions, and decreased contractility (Fernandez et al, 1990). This suggests that regulation of PP1 and PP2 may be involved in cell motility. The role of PP1 and PP2 in keratinocytes spreading and migration has not been studied.  5.3  Genistein Genistein is a broad inhibitor of protein tyrosine kinase (phosphotyrosine kinase;  PtyrK). PtyrK activity is essential for cell spreading in some cell lines. This is illustrated by the experiment where genistein blocked adhesion-induced E R K kinase activation and at the same time decreased the monocyte spreading (Mondal et al, 2000). Genistein also reduced spreading of B16-BL6 melanoma cells (Yan and Har, 2000), arterial smooth muscle cells (Hedin et al, 1997), and keratinocytes (Benedicte et al, 1997).  31  5.4  Sodium metavanadate Sodium metavanadate is an inhibitor of protein tyrosine phosphatases (PTP)  which can localize to focal adhesions where they may play a role in modulating cell attachment and spreading. PTP inhibition was found to reduce intracellular phosphotyrosyl proteins, as well as NIH 3T3 cell spreading on F N (Siobhan et al, 1997). Effects of phosphotyrosine inhibition by PTP on spreading of keratinocyte has not been studied.  5.5  Bombesin Bombesin is part of a large family of neuropeptides that bind to G protein-  coupled receptors and lead to activation of a variety of intracellular signaling pathways (Ji et al, 1998) such as the M A P kinase pathway. In Swiss 3T3 cells, M A P K activation is through a P K C dependent pathway (Seufferlein et al, 1995), while in Rat-1 cells, this is done through a PKC-independent, ras-dependent pathway (Charlesworth and Rozengurt, 1997). This suggests that mechanism of M A P K activation by bombesin (hence G protein-coupled receptor) is cell type specific. One thing is for certain though, that bombesin linked with the G protein-coupled receptor leads to activation of M A P kinase. They have also been shown to lead to rapid increase in the tyrosine phosphorylation of variety of proteins including the F A K (Rozengurt, 1995).  32  5.6  Herbimycin A Herbimycin A is a tyrosine kinase-specific inhibitor. Masson-Gadais et al.  (1997) found that herbimycin inhibits phosphorylation of proteins at the 125kDa range (most likely F A K ) , along with other proteins expressed in the ranges 120 to 180kDa. Herbimycin also inhibited keratinocyte spreading and migration on collagen type I (Masson-Gadais, 1997). This suggests that protein tyrosine kinase activity (specially FAK) is crucial in spreading and migration of keratinocytes.  33  6.  THE CYTOSKELETON  The  cell cytoskeleton  is an integrated array of interconnected  filament-  assemblies, each with a defined architectural organization which define cell shape and polarity. Modulation of the cytoskeletal organization is believed to be crucial in formation of lamellipodia associated with cell spreading and migration. There are three components to the cell cytoskeleton  namely actin, intermediate filaments and  microtubules. Interaction between actin and MTs has been suggested based on the observation that both have the ability to bind common protein such as adenomatous polyposis coli (Ape) (Munemitsu et al, 1994). Also, when actin assembly is blocked in actively migrating cells, MTs loose their alignment and become bent and random in orientation.  6.1  Actin Cytoskeleton Of the three, actin is the most important component in maintaining the structural  framework around the cell (Small et al, 1999). In some mouse models, the cell develops normally in the absence of microtubules and intermediate filaments but not without the actin (Fuchs and Cleveland, 1998).  34  The actin cytoskeleton is a highly dynamic structure (Alpin and Juliano, 1999). Upon stimulation by extracellular factors, actin monomers are polymerized in an organized fashion. Actin polymers can then communicate with the cytoplasmic subunit of integrins through their interaction with proteins of the focal adhesion sites, such as vinculin, a-actinin and F A K (Burrige and Chzanoska, 1999). Polymerization of actin and their interactions with integrins are essential in cell signal transduction cascade such as M A P K pathway, since their disassembly by cytochalasin D has shown to inhibit such pathways (Chen et al, 1994). Actin filaments cross-link with myosin filaments, which results in a force leading to aggregation of integrins and formation of stress fibers. Stress fibers have the ability to contract and exert tension (Small et al, 1999). Aside from stress fibers, the most abundant concentration of actin in cultured cells are in lamellipodia (reviewed in Small et al, 1999). Lamellipodia are comprised of planar meshwork of unipolar actin filaments whose growing ends are oriented outwards. Migrating cells in culture are polarized with a broad flat lamella that terminates in ruffling lamellipodia facing the direction of migration (i.e. the leading edge) (Waterman-Storer and Salmon, 1999). Myosin II, which binds actin filaments, is concentrated at the posterior region of motile cells and along actin stress fibers in the leading lamella, where it is thought to play a role in providing cell contraction and modulating adhesion contact. In fact, experiments show that spreading and migration of PtK2 kidney cells are blocked by 35  butanedione monoxime (BDM), a known inhibitor of muscle myosin II, suggesting that myosin II contractility is required for cell spreading and migration (Cramer and Mitchison, 1995). Therefore, at the leading edge, a combination of actin filament assembly, myosin activity, and the formation of focal adhesion contacts direct the protrusive ruffling activities of the cell, and provide contractility which is thought to be the driving force for cell motility. Filopodia, or microspikes, are another actin-containing structures that may be involved in the process of cell spreading and migration. Filopodia are finger-like protrusive elements that extend from lamellipodia (Zigmond, 1996). They are expressed in variable degrees depending on the cell type and condition. Filopodia are thought to be important sensory structures which monitor the environment and direct polarized cell growth in fibroblasts (Waterman-Storer and Salmon, 1999) and in neural growth cones (Verkhovsky et al, 1995). For instance, stimulation of a single filopodium can result in reorientation of the whole growth cone (Hall, 1998), while growth cones lacking filopodia are no longer able to navigate (Takada, 1997). Furthermore, filopodia are believed to play a role in initiating changes in cell shape and migration (WatermanStorer and Salmon, 1999).  36  6.2  Microtubules Although actin is essential for the protrusive movements of the cell edge,  microtubules are necessary for the regionalization of this activity, leading to cell polarization and locomotion (Kark et ah, 1998). Regulation of these processes by MTs is not well understood. There is also a lot of opposing evidence, which again suggests that this process is dependent on the cell type and experimental conditions. MTs act as positive or negative regulators, and exert their influence on the actin cytoskeleton through removal as well as localization and delivery of regulatory components to contact sites (Terasaki and Reese, 1994). Precise interaction of MTs with adhesion sites is required for proper contact modulation. The spreading and locomotion of cells under normal conditions entails a cycle of contact formation, whereby new focal contacts are formed beneath the leading edge while the old focal contact become uncoupled at the trailing edge (Izzard and Lochner, 1980). This turnover mechanism is influenced by M T s since their complete depolymerization by colchicine stops cell migration. In migrating cells, focal contact turnover coincides with an actin-driven retrograde flow of MTs in the lamellum, where microtubule growth predominates for M T plus end at the leading cell edge, and the M T shortening predominates at the minus ends in the trailing cell body. This process called M T dynamicity is thought to be the  37  driving force for polarized cell locomotion.(reviewed in Waterman-Storer and Salmon, 1999). Experiments with low (nMoIar) concentrations of microtubule inhibitors, taxol and nocodazole , have shown that decrease in M T dynamicity leads to loss of 2  polarization and directional movement of various cell lines (Liao et al, 1995). This event coincides with a reduction in lamellipodium area (Kolodney and Elson, 1995), which suggest that M T dynamicity may regulate cell locomotion through its effects on the lamellipodium. Although MTs are important in extension of lamellipodia and motility, they do not need to enter the lamellipodium (Gunderson and Cook, 1999). This suggests that MTs at the leading edge could regulate lamellipodia production through release of diffusable factors at the base of the lamellipodium. This theory is supported by the observation that rapid (but not complete) depolymerization of M T s by high concentrations of nocodazole leads to large cell protrusions (Mikhailov and Gunderson, 1998). M T regulation of cell locomotion is explained by another theory, where MTs directly activate actomyosin contractility, and cell motility through their interaction with myosin light chain kinase ( M L C K ) (Klemke et al, 1997). M L C K is the main kinase responsible for the phosphorylation of the regulatory light chains which increase the  2  Nocodazole is a MT antagonist, while Taxol is a MT stabilizer; both these drugs have the same effect  at nMolar concentrations, where they both decease the retrograde flow of MTs.  38  actin-activated ATPase activity of both smooth and non-muscle myosin II, and is required for myosin-driven actin filament movement in in-vitro mobility assays (de Lanerolle and Paul,  1991). Kolodney and Elston (1995) demonstrated  that  depolymerization of MTs with nocodazole leads to an induction of M L C K activity and M L C phosphorylation along with induced contractility. This contraction was blocked or reversed by addition of the M T stabilizing drug Taxol, suggesting that depolymerization of MTs may release associated proteins that organize actin filaments, possibly resulting in increased tension (Watermann-Storer C, and Salmon E, 1997). These findings suggest that regulation of M L C phosphorylation by MTs may be a key event in stabilization of stress fibers, and regulation of nonmuscle actin-myosin interactions and contractility. Ingber (1993) has proposed another mechanism for induction of contractility. In this model, microtubules act as opposing force to stress fibers, where MTs are the compression-resistance  elements and actin filaments are the tension generating  elements. The collapse of the compression-resisting elements (the MTs) leads to loss of polarization, which in turn leads to increased contractility, and FA/stress fiber formation. (Danowski, 1989; Ingber, 1993).  39  AIM OF THE STUDY: Keratinocyte migration in human wounds requires extensions of cellular processes called lamellipodia. Tremendous volumes of research has been conducted to understand the signaling pathways leading to cell spreading, lamellipodia extension and migration, yet many questions remain to be answered regarding specific signaling pathways leading to lamellipodia extensions and intracellular protein kinases that control the organization of cytoskeletal actin and microtubules. A number of kinase modulators were used to target various signaling pathways in keratinocyte spreading in wound provisional matrix protein, fibronectin. Of particular interest among these modulators was staurosporine, which was found to promote extension of lamellipodia during keratinocyte spreading on fibronectin. Staurosporine was then further used to ask specific questions about the signaling pathway involved in lamellipodia extension in keratinocytes. The specific aims of this study were to investigate role of the following cellular cascades in staurosporine-induced lamellipodia extension. 1.  Role of other protein kinase and phosphatase inhibitors.  2.  Role of actin and microtubule cytoskeleton using cytochalasin-D and colchicine.  3.  Role of microtubule dynamics using nocodazole and taxol.  4.  Role of M A P K activation using Western blotting technique.  40  MATERIALS AND METHODS 1.  Cell line and cell culture Epidermal keratinocyte cell line HaCaT (Dr. Hubert Fusenig, Germany Cancer  Center, Heidelberg, Germany) are spontaneously  immortalized keratinocytes and  represent normal epidermal keratinocytes in most of their properties. They are not invasive and are able to differentiate under proper conditions (Boukamp et. al., 1988). HaCaT cells were routinely maintained in 25 cm tissue culture flasks (Falcon®; Becton 2  Dickinson Labware, France) in presence of Dulbecco's modified Eagle's medium (DMEM; Flow Laboratories, Irvine, UK) supplemented with 23 m M sodium bicarbonate, 20 m M Hepes (Gibco Biocult, Paisley, UK), antibiotics (50 jug/ml streptomycin sulfate, 100 U/ml penicillin) and 10% heat-inactivate fetal bovine serum (FBS). Cells were incubated at 37°C under 5% C O . The medium was changed every 2-3 days. Cells were 2  allowed to grow until early confluence, where they were detached with a brief incubation with 0.25 % trypsin (in E D T A , in Hanks Solution) at 37°C. Once cells were detached, trypsin activity was blocked immediately with FBS containing D M E M . Cells were recovered by centrifugation and were seeded in sterile culture flasks (approximately 800,000 cells/flask).  41  2.  Antibodies The following antibodies were used. The concentrations of all antibodies used were  previously tested to produce a maximal response (Koivisto et al, 1999). a) Inhibitory rabbit monoclonal antibody against the extracellular domain of Bl integrins (mAB13; Akiyama et. al., 1989) were used at the concentration of 20 jixg/ml for the spreading assays. b) Inhibitory mouse monoclonal antibody against the extracellular domain of ocv integrins (L230; Houghton et. al., 1982) was used at the concentration of 20 /xg/ml for the spreading assays. Antibody L230 was purified from cell culture supernatant of hybridoma cells grown in our laboratory (ATCCHB8448). c) Mouse anti-P tubulin antibody (No. 1111 876, Boehringer Mannheim GmbH, Mannheim, Germany) at a final working concentration of 3 jUg/ml was used in the immunohistochemical staining of HaCaT cells. Alexa Flour™ 546 goat anti-mouse IgG (H+L chains, F(ab') fragment conjugate) was used as the secondary antibody at 2  the concentration of 20 /xg/ml. d) Rabbit polyclonal anti-ACTIVE® M A P K (ERK1 and ERK2 at 42/44 KDa) antibody (Promega, U S A ) was used at the final concentration of 25 ng/ml. Peroxidase conjugated anti-rabbit swine IgG (1:1000 dilution; Dimension Laboratories Inc., Ont., Can.) was used as the secondary antibody.  42  3.  Toxicity tests Drugs were purchased from Sigma and were diluted under Sigma guidelines  (Appendix. A). The toxicity tests were done using Promega 96™ Non-Radioactive Cell Proliferation Assay according to the manufacturer's standard procedure. The cells were trypsinized with 0.25% trypsin two days prior to the experiment. This was done consistently with all experiments to ensure all cells are at approximately the same growth phase. 200 fiL of D M E M (with 10% serum) containing 30,000 HaCaT cells were incubated in triplicates in 96-well culture plates overnight at 37°C. Following overnight attachment medium was removed and cells were rinsed with 200 /xL of serum-free D M E M . Then 50 juL of the required drug concentration was added to the cells. The drugs were diluted in serum-free D M E M , , each with a different range of concentrations (Appendix. A), all prepared at their IX concentration. Control wells were included with cells in serum-free D M E M and no drugs. For drugs that were diluted in D M S O a set of parallel experiments with the appropriate D M S O concentrations were carried. After 24 hours the experiments were stopped and the absorbance (equivalent to the amount of living cells) were detected with ELISA plate reader at 570 nm. The amount of living cells were recorded as the mean absorbance value from triplicate wells, and were graphed using the Microsoft® Excel (version 5.1) program.  43  4.  Cell spreading assays A set of spreading assays were performed on fibronectin (from bovine plasma;  Chemicon) in the presence of the appropriate concentration of each drug. 96-Well plates (Nunc, Denmark) were coated by adding 30 /xL of fibronectin in PBS (20 /xg/ml; diluted +  in Phosphate-buffered saline containing C a , M g and N a +  +  +  ) for 60 minutes. The  unoccupied sites were blocked by 65 /xL of l%heat denatured bovine serum albumin (BSA) in PBS for 30 minutes. Also to measure the amount of non-specific binding to the plates, controls were carried with wells that were only exposed to B S A , and PBS . B S A +  was removed and the wells were washed five times with 150 piL of PBS . Cells in culture +  flasks were trypsinized and mixed with serum-free D M E M and were centrifuged twice to remove all the serum. During seeding, cells were exposed with cycloheximide (final concentration of 50 uM) to prevent de novo protein synthesis in cells. 5000 cells in 25 /xL of serum-free D M E M were added on top of 25 [xL of the each drug diluted in D M E M (prepared at 2X concentration), giving a final IX concentrations of 100 uM for genestein, 50 n M for staurosporine, 8.7 [iM for herbimycin A, 25 n M for okadaic acid, 200 /xM of bombesin, 100 juM for sodium-meta-vanadate and 100 nM for wortmannin. In addition two D M S O controls, one at 0.02 % and one at 0.5 %, were included which reflect the amount of D M S O present in okadaic acid and herbimycin A , respectively. All drug treatments were carried in triplicates. Cells were incubated at 37°C until obvious  44  spreading in F N was seen (approx. 75 minutes). At this point cells were fixed by carefully adding 100 juL of 2X fixative on top of the wells (8% formaldehyde,  % 0 1  sucrose in PBS ). Wells were then filled with distilled water to the top and covered with +  glass plate for observation with a phase-contrast microscope. Three fields in each well were randomly observed and the percentage of the cells spread was determined. Only cells that were completely surrounded with lamellar cytoplasm were considered as 'spread'. After counting, plates were washed and dried and stained with 0 1.%  crystal  violet. Stained cells were photographed under the 20X objective using a camera (Zeiss) attached to a phase contrast microscope. Cell spreading experiments involving the drugs taxol, nocodazole, colchicine, and cytochalasin-D were done according to the procedure described above. In experiments involving a combination of drugs with staurosporine, drugs at their 2X concentration were diluted in the same vial with 2X concentration of staurosporine (final IX concentration: 0 nM, 10 nM, and 50 nM). In spreading assays involving inhibitory anti-integrin antibodies, cells were pretreated on ice with either 81 (mAB13;  20jUg.m / )l  antibody, ccv (L230; 20ug/ml) antibody,  or both antibodies for 30 minutes to allow attachment of antibodies onto the cell surface. Antibody concentrations used were tested to produce maximal inhibition in cell spreading  45  (Koivisto et al, 1999). Following pretreatment with antibodies, cells were allowed to spread in 50 nM STP according to the procedure described above. HaCaT cells spreading assays were also conducted in presence of 50 n M staurosporine on type I collagen (20 ^g/ml; from bovine tendon; Upstate Biotech), type IV collagen (20 /t.g/ml; diluted in 0.1M acetic acid; Sigma), and laminin-5 (culture medium; gift from Dr. Quarants) using the method described above. To examine detailed morphology of keratinocyte spreading, HaCaT cells were allowed to spread on fibronectin-covered cover slips placed inside 24 well plates. 250 a\ of serum-free D M E M containing 50,000 cells, and 250 jiil of the 2X concentration of the drug staurosporine (IX concentrations: 0 nM, lOnM, 50nM) were added to each well. Cells were allowed to spread for 75 minutes, after which they were fixed with glutaraldehyde (2.5 % in 0.1 M PBS), and were treated for scanning electron microscopy (Aoki andTavassoli, 1981; Firth et al, 1997).  5.  Immunocytochemical staining HaCaT cells were allowed to spread on fibronectin-coated cover slips (20 jug/ml)  as stated above for 75 minutes in presence of serum-free D M E M control, 10 n M staurosporine, and 50 n M staurosporine. Coverslips were rinsed in serum-free D M E M  46  (no antibiotics). Samples were then fixed for 10 minutes in freshly made 3.7% formaldehyde (containing 4% sucrose) (BDH Inc.) in warm (37°C) PBS, followed by rinses with PBS. After fixation, cells were permeabilized for 3 minutes in 0.5 % Triton-X (Sigma) in CS buffer (Cytoskeletal Stabilizing buffer; 0.1 M pipes, 1 mM E G T A , 4 % P E G 8000, at pH 6.9) followed by a wash in CS buffer. Samples were then blocked with 1 % bovine serum albumin (BSA; Sigma) in PBS at 37°C for 30 minutes. Microtubules were stained using indirect immunofluorescence. Primary mouse anti-p tubulin antibody was used at the concentration of 3 /xg/ml. Alexa Flour™ 546 goat anti-mouse IgG was used as the secondary antibody at the concentration of 20 /xg/ml. Incubation of primary antibodies were performed for one hour at 37°C. 30 fi\ of antibody solution were dispensed into culture dishes. After they had been carefully blotted, the cover slips were placed, cell-side down, onto the droplets of antibody. In order to maintain a humid environment, moist filter paper was placed around the culture dish. Coverslips were incubated with the primary antibody at 37°C for 1 hour. After incubation, they were washed in 1% B S A , and were incubated with 50 fi\ of secondary antibodies, as it was done with primary antibodies. After one hour cells were rinsed and mounted, where they were viewed using the 63X oil immersion objective of fluorescence microscope.  47  6.  Western blotting To examine the possible activation of mitogen activated protein kinase (MAPK) in  keratinocytes, 300,000 HaCaT cells were seeded on fibronectin-coated 35 mm culture dishes in presence of serum-free D M E M , 10 nM staurosporine, or 50 nM staurosporine. A parallel spreading assay was also carried under the same conditions as described above. Cells were collected at 30, 60, 90, and 120 minutes. After each time points, unattached cells were collected into a 1.5 ml micro centrifuge tube where they were centrifuged at 1000 rpm for 1 minute. Attached cells were lysed and collected using the SDS reducing buffer prepared according to Laemmli guidelines (1970). The cells were then added into the pellet of unattached cells, so that samples at all time points had the equal number of cells. Sample separation was carried out by electrophoresis using 4% stacking and 10% resolving gels all according to guidelines stated by Towbin et al (1979). After electrophoresis, samples were transferred onto Hybond E C L membrane (Amersham, Little Chalfont, Bucking Hamshire, U K ) and immunoblotted with antiactive® M A P K antibody as primary, and peroxidase-conjugated swine anti-rabbit IgG antibody as secondary antibody. Transfer and immunoblotting was done according to Amersham's guidelines. Detection was also done according to Amersham's guideline using the Amersham's E C L kit.  48  7.  Cell Migration Assays: Cell migration assays that require cell body translocation (haptotaxis) were  performed using fibronectin coated (20 jiig/ml; coated on both sides) Polycarbonate membrane filters (pore size 8.0 [Jim, 13mm; Poretics Corp.®, Livermore, C A ) placed inside Boyden chambers. 100,000 HaCaT cells were seeded in the upper chamber on the porous membrane filters in presence of keratinocyte growth medium ( K G M ; Clonetics, San Diego, C A ) , and staurosporine (IX concentrations: control, 10 n M , 50 nM). The same concentration of staurosporine was present on both sides on the membrane. Cells were allowed to migrate across the membrane for 8 hours, after which membrane filters were fixed with glutaraldehyde (2.5 % in 0.1 M PBS), and were treated for scanning electron microscopy (Aoki and Tavassoli, 1981; Firth et al, 1997). The number of cells in the lower side of the membrane (cells which migrated across the membrane) were counted in ten random fields at the 100X magnification. Only cells that had fully migrated through the pores were counted as "migrated".  49  RESULTS 1.  Effects of protein kinases and phosphatases on keratinocyte spreading on fibronectin To study the role of protein kinases and phosphatases in lamellipodia formation in  keratinocytes, a series of drugs aimed at modulating activities of these intracellular proteins were used. The drugs with their respective targets are listed in table 2.  Table 2. Protein kinase and protein phosphatase modulators and their respective targets used in spreading assay experiments. NAME  FUNCTION  CONCENTRATION USED [IX]  Staurosporine (STP)  Broad kinase inhibitor  10 nM, 50 nM  Wortmannin  Specific inhibitor of PI-3Kinase  100 nM  Bombesin  Activation of tyrosine phosphorylation through  200 /xM  binding to G protein-coupled receptors. Okadaic Acid  Specific inhibitor of protein phosphatase type I  20 /xM  and II Specific tyrosine kinase inhibitor  8.7/xM  Genistein  Specific inhibitor of tyrosine protein kinase  200 /xM  Sodium metavanadate  Inhibitor of tyrosine protein phosphatase  100 /xM  Herbimycin A  Before conducting the spreading experiments, a series of toxicity tests were done, where the highest non-toxic concentration of each drug was determined. Graphs for these toxicity tests are included in appendix B.  50  1.1.  Staurosporine, Wortmannin and Bombesin induce keratinocyte spreading on fibronectin. HaCaT keratinocytes were seeded on fibronectin (FN) in the presence of the  selected drug concentration in serum free D M E M for approximately 75 minutes. Since okadaic acid and herbimycin-A were solubilized in 0.02%  and 0.5%  DMSO,  respectively, a set of controls containing the two vehicle solvent D M S O concentrations were included. D M S O (0.001%) control for staurosporine (STP) was used initially but found to be have no significance on cell spreading (appendix B). The percentage of cells that were 100% spread were determined using the 40X objective of the light microscope. The spreading of cells on F N is indicated by the formation of a curtain-like extension all around the cell (Fig. 4, open arrows). Only cells with this lamellipodium all around were considered as "spread". The results illustrate that relative to the control, cell spreading was induced by 240% in presence of 50 n M STP, 330% in the presence of bombesin, and by 190% in the presence of wortmannin (Figure 3). Herbimycin-A on the other hand was found to inhibit cell spreading, although when compared to the 0.5% D M S O control, this inhibition is considered insignificant. Okadaic acid, genistein, and sodium metavanadate were found to have no effects on cell spreading. No spreading was observed on B S A coated wells, indicating that cells spread specifically on FN.  51  u o  c u E o a.  B  r3 'EL >. U  T3 c 3 60 « •a ^ '<- 'C c 17)  >» Z u E o  c C U  u -o s  c  c o 8 T3  1-  C  c ed U C3  u u a o o Clj  c  o  c X)  o  3 C  Q  T3  C g.  "53  00  E -  a.  2} £  Q >, B  X! 5 c p >+t£ ^ O w u  JS  -a e  ° u c  u c1>d  <U u ed ^3 c 'S uu T3o a B 4 — -> o  o  a,  o  U  C B  -O  s •a CU CU  en o 3 U  s II  s  c c  Q t/i + c  s  3 d u V c  c  52  1.2.  Staurosporine induces formation of extended lamellipodia  The morphology of HaCaT keratinocyte spreading in the presence of various drugs was carefully monitored to find out any specific effects on lamellipodia formation (Fig. 4). In serum free D M E M , genestein, okadaic acid, and sodium metavanadate, the cells spread similarly to control cells (Fig. 4 A, C, E , and G respectively); they are either non-polar (lamellipodium extends circularly all around the cell), bi-polar (two lamellipodia stretching towards two opposing directions), or multi-polar (lamellipodia are extended towards multiple directions). Keratinocytes in the presence of herbimycin-A and bombesin appear to have spread somewhat more in a non-polar fashion (Fig. 4 D, and F respectively). Interestingly, STP appeared to have a unique effect on keratinocyte cell morphology. Cells formed very thin but long lamellipodia extending in multiple direction (Fig. 4 B, black arrows). We will refer to these as "extended lamellipodia", or "E-Lams". At the tip of the E-Lam, small curtain-like lamellipodium is formed. Because of the unique effect of STP on keratinocyte lamellipodia formation and its ability to stimulate cell spreading on F N , it was selected for further studies on regulation of lamellipodia formation in keratinocytes.  53  A  2  #v  • V . ; J D  c  F  ^ 4  G  N  g  «  •  •  •  *  Figure 4. Morphology of HaCaT keratinocytes seeded on fibronectin in presence of protein kinase and phosphatase modulators. Cells were allowed to spread on F N coated wells for 75 minutes in presence of serumfree D M E M (A), 50 n M staurosporine (B), 200/iM genistein (C), 8.7 ixM herbimycin-A (D), 20 n M okadaic acid (E), 200 /xM bombesin (F), 100 / i M sodium metavanadate (G), and 100 n M wortmannin (H). Open arrows indicate lamellipodium. Solid arrows indicate extended lamellipodia.  54  1.3.  The morphology of staurosporine-induced formation of  extended  lamellipodia is different on different extracellular proteins. To determine whether effect of STP was specific to F N matrix alone, keratinocytes were seeded on collagen type I, collagen type IV, and laminin-5 (each at 20 /Ltg/ml) in presence and absence of 50nM STP, for 75 minutes. The results were plotted based on three experiments except laminin-5 where only one experiment was done. According to the results, keratinocytes seemed to spread faster on collagen type I, and collagen type IV, when compared to F N or laminin-5 (Fig. 5). This appears to be the case both in control and STP-treated cells. Extension of keratinocyte lamellipodia appeared to be different on different matrix proteins (Fig. 6). STP-treated HaCaT cells on collagen type I and collagen type IV (Fig. 6D, F respectively) appear to form multiple short E-Lams, while on F N (Fig. 6B) cells form fewer but longer E-Lams. Cells on Laminin-5 also developed E-Lams (Fig. 6H); however, they appeared to be somewhat shorter than those formed on FN. Further studies on STP-induced E-Lam formation were conducted on F N matrix, because 1)- it is the main component of wound bed provisional matrix, and 2)- the morphology of the E Lam was found to be simple and unique.  55  1 00% • No S T P  90%  • S T P 50 nM  80%  73 a  r-r—i  70%  60%  H  50% , ^  w  40% 30% 20% 10% 0% Fibronectin  Figure 5.  Laminin-5  Collagen type I  Collagen type I V  Staurosporine-induced cell spreading on different matrixes.  Wells were coated in triplicate with fibronectin, laminin 5, collagen type I, and collagen type IV, each at the concentration of 20 /xg/ml. Prior to seeding, keratinocytes were treated with 50 /xM of cycloheximide to prevent de novo protein synthesis. Cells were allowed to spread for 75 minutes in presence and absence of 50 nM STP. Cells spreading was calculated based on three random fields from each well. (Mean±S.D.);n=9. * (p < 0.05) to FN  56  B  V c  • • •  >  •  D  /  " •  •  w- * G  •  * • • Figure 6. Morphology of HaCaT cells spreading on fibronectin, collagen type I and IV, and laminin-5. Cells were allowed to spread on F N (A, B), collagen type I (C, D), collagen type IV (E, F) , and laminin-5 (G, H), for 75 minutes in the presence (B, D , F, H) or absence (A, C , E , G) of 50 n M staurosporine.  57  1.4.  Staurosporine stimulated keratinocyte spreading is mediated through ccv and p i integrins. To investigate, whether STP- stimulated E-Lam formation on F N was integrin  dependent, a set of spreading assays were conducted in the presence of integrin blocking antibodies. Cells were seeded on FN-coated wells (20 /xg/ml) for 75 minutes in the presence and absence of 50 nM STP and also in the presence or absence of monoclonal antibodies against pi integrin (Mabl3, 20 jiig/ml) or ocv integrin (L230, 20 ptg/ml) either together or separately. It was found that mAb against Pi decreased cell spreading in control by 67%, while it decreased spreading of STP-treated cells by 62%. Similar inhibition was observed when cells were treated with anti-av antibodies alone. When the two antibodies were combined, they totally abolished cell spreading on F N . In partially inhibited wells, the single integrin antibody did not change the morphology of STPinduced E-Lams, indicating that both STP-induced cells spreading and E-Lam extension are dependent on  pi and ccv integrins,  previously shown to be cc5pi, ocvpi and avP6  (Koivisto etal. 1999).  58  Lam E-Lam  +  +  +  +  +  +  —  +  —  +  —  +  Figure 7. Effect of inhibitory anti-integrin antibodies on H a C a T cells on fibronectin. Cells with or without 50 nM STP-treatment were allowed to spread on wells coated with fibronectin (20 /xg/ml) for 75 minutes. Blocking antibodies against ocv (L230; 20 /Jg/ml) and (31 (Mabl3; 20 /xg/ml) were used together or separately along with STP. The mean value of triplicate wells were used. The morphology of the cells (i.e. presence or absence of Lamellipodia, and Extended Lamellipodia) was also evaluated. (Mean±S.D.); n=9. * P< 0.05 to no mAbs control  59  1.5.  Promotion of keratinocyte extended lamellipodia by staurosporine is concentration-dependent. The effect of S T P on cell spreading and cell morphology was further investigated  using different concentrations of S T P . Cells were seeded on F N (20 jiig/ml) in serum-free media in the presence of 10 n M and 50 n M S T P for 75 minutes. Although cell spreading was stimulated to the same level at both 10 n M and 50 n M S T P , cells at 10 n M S T P (Fig. 9B) did not form extended lamellipodia (E-Lam) (black arrows) as they did at 50 n M STP (Fig. 9C). The size of lamellipodia (open arrows) however, was larger than the ones in control cells (Fig. 9 A ) . The difference in the shape of the lamellipodia is clearly observed when cells are viewed using the Scanning Electron Microscope ( S E M ) . Another unique feature in 50 n M S T P treated cells is the expression of short, hair-like structures called filopodia (arrowheads), which are not visible under light microscope, and are only viewed here with the aid of the S E M microscopy. Filopodia are projected from the lamellipodia, and do not exist in control cells, or cells treated with 10 n M S T P .  60% M 50%  Figure 8. Effect of concentrations  i  of  different stauro-  sporine on cell spreading on  a 40%  fibronectin.  & 30%  Cells were allowed to spread on FN  g  or BSA for 75'  $  20%  in presence or  absence of 10 nM STP, and 50 nM STP.  10%  (Mean + S.D.); n=9. 0% Control  STPIOnM  STP50nM  BSA  *p < 0.05 over control  60  1.6.  Formation  of  extended  lamellipodia  is  not  blocked  by  other  kinase/phosphatase inhibitors. To determine whether the formation of E-lams could be prevented with protein kinase inhibitors (wortmannin and genistein), or protein phosphatase inhibitors (okadaic acid and sodium metavanadate), cells were seeded on F N (20 jUg/ml) for 75 minutes in presence of serum-free D M E M control, 10 nM STP and 50 n M of STP (Fig. 10 left, middle, and right column, respectively), in combination with genistein (200 uM) (Fig. 10 D-F), okadaic Acid (20 nM) (Fig. 10 G-I), sodium metavanadate (100 nM) (Fig. 10 J-L), or wortmannin (100 nM) (Fig. 10 M-O). None of these drugs were able to block the STPinduced formation of E-Lams. The only change in cell morphology was observed at the combination of 10 n M STP with 100 n M sodium metavanadate (Fig. 10 K) which resulted in the formation of spindle-shaped cells, some with small lamellipodia at the ends. Also enhanced spreading by STP was not changed by any of the protein kinase or phosphatase modulators tested (Fig. 11). The only difference that was statistically significant was the stimulation of cell spreading by wortmannin in control cells.  62  1^  * i  •  *  4ir^^Sk.  V  D t  1 H  * W  C* K  "  <\ 1  r  0  ^  *-  1 1  Figure 10. Morphology of HaCaT cells spreading on fibronectin in presence of STP 10 and STP50, in combination with various protein kinase and phosphatase modulators.  Cells were allowed to spread for 75 minutes on FN-coated wells (20 /ig/ml) in presence of no serum D M E M control (left column), 10 n M STP (middle column), and 50 n M S T P (right column), in combination with 200 liM genistein (D-F), 20 n M okadaic acid (G-I), 100 fM sodium metavanadate (J-L), and 100 n M wortmannin (M-O). Cells were stained with crystal violet and photographed using the 20X objective.  63  c o  S  &  c o  o  u o  u o  c o  00  c  H3  a, O  H  M  u  a  X c o o  H T3  c c c u  DH  H oo  c  a. r-  C/3  II  w  c  o  1=  u >  Q  3  DO  GO  in  c  ° v  ca «3  Z.  64  2.  The role of actin and microtubule cytoskeleton in staurosporine-induced keratinocyte cell spreading  2.1.  Actin polymerization is required for cell spreading and E-Lam formation. To determine the involvement of actin cytoskeleton  on the formation of  lamellipodia in STP-treated cells, keratinocytes were simultaneously exposed to the actin-disrupting agent, cytochalasin-D. Based on the literature (Small et al,  99)  concentration of 1 fxM was used to completely depolymerize the actin filaments. The control cells were treated with 0.01% ethanol vehicle. Cells were allowed to spread on 20 LMg/ml F N for 75 minutes. The results show that control cells, and STP-stimulated spreading was completely blocked by cytochalasin-D (Fig. 12). The ethanol control had no effect.  2.2.  Microtubule polymerization is required for the formation of extended lamellipodia. To find out the involvement of microtubule (MT) filaments on the formation of  lamellipodia in STP-treated cells, keratinocytes were simultanously exposed to the M T depolymerizing agent, colchicine. According Mikhailov and Gundersen (1998), colchicine at 1 uM concentration completely depolymerizes microtubules. Cells were allowed to spread for 75 minutes  on F N . It was  found that colchicine at  65  cCu3  IT3 ft  09 05 ga  • No STP • STP 1 0 nM • STP 5 0 nM  60%  5 0 %  1  40%  1  CU | CM  30%  O |  $  20%  10%  0%  Control (0.01 % B O H ) Figure 12.  CC-D 1 /jM  Effect of cytchalasin-D on H a C a T cell spreading on fibronectin.  Cells were allowed to spread for 75 minutes on Fibronectin (20ug/ml) coated wells is presence or in absence of staurosporine (10 nM, 50 nM) and cytochalasin-D (1 /xM). The results are based on triplicate wells. Cell did not spread on BSA coated wells. Control cells contain 0.01% ethanol, equivalent to the amount of ethanol in 1 /iM cytochalasin-D. nvTean + S.D.I: n=9.  66  concentration of 1 /xM completely inhibited spreading of control cells and 10 nM STPtreated cells. Cell spreading in the presence of 50 nM STP was reduced by 17% (Fig. 13). Interestingly, colchicine blocked the formation of E-Lams, and appeared to reduce the size of the lamellipodia (Fig. 14). S E M pictures of these cells demonstrate that although the formation of E-Lams was blocked by colchicine, the finger-like projections (filopodia) continued to form (Fig. 14 B).  2.3.  Microtubules accumulate to the tip of the extended lamellipodia in STP-stimulated keratinocytes. Cells were grown in presence and the absence of STP 10, and STP50 on FN for 75  minutes, fixed and processed for immunolocalization of P-tubulin, a component of microtubules. It was found that P-tubulin accumulated mainly around the nucleus in control and STPlO-treated cells (Fig. 15 A, and B, respectively). In STP50-treated tubulin accumulated at the tips of the E-Lams in addition to the area around the nucleus (Fig. 15 C) (arrowheads).  67  • No STP  70% -  • STPIOnM  60% -  S U  STP 50 nM  50%  w 40% 30%  «g 20% ^  ±  10% 0%  Control  Lam  +  E-Lam Figure 13.  + +  Colchicine  +  Effect of colchicine on HaCaT cell spreading on fibronectin.  Cells were allowed to spread for 75 minutes on FN (20 jug/ml) coated wells is presence or in absence of staurosporine (10 nM, 50 nM) and colchicine (1 juM). The results are based on triplicate wells. Cell did not spread on BSA coated wells (results not shown). One typical experiment out of three is shown. (Mean ± S.D.); n=9. *p < 0.05 over STP 50 control  Figure 14. Morphology of HaCaT cells treated with 1 uM colchicine and 50 n M staurosporine on fibronectin. Cells were allowed to spread on FN-coated coverslips for 75 minutes in presence of 50 nM STP and 1 nM colchicine. Crystal violet stained cells (A); SEM view was taken at 1000X magnification (B). lamellipodia are labeled with arrow, and filopodia with arrowheads. For control cells refer to figure 9.  68  Figure 15.  Immunolocalization of tubulin in HaCaT cells  in response to staurosporine. Cells were allowed to spread on FN-coated coverslips for 75 minutes in presence of no serum DMEM control (A), 10 nM staurosporine (B), and  50  nM  staurosporine  permeabilization,tubulin flourescence.  (C).  After  was immunolocalized  fixation  and  by immuno-  Figures were taken with the 63X objective. The  arrowheads demonstrate accumulation of tubulin at the tips of the extended lamellipodia.  3.  Role of microtubule dynamics on STP-stimulated keratinocyte spreading on fibronectin  3.1.  Taxol and nocodazole  have no effect on the formation of extended  lamellipodia Because microtubules appear to be important for the formation of extended lamellipodia, we investigated further whether the microtubule dynamics play a role in E Lam formation. For this purpose, the M T inhibitors taxol and nocodazole (modulators of microtubule dynamics) were tested for their toxicity on HaCaT keratinocytes (Appendix A). The highest non-toxic concentration along with a higher (and slightly toxic) concentration of each drug was used for further experimentation. Both doses of 20 and 200 n M of nocodazole were found to have no effect on cell spreading in control wells (Fig. 16). The spreading of 10 n M STP-treated cells however, were reduced to control levels. Cells treated with 50 n M STP were not affected by nocodazole at 20 nM, but their spreading was reduced by 35% when treated with 200 nM of nocodazole. Similar effects were observed with 10 n M taxol, where control cells remained unchanged while in 10 nM STP-treated cultures, cell spreading was reduced to the level of control cells. With 10 nM taxol, cell spreading in 50 n M STP-treated cultures remained unchanged. High concentration of taxol reduced cell spreading by 50% in both control and STP-treated cells. Despite the reduction in cell spreading at high concentrations of taxol and nocodazole, the formation of E-Lams were not blocked.  70  a,  T3  t/5  0  *H 0 B  2  o  1 2  u  T3 OJ  -3 B ty  8f  X  u  5  £  o <u C  r3  o  X)  c o  3  -  O  2 3  p  X  3  E  •g -5  B  _ |  in  £ o ^ o E  B  el  u U E  0  XT  °!  =1 c  o  o  ==  o  «  O  <3 o c  O  -C 3  0  r3  a  u  B  c  a w c 'S  H  <  m  c  o o  •g c  O.  c  M  -a B -  g ed u c o 6 z 1 •d o a. c c  o o  •a  u  S .£ «  •d 2u  si o  c c u 8 Jo  B B  E  c  3  2  ll  B  d  2  >  u  o g B  B C  u  vi c +1 uon B © v c  71  4.  M A P K activation in response to Staurosporine  Mitogen activated protein kinase pathway (MAPK; ERK1/ERK2) regulates many cellular events including cell morphology and spreading (reviewed in Alpin et al, 1998). The function of the M A P K is thought to be time-dependent (Marshal, 1995). We therefore tested whether phosphorylation of M A P K is induced by STP using antibodies against phosphorylated E R K 1 and E R K 2 (MAPK) at different time points of spreading on FN. Cells were treated with STP10 and STP50 and were collected at 30, 60, 90 and 120.minutes. A parallel spreading assay (Fig. 17C) was performed to correlate the formation of lamellipodia and E-Lams with activation of M A P K . The reduced sample was separated by electrophoresis, transferred onto Ffybond E C L membrane and immunoblotted with an antibody against phosphorylated M A P K . Phosphorylated E R K 1/2 (Fig. 17 A) were quantified using the NIH Image program (ver. 1.62). The level of phosphorylated E R K 1 and E R K 2 in STP50-treated cells was 56% higher than the control cells, and 110% higher than the STPlO-treated cells at 30 minutes (Fig. 17B). This activation appeared to precede significant cell spreading. Phosphorylation of M A P K continued to increase along with keratinocyte spreading on F N . Formation E-Lams induced by STP50 was evident already after the 30 minute time point. STP50-treated cell spreading rate was similar to STPlO-treated cells. M A P K phosphorylation however, was higher for STP50-treated cells at all time points.  72  (A)  (B)  a  30000 25000 20000  • No STP • STP 10 nM • STP 50 nM  15000 T5 C  I  10000  33  5000 0 30'  (C)  cs CU  a  ft  1oo % 75 %  60'  Time (min)  90'  1 2 0'  90  1 20  • No STP • S T P 1 0 nM • STP 5 0 nM  en  5 0% 2 5 % 0 % 30  60 '  Time (min)  - - +  E-Lam  Figure 17.  Activation of M A P K (ERK1/2) in STP-treated HaCaT cells.  Cells were allowed to spread in F N - coated (20 jUg/ml) wells in presence of no serum D M E M control, 10 n M STP and 50 n M STP. Cells were collected at four different time points (30', 60', 90', and 120'). Reduced samples were separated by electrophoresis, transferred onto Hybond E C L membrane and immunoblotted with antibodies against phosphorylated M A P K protein. Bands were quantified using N I H Image program (Ver. 1.62). A parallel spreading experiment was carried out under identical conditions, where cells were fixed and counted at each time point. The morphology of the cells (i.e. presence of extended lamellipodia) at each time point was also examined. (Mean±S.D.); n=10.  73  5.  Effect of staurosporine on cell migration  5.1.  Staurosporine stimulates cell migration on fibronectin. Extension of lamellipodium is a first sign of cell movement. W e investigated,  therefore, whether STP-induced cells are more migratory. T o determine the effects of S T P on cell migration, H a C a T cells were allowed to migrate in Boyden chambers using membranes coated with 20 jiig/ml of fibronectin on both sides in presence of STP10 and STP50 diluted in keratinocyte growth medium ( K G M ) for 8 hours. Both the upper and lower chamber contained the same S T P concentration. Membranes were fixed and subsequently processed for S E M (Fig. 19). The results demonstrated that cell migrating across the membrane pores was increased by almost 100% in presence of STP50 and STP10, when compared to control (Fig. 18).  Figure 60.0 T  18. Effect  of S T P on  H a C a T cell migration. Cells were seeded onto F N (20 /xg/ml) coated Polycarbonate membrane filters  *  placed presence  in  Boyden of  Chambers, in  keratinocyte  growth  medium ( K G M ) in presence or absence  •£ 20-0 J £ z  of STP10 n M , and 50 n M . Cells were  1  allowed  10.0 • o.o  4  no STP  to  migrate  across  the  membrane for 8 hours. Filters were processed for S E M , and the number of cells migrated across the membrane per  STPIOnM  STP50nM  field were counted. (Mean±S.D.); n=10. *P < 0.05 over no STP control  74  Figure 19. Morphology of migrating HaCaT cells treated with different concentrations of staurosporine on FN viewed by scanning electron microscopy. Cells were allowed to migrate through FN-coated membranes for 8 hours in presence of no serum D M E M (A), 10 n M S T P (B), and 50 n M S T P (C). After fixation with glutaraldehyde, the membranes were treated for scanning electron microscopy. Pictures were taken at 480X magnification.  DISCUSSION Keratinocyte migration on wound provisional matrix starts with the formation of long lamellipodia extending into the wound (Larjava et al, 1996). The rest of the cell will eventually follow, leading to migration of the keratinocyte. During this process, cell adhesions are constantly formed and dissolved (reviewed in Mitchison and Cramer, 1996). Formation of lamellipodia leading to cell migration also involves regulation of the cytoskeletal actin and microtubule organization (Small et al,  1999; Mikhailov and  Gunderson, 1999). It is believed that intracellular protein kinases and protein phosphatases control integrins affinity and cytoskeletal structures (O'Tool et al, 1994). Although a large volume of research has been done to elicit these intracellular proteins, many questions remain to be answered with respect to the pathways involved in the formation of extended lamellipodia and cell migration in keratinocytes.  1.  S T P stimulates cell spreading and formation of extended lamellipodia on fibronectin through av and (31 integrins In this research we used a number of protein kinase and phosphatase inhibitors to  target different signaling pathways in HaCaT keratinocytes. Of all the drugs used, staurosporine appears to promote the most unique effect; when cells are treated with 50  76  nM STP, not only did they spread and migrate more, but also they formed long extended lamellipodia similar to the ones expressed in keratinocytes at the edges of the wound in vivo (Larjava et al., 1996). More interestingly, cells treated with 10 n M STP, although they showed equally enhanced cell spreading and migration, do not form extended lamellipodia. This suggests that the signaling pathways activated in keratinocytes at 50 nM STP are different from the ones at 10 n M STP. The formation of extended lamellipodia by STP was previously observed in rat epidermal cells (Sako et al, 1988), rat pheochromocytoma PC12 cells (Rasouly 1994) and megakaryoblastic cells (Yamazaki et al, 1999); however, the intracellular mechanism by which this occurs has not yet been determined. Formation of long E-Lams, is unique to the F N matrix. STP-treated cells seeded on other extracellular matrix molecules (i.e. laminin-5, collagen type I, and collagen type IV) showed extended lamellipodia which are shorter than those formed on F N . This suggests that the intracellular signals caused by STP which result in E-Lam formation, are dependent on F N matrix and therefore likely to involve the keratinocyte specific integrins, ccv|3l, a5|3l and av(36 (Koivisto et al,  1999). This hypothesis is further  supported by our results where both control and STP treated cells showed decreased cell spreading in presence of inhibitory anti-av and anti-Pi antibodies. When both antibodies were present at the same time, cell spreading was completely inhibited suggesting that  77  signaling cascades leading to cell spreading and E-Lam formation require adhesion of cells to F N via the av and (31 integrins. These results are consistent with Koivisto et al. (1999) that showed keratinocytes can use av(3l, a5(3l and ocv(36 integrins to attach and migrate on FN. How STP modulates intracellular protein kinases has not been elucidated. STP is a very potent drug that has the ability to either stimulate or inhibit multiple protein kinases in a cell-type specific manner (Tamaoki and Nakano, 1990), thus it offers many potential targets that may play an active role in the STP-induced cell spreading and E Lam formation in keratinocytes. To test some of these proteins, HaCaT cells were treated with STP in combination with tyrosine protein kinase and phosphatase inhibitors (genistein and sodium metavanadate, respectively). The results show that none of the drugs affected STP-induced cell spreading, or STP-induced E-Lam formation, suggesting that these processes are not mediated by tyrosine phosphorylation. In addition to genistein and sodium metavanadate, specifc inhibitors of phosphoinositide 3-Kinase (PI3K) (wortmannin) and protein phosphatase type I and II (PP I, PP II) (okadaic acid) were tested with similar results, suggesting that activation of PI-3K, or extended phosphorylation of PP I and PP II were also not involved in the formation of E-Lams in keratinocytes.  78  The only combination that appeared to have an effect in the morphology of keratinocytes was with the drug sodium metavanadate and 10 n M STP, where keratinocytes demonstrated spindle shaped morphology. It is therefore likely that sodium metavanadate-induced phosphorylation/de-phosphorylation of intracellular kinases is involved in regulation of spindle shape cell morphology. Since sodium metavanadate is an inhibitor of protein tyrosine phosphatase, the spindle shape morphology might be as a result of extended phosphorylation of tyrosine. The transformation of round keratinocytes to spindle shape has been observed previously (Caulin et al,  1995), where TGF-fH  treated mouse epidermal keratinocytes expressed a fibroblast-like phenotype that was able to induce spindle cell carcinoma upon transplantation in athymic nude mice. Fibroblastic spindle shape morphology is unusual for keratinocytes, and artificial promotion of such phenotype with kinase modulators might provide a useful tool to investigate the signaling pathways involved in this process, as well as the potential pathological consequences of the fibroblastic phenotype. When treated alone, bombesin and wortmannin, show induced cell spreading, but no E-Lam formation, suggesting E-Lam formation is not a simple addition to cell spreading, and it is probably regulated through a specific pathway. Wortmannin induces cell spreading by specifically inhibiting the PI-3K protein in keratinocytes. This is contrary to the previous data on platelets and osteoclasts, where cell spreading was  79  inhibited by wortmannin (Lakkakorpi et al,  1997), suggesting that PI-3K plays a  different role depending on the cell type. When cells are treated with both wortmannin and STP, the spreading effect was not synergistic, suggesting that PI-3K inhibition may not be involved in the STP-induced E-Lam formation. Bombesin-induced keratinocyte spreading is likely to be stimulated through activation of the Ras pathway by the G protein-coupled receptors (Fig. 3) (Charlesworth and Rozengurt, 1997). According to the literature, bombesin/G protein-coupled receptor could also induce spreading through activation of PI-3K (Fig. 3) (Seufferlein et al,  1995), but this is not likely in  keratinocytes since it would be contrary to the results we found with wortmannin. What major proteins then might be involved in STP-induces cell spreading and E Lam formation in keratinocytes? Some researchers have shown that in keratinocytes, STP leads to the activation of the protein kinase C (PKC) (Jones and Sharpe, 1994), which is downstream from PI-3K (Fig. 3) (Mattias et al, 1998). This is contrary to the belief that STP is purely a P K C inhibitor (Ruegg and Burgess, 1989). Previous data has shown that STP acts as a P K C agonist to give similar effects as TPA (tetradecanoylphorbol acetate) such as increase in number of cornified envelopes (Jones and Sharpe, 1994). Also PKC activation has been correlated with actin cytoskeleton remodeling in keratinocytes, where stress fibers were induced, and cortical actin filaments were disassembled (Marias et al, 1998). PKCs have also been shown to play a crucial role in the regulation of various  80  integrin-dependent cellular functions such as cell adhesion, spreading and motility by increasing affinity of integrins (reviewed in Clark and Brugge, 1995). Previous results on colon carcinoma cells demonstrate that cell spreading and migration can occur as a result of an increased activation of mitogen activated protein kinase (MAPK), through PMAinduced activation of P K C (Rigot et al, 1998). Thus, it is possible that induced cell spreading and E-Lam formation requires direct activation of P K C and M A P K by STP (thus bypassing PI-3K), and is dependent on integrin receptors (Fig. 20C).  2.  MAPK  activation precedes STP-induced cell spreading and E - L a m  formation M A P kinase (ERK 1 and E R K 2) have been recognized as a major system which cells transduce a variety of extracellular signals (Fig. 2) (reviewed in Alpin et al, 1998; Longhurst et al, 1998; Takada et al, 1997; Seger and Kreb, 1995). While M A P K has been associated with the transcriptional control of genes important for cell proliferation and differentiation, it is now clear how M A P kinase can also promote cell spreading and migration on the E C M in a transcription independent manner (Alpin et al 1998; Klemke, 1997). This is supported in a recent paper, where PD98059, a specific M A P K inhibitor, reduced cell migration (Gundersen and Cook, 1999). We examined the effects of STP on M A P K activation using Western blotting technique, and the results demonstrate that  81  keratinocytes treated with STP10 and STP50 show different levels of M A P K activation. STP50 treated cells exhibit almost twice as much M A P K phosphorylation as the STP10 treated cells. The effects of M A P K signaling strength has been demonstrated before, in Swiss 3T3 cells, where weak stimulation of M A P K is PI-3K dependent but strong stimulation is PI-3K independent (Duckworth and Cantley, 1997). Also the duration of M A P K activation was found to lead to different M A P K functions. For example, in keratinocytes, sustained activation of M A P K leads to cell migration while transient activation of M A P K does not (McCawley et al., 1999). Therefore, it is reasonable to hypothesize that the difference in level of M A P K phosphorylation may be the reason why E-Lams are formed at 50 nM STP and not 10 nM STP. The difference in activation of M A P K is evident before obvious cell spreading is present, which suggests that STP-induced M A P K activation precedes cell spreading and E-Lam formation. This result supports the hypothesis that induction of cell spreading and formation of E - L A M by STP is by an "inside-out" mechanism, where integrin function is modulated by M A P K from inside the cell (Fig. 2)(reviewed in Longhurst and Jennings, 1998). Further experiments should be conducted in keratinocytes with the use of specific M A P K inhibitors to further elicit the degree of involvement of M A P K in cell spreading and E-Lam formation.  82  3.  Actin and microtubule cytoskeleton are required for the formation of extended lamellipodia STP-induced changes in cytoskeletal actin microfilaments and MTs, as well as  their involvement in cell migration and spreading have been reported by several groups (Miyamoto and Wu, 1990; Maroney et al, 1995; Rasouly et al 1994); however, little is known about their relation with extended lamellipodia formation, especially in keratinocytes. Our results confirm previous studies (reviewed in Alpin and Juliano, 1999; Yamazaki et al, 1999) where actin depolymerization by cytochalasin-D was found to completely block cell spreading and lamellipodia formation, suggesting that actin polymerization is an integral part of these processes. Past research has shown actin polymerization, and specially its association with focal adhesions and contractility are regulated by M A P K (reviewed in Alpin and Juliano, 1999). According to Kelmke et al (1997), M A P K regulates actin-myosin interactions, focal adhesion formation and cell contractility necessary for cell migration (Klemke et al, 97) through activation of the myosin light chain kinase (MLCK) (Fig. 2). Studying phosphorylation of M L C K in connection with differential M A P K activation by STP, may provide valuable insights on mechanisms by which E-Lams are formed. Similar results to actin were observed in depolymerization of microtubules by colchicine, where spreading of the control and STP 10-treated cells were completely  83  blocked. While total cell spreading was only partially inhibited at 50 nM STP, formation of E-Lams was blocked completely, suggesting that MTs also play a crucial role in formation of lamellipodia and extended lamellipodia. Our immunostaining experiments clearly indicate that microtubules are localized to the tips of the extended lamellipodia as well as the cell body around the nucleus in the STP 50 treated cells. The "diffusable factor" theory  (Mikhailov and Gunderson, 1998) suggests MTs at the edges of the  expanding lamellipodium might play a role in modulation of cell migration and lamellipodia formation by releasing diffusable factors into the lamellipodium. This theory is further supported by our spreading experiments where taxol, a M T hyperstabilizer, reduce spreading by 50%. It is possible that taxol is reducing cell spreading and lamellipodium formation by inhibiting the release of M T diffusable factors. High concentration of nocodazole, a M T polymerization inhibitor, also decreased STP-induced cell spreading and lamellipodia formation, but this is mostly due to depolymerization effect (characteristic of nocodazole at high concentration) similar to colchicine. It has been suggested that MTs regulate cell shape and cell migration through their interactions with M A P K (reviewed in Gunderson and Cook, 1999). In neutrophils, treatment with taxol leads to inhibition of M A P K (Jackson et al, 1997). Furthermore, M A P K has been shown to associate with a number of MT-specific proteins such as tubulin, and microtubule-associated protein 1 (MAPI) and microtubule-associated  84  protein 2 (MAP2) in a veriety of cell lines, including neurons (Morishima-Kawashima and Kosik, 1996), Chinese hamster ovary cells (Hoshi et al,  1992), and PC12 cells  (Reszka et ah, 1997). These proteins are thought to be active elements in M T cytoskeleton organization. However, they are not the only players involved since some M A P K mutations which do not affect M T binding, still cause defects in M T organization (Reszka etal, 1997). Another pathway that leads to regulation of actin cytoskeleton is by the family of small GTP-binding proteins, comprising of Rho, Rac, and Cdc42 (reviewed by Tapon and Hall, 1997). Numerous reviews have described these proteins as key players in the formation of integrin-mediated stress fibers, lamellipodia, and filopodia formation. Our preliminary results with dominant negative Rho A , Rac and Cdc42 (data not shown), suggest that RhoA is crucial in keratinocyte spreading and lamellipodia formation since 100% of the cells expressing the dominant negative Rho A construct, showed no spreading. No difference was observed with Rac and Cdc42; however, further experiments with dominant-negative as well as dominant-positive forms of these GTPases need to be conducted to provide a better understanding of some of the pathways involved in the formation of E-Lams in migrating keratinocytes in wounds.  85  4.  Summary: Formation of extended lamellipodia is a unique characteristic of keratinocyte  migration in the wound. Integrin function, and cell attachment to substrate molecules, as well as organization of actin and microtubule filaments are crucial in formation of extended lamellipodia. Although the intracellular proteins involved in this process are not fully understood, our results indicate that M A P kinase might be involved as a key regulator. A hypothetical mechanism for the regulation of E-Lams and cell spreading is illustrated in Figure 20. According to our results, normal attachment of cells to F N leads to activation of M A P K . This in turn could lead to an inside-out activation of integrins and cell spreading (Fig 20 A). A higher activation of M A P K induced by 10 n M STP (in addition to attachment to FN) might lead to increased integrin affinity and induced cell spreading (Fig 20 B). The highest level of M A P K activation, induced by 50 n M STP, triggers formation of extended lamellipodia as well as enhanced cell spreading (Fig. 20 C). Thus, suggesting that M A P K activation has to reach a certain threshold level before E-Lams can be formed. Since actin and microtubule filaments are required for E-Lam formation and cell spreading, M A P K interaction with cytoskeletal elements as well as proteins such as M L C K might be an integral part of this process. Aside from the M A P K pathway, M L C K and the cytoskeleton could be regulated through the small Ras related GTP-binding proteins, RhoA, Rac, and Cdc42. Although  86  our preliminary results support this hypothesis, more research has to be done to fully understand this pathway in keratinocytes.  87  g>"8  .E •a co a> o. co  o CO c co *- o x: aj co 'c  03  O  CD CD  "D Q-  .9 CD £ o a. co  -E  CD -Q  5  C O  s y o x:  Q— CD  C) 3 O  CD  3  o c £  5  £I* lol  |£ -a  CD  -a  co  cu  c  2s  "D D) C CD ®  C  x— CD HO  ° s .1 s  3  en >>CD £  CD  CO  1 <•>  2 c  Q . CO CO  CD  CO  E  CD CO O  CO  CD  0) «^  CD  H— "S i fc— o CCDD o S o 3 co c • •a c c .2c To —  c  'T Q.  C CO  te >- co  O *• _  CD  73  CO  •n  c  g CD  CD  >< O O Q. ^ T3  ™ 5  8 j= 5  te  ?5  ~  CD  «  o  <  o  JZ _ 0 ° c—  m  ®  «  .y 5 2  CD —  O  •= .O «  2a  H-  ca O o w>  CO CD -  V£  co  E co  jd CD"  £ E E L >- c C CO _3 J O < 1 CD E Li ill * x: o O O O CO  o re d e c  O  o  CM  _  O  CD  ^  CO CO CO  L. z  - E E o o  88  CONCLUSIONS AND SUGGESTIONS FOR FUTURE RESEARCH 1. Staurosporine induces the formation of lamellipodia and extended lamellipodia in keratinocytes. 2. Formation of lamellipodia and extended lamellipodia requires organization of actin and microtubule filaments. Confocal microscopy can be used to further illustrate the organization of actin cytoskeleton in E-Lam formation. 3. Differential activation of M A P K is associated with different cell morphology, where high activation may lead to formation of extended lamellipodia while low activation has lead to formation of lamellipodia. To further test this hypothesis spreading assays with the specific M E K inhibitor (i.e. PD 58059), which works upstream of M A P K could be conducted. 4. Lamellipodia formation and cell spreading in keratinocytes might involve diffusing factors released by microtubules. To further test this hypothesis experiments using fluorescence video microscopy (Mikhailov and Gundersen, 1998) can be conducted where we could observe M T behavior in relation to E-Lam formation. 5. Modulation of tyrosine phosphorylation/dephosphorylation, or PI-3 kinase plays a minor role in staurosporine-induced keratinocyte spreading and E-Lam formation. Specific inhibitors against intracellular protein kinases such as calphostine (against 89  PKC) and KT5926 (against M L C K ) could be used to identify other protein kinases and phosphatases involved in the process of E-Lam formation. Our preliminary experiments with dominant-negative RhoA, Rac and Cdc42 genes constructs offered exciting insights on the role of Ras-related GTPase pathway in cell spreading. Further experiments need to be conducted using dominant-positive as well as dominant-negative gene constructs to understand the role of Ras-related GTPase proteins in cytoskeletal organization, cell migration and E-Lam formation.  90  REFERENCES Adams JC, Watt F M (1991) Expression of (31, (33, (34, and (35 integrins by human epidermal keratinocytes and non-differentiating keratinocytes. Journal Cellular Biology  of  115:829-841.  Akiyama SK, Yamada SS, Chen W T , Yamada K M . (1989) Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. Journal of Cell Biology  109(2): 863-75.  Alberts B, Bray D, Lewis J, Raff M , Roberts K , Watson JD (1993) Molecular biology of the cell, third ed., pp. 284-286. Garland publishing co, N Y . 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Current Opinions in Cell Biology 8: 66-73.  107  APPENDICES  APPENDIX A - Drug Dilutions DRUG  SOLVENT  STOCK  CONCENTRATION  CONCENTRATION  USED; [IX]  5 mM  10 nM, 50 nM  Staurosporine  DMSO  Genistein  0.25 M NaOH  100 mM  200 fiM  Herbimycin A  DMSO  1.74 m M  8.7  Okadaic acid  DMSO  Bombesin  dH 0  Sodium metavanadate  d H 0 (95 °C)  Wortmannin  100  MM  MM  20 nM  1 mM  200 ixM  100 m M  100 uM  DMSO  10 mM  100 nM  Taxol  DMSO  10 mM  10 nM, 50 nM  Nocodazole  DMSO  10 mM  20 nM, 200 nM  Colchicine  dH,0  25 mM  1  Cytochalasin D  Ethanol  Cycloheximide  DMEM  2  7  lOmM 5mg/355ml (fresh)  MM  1 uM 50/iM  108  A P P E N D I X B-Toxicitv Experiments  DMSO  DMSO CONCENTRATION [1X1 (%) 5 2 1.5 1 0.5 0.1 0.05 0  ABSORBANCE  Std. Dev.  0.145 0.325 0.515 0.588 0.755 0.789 0.718 0.725  0.017 0.119 0.0549 0.038 0.047 0.0407 0.056 0.05  2  Herbimycin A  Herbimycin A CONCENTRATION n x i UJM) 69.6 34.8 17.4 8.7 1.74 0.87 0.435 0  3  Concentration  ABSORBANCE  Std. Dev.  0.244 0.435 0.683 0.625 0.624 0.665 0.723 0.725  0.055 0.0353 0.0154 0.011 0.025 0.003 0.05 0.05  30  40  Concentration  Genistein  Genistein  CONCENTRATION  ABSORBANCE  Std. Dev.  [1X1 UiM) 500 400 250 200 100 50 10 0  0.304 0.232 0.24 0.44 0.954 1.382 1.785 1.799  0.011 0.09 0.006 0.064 0.154 0.014 0.044 0.047  1 oo  200  300  400  500  Concentration  [09  Na metavanadate CONCENTRATION [1X1 W) 5000 2000 1000 500 200 100 50 0  Na m e t a v a n a d a t e 2.5  ABSORBANCE  Std. Dev.  0.384 0.667 1.076 1.414 1.884 1.787 1.819 1.805  0.005 0.09 0.006 0.064 0.154 0.014 0.044 0.047  Okadaic acid CONCENTRATION [1X] (nM) 200 100 50 40 25 20 10 0  2000  3000  4000  5000  80  1 00  200  250  Concentration  Okadaic acid 2.5  ABSORBANCE  Std. Dev.  0.337 0.266 0.247 0.417 1.034 1.62 1.913 1.668  0.005 0.09 0.006 0.064 0.154 0.014 0.044 0.047  Staurosporine CONCENTRATION [1X1 (nM) 400 250 200 120 100 50 1 0 0  1 000  20  40  60  Concentration  Staurosporine 2.5  ABSORBANCE  Std. Dev.  0.177 0.222 0.399 0.646 0.79 1.244 1.916 1.705  0.005 0.09 0.006 0.064 0.154 0.014 0.044 0.047  50  100  150  Concentration  110  Bombesin CONCENTRATION [1X1 (fjM) 50 40 25 20 15 10 5 0  Bombesin  ABSORBANCE  Std. Dev.  0.741 0.634 0.797 0.662 0.733 0.799 0.802 0.801  0.067 0.035 0.055 0.083 0.075 0.075 0.04 0.08  1o  Wortmannin CONCENTRATION [1X1 (nM) 1000 500 250 100 50 25 1 0 0  20 30 Concentration  40  50  200  250  20  25  Wortmannin  ABSORBANCE  Std. Dev.  0.922 1.11 1.148 1.108 0.995 1.016 1.071 1.416  0.427 0.347 0.21 0.25 0.126 0.083 0.166 0.047  Colchecine  50  100 150 Concentration  Cholchicine  CONCENTRATION  ABSORBANCE  Std. Dev.  [1X1 (uM) 100 50 25 10 5 2.5 1 0  0.567 0.51 0.511 0.649 0.537 0.535 0.802 1.14  0.067 0.187 0.145 0.083 0.075 0.075 0.28 0.427  10 15 Concentration  111  Taxol CONCENTRATION [1X1 (nM) 1000 500 250 100 50 25 1 0 0  Taxol  ABSORBANCE  Std. Dev.  0.459 0.331 0.521 0.938 0.841 1.023 1.125 1.191  0.051 0.05 0.106 0.309 0.108 0.095 0.11 0.132  100  300  400  500  Concentration  Nocodazole CONCENTRATION [1X1 (nM) 1000 500 250 100 50 25 10 0  200  Nocodazole  ABSORBANCE  Std. Dev.  0.305 0.307 0.391 0.602 0.817 0.894 0.944 1.036  0.195 0.222 0.131 0.218 0.051 0.035 0.036 0.043  1 00  200  300  400  500  Concentration  112  APPENDIX C - R A W D A T A F O R T H E G R A P H S APPENDIX C I - Effect of protein kinase and phosphatase modulators on HaCaT keratinocyte spreading (Figure 3). % Spread  Std. Dev.  Control  17.92%  8.80%  Genestein 200 uM  18.37%  5.43%  Herbimycin-A 8.7 u M  7.00%  5.07%  Staurosporine 50 n M  43.37%  9.43%  Okadaic Acid 20 nM  18.94%  11.78%  Bombecin 200 u M  59.37%  7.40%  Na-Vanadate 100 uM  15.11%  11.78%  Wortmannin 100 n M  35.50%  7.40%  0.50%  7.49%  D M S O 0.02%  18.51%  2.87%  D M S O 0.5%  8.80%  3.20%  Treatment  BSA  APPENDIX C2- Staurosporine-induced cell spreading on different matrices (Figure 5) Treatment  % Spread  Strd. Dev.  Fibronectin  20.00%  9.70%  Laminin-5  24.58%  5.32%  Collagen I  39.69%  10.53%  Collagen IV  48.00%  10.26%  113  APPENDIX C3- Effect of inhibitory anti-integrin antibodies on HaCaT cells on fibronectin (Figure 7) Treatment  Control  Strd. Dev.  No mAbs  15.33%  5.77%  anti-61 mAbs  4.98%  3.36%  anti-av mAbs  2.34%  2.71%  anti-61 and anti-av mAbs  0.30%  0.00%  BSA  0.03%  0.0%  APPENDIX C4- Effect of different concentration of staurosporine on cell spreading on fibronectin (Figure 8) Treatment  % spread  Std. Dev.  Control  27.95%  4.78%  STP 10 nM  45.85%  8.56%  STP 50 nM  49.02%  7.90%  BSA  0.40%  0  APPENDIX C5- Effect of STP10 and STP50 on HaCaT cell spreading on fibronectin in combination with protein kinase and phosphatase modulators (Figure 12) STP 50 nM  STP 10 nM  N O STP  SD  Treatment  % spread  SD  % spread  SD  Treatment  Control  20.5%  9.1%  STP 10  40.5%  9.9%  STP 50  49.9%  11.5%  Genestein 200  18.5%  8.2%  Genestein 200  37.5%  8.1%  Genestein  45.8%  11.0%  25.0%  8.5%  Okadaic Acid  39.7%  9.2%  Okadaic Acid  41.9%  11.1%  22.3%  9.0%  Na-Vanadate  38.2%  9.5%  Na-Vanadate  51.3%  10.2%  30.1%  12.5%  56.4%  9.5%  Treatment  uM Okadaic Acid 20 nM Na-Vanadate  100 nM  uM 20 nM  Wortmannin 100 nM  200 uM 20 nM 100 uM  100 uM  100 uM Wortmannin  % spread  42.5%  7.8%  Wortmannin 100 nM  114  APPENDIX C6- Effect of cytochalasin-D on HaCaT cell spreading on fibronectin (Figure 12) NO STP  % spread  SD  Control  22.5%  8.2%  C C - D luM  0.5%  0.0%  Treatment  STP 50 nM  STP 10nM  % spread  SD  STP 10  50.2%  9.9%  CC-Dl/iM  0.5%  0.0%  Treatment  % spread  SD  STP 50  52.8%  11.5%.  CC-DljttM  0.5%  0.0%  Treatment  APPENDIX C7- Effect of colchicine on HaCaT cell spreading on fibronectin (Figure 13) NO STP  Treatment Control Colchicine 1000 nM  % spread  SD  20.5%  7.6%  STP 10  4.1%  Colchicine  3.3%  STP 50 nM  STP 10 nM  Treatment  Treatment % spread  SD  % spread  SD  40.7%  9.9%  STP 50  49.9%  11.5%  2.3%  Colchicine  41.9%  5.6%  1.3%  1000 nM  1000 nM  APPENDIX C8- Effect of microtubule modulators on HaCaT cell spreading on fibronectin (Figure 16) NO STP  STP 50 nM  STP 10 nM  % spread  SD  Treatment  % spread  SD  Treatment  % spread  SD  Control  20.5%  7.6%  STP 10  40.7%  9.9%  STP 50  49.9%  11.5%  Nocodazole  21.9%  3.7%  Nocodazole 20  23.7%  6.0%  Nocodazole  51.3%  4.2%  20.8%  8.3%  Nocodazole  19.6%  7.0%  32.8%  4.3%  21.8%  4.9%  Taxol  15.6%  5.8%  Taxol  48.8%  13.3%  10.2%  5.8%  Taxol  16.7%  6.2%  Taxol  25.7%  7.5%  Treatment  20 nM Nocodazole  10 nM Taxol 50 nM  10 nM 50 nM  20 nM Nocodazole 200 nM  200 nM  200 nM Taxol  nM  10 nM 50 nM  115  APPENDIX C9A-  Activation of M A P K (ERK1/2) in STP-treated HaCaT cells. (Figure  17B)- Band intensity.  STP ] OnM  NO STP Length of  % spread  Length of  STP 50 nM  % spread  % spread  Treatment  Treatment  Treatment  Length of  30'  8,632  30'  6,398  30'  13,433  60'  10,666  60'  8,819  60'  17,905  90'  10,832  90'  15,194  90'  23,072  120'  14,615  120'  21,415  120'  25,466  APPENDIX C9B-  Activation of M A P K (ERK1/2) in STP-treated HaCaT cells. (Figure  17C)- % cells spread.  30'  3.0%  3.3%  30'  3.2%  3.5%  60'  16.2%  4.5%  60'  17.0%  4.1%  9.10%  90'  65.5%  11.8%  90'  71.0%  11.0%  8.80%  120'  69.5%  10.5%  120'  74.0%  10.0%  1.0% 9.1%  2.60%  60'  5.60%  90'  22.3%  120'  26.7%  30'  APPENDIX C10-  Treatment  SD  SD  SD  Treatment  % spread  % spread  % spread  Treatment  STP 50 nM  STP 10 nM  NO STP  Effect of STP on HaCaT cell migration (Figure 18)  Treatment  # of cells migrated  Std. Dev.  Control  22.1  7.5  STP 10 nM  34.8  6.8  STP 50 nM  37.9  1 0.4  116  


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