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Smad2 overexpression and the progression of periodontal disease Mazen, Alotaibi Kitab 2015

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SMAD2 OVEREXPRESSION AND THE PROGRESSION OF  PERIODONTAL DISEASE  by  Mazen Kitab Alotaibi  BDS, King Saud University, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Craniofacial Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   July 2015  © Mazen Kitab Alotaibi, 2015    ii Abstract Periodontitis is a chronic inflammatory disease, characterized by destruction of the periodontal attachment apparatus including the alveolar bone.  Previous studies have provided evidence for the involvement of transforming growth factor beta (TGF-β) signaling in periodontitis progression. TGF-β signaling is responsible for a variety of cellular processes including proliferation, differentiation and apoptosis. The SMAD2 transcription factor lies at the heart of TGF-β intracellular mediators. Previous authors have reported the effect of Smad2 overexpression on multiple mouse tissues (Ito et al 2001), but did not report the role of Smad2 overexpression on the progression of periodontal disease.  We hypothesized that Smad2 overexpression alters apoptosis, cell proliferation, and inflammatory cytokine secretions in the junctional epithelium (JE), leading to periodontal attachment loss. A mouse model that overexpresses Smad2 in epithelial cells driven by the cytokeratin 14 promoter (K14) was used to test the hypotheses. The K14-Smad2 mice findings were compared to those observed in wild type (WT) mice that served as controls. The results of the study showed that Smad2 overexpression reduced the histological surface area of JE when compared to WT mice. The reduction of the JE surface area in K14-Smad2 mice was attributed to an increased apoptotic index and a reduced proliferation rate. The overexpression of Smad2 increased the apoptotic index by down regulating Bcl2, an antiapoptotic molecule. Smad2 overexpression also reduced the proliferation rate of the JE cells in K14-Smad2 mice by upregulating c-Myc, which in turn upregulates phosphorylated retinoblastoma P15, and P27. The overexpression of Smad2 resulted in severe alveolar bone loss in the K14-Smad2 mice when compared to the WT controls.  Smad2 overexpression resulted in a reduction in the bone density and bone volume in the K14-Smad2 mice when   iii compared to their WT counterparts. The severe alveolar bone loss in K14-Smad2 mice was attributed to an upregulation in tumor necrosis factor alpha (TNF-α) , RANKL and increased osteoclast numbers.  In summary the overexpression of Smad2 reduced the histological surface of JE and resulted in severe bone loss that follows a chronic disease pattern in K14-Smad2 mice.   iv Preface  Chapter 2 has been published. Fujita, T., Alotaibi, M., Kitase, Y., Kota, Y., Ouhara, K., Kurihara, H., & Shuler, C. F. (2012). Smad2 is involved in the apoptosis of murine gingival junctional epithelium associated with inhibition of BCL-2. Archives of Oral Biology, 57(11), 1567–1573. The work presented in Chapter 2 was carried out in collaboration between all authors. Dr. Fujita, Dr. Kurihara and Dr. Shuler defined the research theme. Dr. Fujita and Dr. Kitase designed methods and experiments. Dr. Aloatibi and Dr. Fujita carried out the laboratory experiments and analyzed the data. Dr. Kitase and Dr. Fujita interpreted the results and wrote the manuscript. Dr. Yuki and Dr. Ouhara interpreted the results, read and commented on the drafts. Dr. Shuler edited the manuscript. All authors have contributed to, seen, and approved the manuscript.  Chapter 3 has been published. Alotaibi, M. K., Kitase, Y., & Shuler, C. F. (2014). Smad2 overexpression reduces the proliferation of the junctional epithelium. Journal of Dental Research, 93(9), 898–903. Dr. Shuler, Dr. Kitase and Dr. Alotaibi defined the research theme and designed the research methods. Dr. Mazen Alotaibi carried out the laboratory experiments and analyzed the data. Dr. Alotaibi wrote the manuscript. Dr. Shuler, and Dr. Kitase reviewed and edited the final manuscript. Chapter 4 has been submitted for publication. Dr. Shuler, Dr. Kitase and Dr. Alotaibi defined the research theme and designed the research methods. Dr. Alotaibi carried out the laboratory experiments and analyzed the data. Dr. Alotaibi wrote the manuscript. Dr. Shuler and Dr. Kitase reviewed and edited the final manuscript. The Animal Care Committee of The University of British Columbia approved the   v studies (A09-0227).    vi Table of Contents Abstract ................................................................................................................................... ii	  Preface ..................................................................................................................................... iv	  Table of Contents .................................................................................................................... vi	  List of Tables ............................................................................................................................ x	  List of Figures ......................................................................................................................... xi	  List of Abbreviations ............................................................................................................ xii	  Acknowledgements ............................................................................................................... xiv	  Dedication .............................................................................................................................. xvi	  Chapter 1: Review of the Literature……………………………………………………….. 1	  1.1	   An Overview of the Research Plan ....................................................................................... 1	  1.2	   Anatomy and Development of the Junctional Epithelium .................................................... 2	  1.3	   The Role of the JE in Maintaining Health ............................................................................ 4	  1.4	   Periodontal Disease and Genetics ......................................................................................... 6	  1.5	   The Role of Apoptosis on the Progression of Periodontal Disease ...................................... 7	  1.6	   TGF-β Superfamily ............................................................................................................... 8	  1.7	   TGF-β Activation ................................................................................................................ 10	  1.8	   The Role of TGF-β in Wound Healing ............................................................................... 11	  1.9	   The JE and TGF-β Receptors Expression in Health and Disease ....................................... 13	  1.10	   The Role of TGF-β on Apoptosis and Mitotic Activity ..................................................... 13	  1.11	   The Role of Inflammatory Cytokines/Chemokines in the Progression of Bone Loss ........ 14	  1.12	   K14-Smad2 Mice Phenotype .............................................................................................. 15	  1.13	   Hypothesis .......................................................................................................................... 17	  1.14	   Aims and Objectives ........................................................................................................... 17	  Chapter 2: SMAD2 is Involved in the Apoptosis of Murine Gingival Junctional Epithelium Associated with Inhibition of BCL-2………………………………………… 19	  2.1	   Overview ............................................................................................................................. 19	  2.1.1	   Objective ...................................................................................................................... 19	  2.1.2	   Methods ........................................................................................................................ 19	    vii 2.1.3	   Results .......................................................................................................................... 19	  2.1.4	   Conclusions .................................................................................................................. 20	  2.2	   Introduction ......................................................................................................................... 20	  2.3	   Materials and Methods ........................................................................................................ 21	  2.3.1	   Animals and Genotyping ............................................................................................. 21	  2.3.2	   Real-time PCR ............................................................................................................. 22	  2.3.3	   Immunohistochemistry ................................................................................................. 22	  2.3.4	   Analysis of Junctional Epithelial Apoptosis Rate ........................................................ 23	  2.4	   Results ................................................................................................................................. 24	  2.4.1	   Activation of Overexpressed SMAD2 Was Associated With an Increase in Endogenous Tgf-β1 Expression in Gingival Epithelial Tissue ................................................ 24	  2.4.2	   Increased JE Apoptosis in K14-Smad2 Mice ............................................................... 25	  2.4.3	   Reduction in BCL-2 Expression in K14-Smad2 Mice ................................................. 27	  2.5	   Discussion ........................................................................................................................... 29	  2.5.1	   Mouse Model System ................................................................................................... 30	  2.5.2	   Overexpression of Smad2 Induces Apoptosis in JE ..................................................... 30	  2.5.3	   Underlying Mechanism by Which Overexpression of Smad2 Induces Apoptosis in  JE……………………………………………………………………………………………..31	  2.6	   Conclusion .......................................................................................................................... 32	  Chapter 3: Smad2 Overexpression Reduces the Proliferation of the Junctional Epithelium…………………………………………………………………………………...33	  3.1	   Overview ............................................................................................................................. 33	  3.1.1	   Objective ...................................................................................................................... 33	  3.1.2	   Methods ........................................................................................................................ 33	  3.1.3	   Results .......................................................................................................................... 33	  3.1.4	   Conclusions .................................................................................................................. 34	  3.2	   Introduction ......................................................................................................................... 34	  3.3	   Materials and Methods ........................................................................................................ 36	  3.3.1	   Animals and Genotyping ............................................................................................. 36	  3.3.2	   Histology and Immunohistochemistry ......................................................................... 37	  3.3.2.1	   Decalcification and Paraffin Embedding of the Samples ..................................... 37	  3.3.2.2	   Hematoxylin and Eosin ......................................................................................... 37	  3.3.2.3	   Immunohistochemistry ......................................................................................... 37	  3.3.3	   Real-time PCR ............................................................................................................. 38	    viii 3.3.4	   Western Blots ............................................................................................................... 39	  3.3.5	   Statistical Analysis ....................................................................................................... 40	  3.4	   Results ................................................................................................................................. 40	  3.4.1	   Smad2 Overexpression Altered the Surface Area of the JE Cells ............................... 40	  3.4.2	   Smad2 Overexpression Reduces the Proliferation Rate of the JE Cells ...................... 42	  3.4.3	   Increased SMAD2 Up-regulates c-MYC ..................................................................... 44	  3.4.4	   Smad2 Overexpression Increased Both P15 and pRB to Inhibit JE Cell  Proliferation ............................................................................................................................ 45	  3.5	   Discussion ........................................................................................................................... 46	  Chapter 4: Smad2 Overexpression Induces Alveolar Bone Loss by Up-regulating  TNF-α………………………………………………………………………………………...49	  4.1	   Overview ............................................................................................................................. 49	  4.1.1	   Background .................................................................................................................. 49	  4.1.2	   Methods ........................................................................................................................ 49	  4.1.3	   Results .......................................................................................................................... 49	  4.1.4	   Conclusion ................................................................................................................... 50	  4.2	   Introduction ......................................................................................................................... 50	  4.3	   Materials and Methods ........................................................................................................ 53	  4.3.1	   Animals and Genotyping ............................................................................................. 53	  4.3.2	   Photographs .................................................................................................................. 53	  4.3.3	   Micro CT Analysis ....................................................................................................... 54	  4.3.4	   Histology and Immunohistochemistry ......................................................................... 54	  4.3.4.1	   Decalcification and Paraffin Embedding of the Samples ..................................... 54	  4.3.4.2	   Hematoxylin and Eosin ......................................................................................... 54	  4.3.5	   Immunohistochemistry ................................................................................................. 55	  4.3.6	   Real-time PCR ............................................................................................................. 55	  4.3.7	   Western Blots ............................................................................................................... 57	  4.3.8	   Statistical Analysis ....................................................................................................... 57	  4.4	   Results ................................................................................................................................. 58	  4.4.1	   Smad2 Overexpression Results in Severe Alveolar Bone Loss ................................... 58	  4.4.2	   Increased SMAD2 Up-regulates Tnf-α and Rankl ....................................................... 61	  4.4.3	   The Overexpression of Smad2 Increased the Number of Osteoclasts ......................... 62	  4.5	   Discussion ........................................................................................................................... 63	  Chapter 5: General Discussion, Conclusions, and Future Directions…………………... 68	    ix 5.1	   General Discussion ............................................................................................................. 68	  5.1.1	   JE to Maintain Health .................................................................................................. 68	  5.1.2	   Recombinant TGFβ1 Mediated Periodontal Regeneration .......................................... 69	  5.1.3	   Smad2 and Bone Loss .................................................................................................. 70	  5.2	   Conclusion .......................................................................................................................... 71	  5.3	   Future Directions ................................................................................................................ 72	  References .............................................................................................................................. 74	     x List of Tables Table 1: The Effects of SMAD2 Overexpression on Healing and Gingival Cells…………..12 Table 2: Primer Sequences for p15, p21, p27, and Gapdh…………………………………..38	  Table 3: Primer Sequences for Tnf-α , Il1-β,  Ifγ, Rankl, Opg and Gapdh…………………..56    xi List of Figures Figure 1: The Dento-Gingival Unit............................................................................................2	  Figure 2: TGFβ Signalling Through SMAD2.........................................................................10	  Figure 3: Activation of Overexpressed SMAD2 was Associated with an Increase in Endogenous Tgf-β1 Expression in Gingival Epithelial Tissue...........................................25	  Figure 4: Increased JE Apoptosis in K14-Smad2 Mice...........................................................27	  Figure 5: Reduction in Bcl-2 mRNA Expression in K14-Smad2 Mice...................................28	  Figure 6: Reduction in BCL-2 Protein Expression in K14-Smad2 Mice.................................29	  Figure 7: Smad2 Overexpression Altered the JE Surface Area...............................................41	  Figure 8: Smad2 Overexpression Reduced the Proliferation Rate of JE Cells........................43	  Figure 9: Smad2 Overexpression Inhibits C-MYC..................................................................45	  Figure 10: Smad2 Overexpression Up-regulates P15, P27 and Increases the Protein Level of p-RB....................................................................................................................................46	  Figure 11: Smad2 Overexpression Induces Chronic Alveolar Bone Loss...............................59	  Figure 12: Smad2 Overexpression Reduces Both Bone Volume and Bone Density...............61	  Figure 13: Smad2 Overexpression Up-regulates Tnf-α and Rankl..........................................62	  Figure 14: Smad2 Overexpression Increased the Number of Osteoclasts in K14-Smad2  Mice ....................................................................................................................................... 63	  Figure 15: Smad2 Overexpression and the Progression of Periodontal Disease ................... 72	       xii List of Abbreviations BAD Bcl-2-associated death promoter  BAX Bcl-2-associated X protein BCL2 B-cell lymphoma 2 BCL-XL B-cell lymphoma-extra large BMP bone morphogenic protein  BID BH3 interacting-domain death agonist BIK Bcl-2-interacting killer BIM Bcl-2-like protein 11 CEACAM1 carcinoembryonic Ag-related cell adhesion molecule  DAPI 4′, 6′- diamidino-2-phenylindole DAT directly attached to tooth ECM extracellular matrix EGF epidermal growth factor  GS the glycine-serine rich region of TGF-β receptor type I ICAM-1 intercellular adhesion molecule-1  IFγ interferon gamma IL-1α interleukin-1 alpha  IL-1β interleukin-1 beta IL-8 interleukin-8 JE junctional epithelium  K14-Smad2 Smad2 overexpression mice  MAP kinases mitogen-activated protein kinases OPG osteoprotegerin PBS Phosphate-buffered saline PCNA Proliferating cell nuclear antigen PI3K phosphatidylinositol 3-kinases  PLS papillon–Lefèvre syndrome  PMN polymorphonuclear leucocyte P-RB phosphorylated retinoblastoma    xiii RANKL receptor activator of nuclear factor kappa-B ligand SLC small latent complex  TβRI TGF-β type I receptor  TβRII TGF-β type II receptor TGF-β transforming growth factor-β TNF-α   tumor necrosis factor alpha  TRAP  tartrate-resistant acid phosphatase  TUNEL terminal deoxynucleotidyl transferase dUTP nick end labelling  WT wild type XIAP X-linked inhibitor of apoptosis protein    xiv Acknowledgements Praise be to Allah the most merciful It is with pride and pleasure that I extend my acknowledgement to a number of people that helped me during my time at UBC. I want to thank my supervisor Dr. Charles Shuler for giving me the opportunity to work with him. Dr. Shuler gave me analytical skills that helped me to think, evaluate data, and most importantly ask the right questions independently. I want to thank Dr. Shuler for his support, guidance, and importantly his patience during my period at UBC. Dr. Shuler has been a teacher, a leader, a mentor, and a father figure for me in Vancouver, so words will not explain my gratitude to him.  I special thanks goes toward Dr. Yukiko Kitase. Dr. Kitase took me under her wing and guided me during my PhD. I thank Dr. Kitase for spending endless hours teaching me the appropriate laboratory techniques and most importantly discussing and developing my research.  I want to thank my advisory committee, Dr. Clive Roberts, Dr. Edward E. Putnins, and Dr. Lari Hӓkkinen, for their support and valuable comments that helped me develop this thesis. I greatly appreciate the support that Dr. Roberts has given me during my time at UBC in both the graduate periodontics program and the PhD program. Dr. Roberts’ guidance as an academic advisor has helped me to develop and progress during the years of my studies. I want to thank Dr. Edward E. Putnins for his wise insight and words of encouragement during my studies at UBC. Dr. Putnins, I see the light at the end of the tunnel and I am running as fast as I can to take my PhD. Many thanks to Dr. Lari Hӓkkinen for opening my vision and   xv enhancing my interpretations of the periodontal literature. The periodontal literature review course directed by Dr. Hӓkkinen helped me to interpret, evaluate, and criticize the data in the literature, which was translated in this dissertation. This work would not have been possible without the support of my family, particularly my wife Siham, who has given me love and support throughout my life. Siham, your love is the light that brightens my life and the power that keeps me going forward. The best way that I can express how I feel is by saying I love you. To my lovely daughters Alhanoof, Dimah, and Lama, thank you for your love and support during my studies. The time has come for me to spend more time with you as we have all sacrificed a lot during the past few years (Daddy is coming home). I owe everything in my life to my parents. My father Dr. Kitab Alotaibi and my mother Miznah have devoted their lives to making me happy and because of them I am the man that I am today.  Special thanks are extended to the Saudi government and to the Saudi Cultural Bureau for their academic and financial support during my studies at UBC. This dissertation work has received a Dr. Joseph Tonzetich Fellowship. Financial support for the study discussed in chapter 2 was provided by a research grant (RO1 DE16296) from the National Institute of Dental and Craniofacial Research to CS and a Grant-in-aid for the Encouragement of Young Scientists (B) (No. 22792087) from the Japan Society for the Promotion of Science, Japan to Dr.Fujita.     xvi Dedication      This dissertation is dedicated to  My Wife Siham  Siham your love is the light that brightens my life and the  power that keeps me going forward   And also to   My parents  for their love and support       1 Chapter 1: Review of the Literature  1.1 An Overview of the Research Plan This study focuses on the effect of Smad2 overexpression on the periodontal apparatus; SMAD2 is a member of a transcriptional complex that is a primary intracellular mediator of the TGF-β signalling pathway. Previous authors have reported the effect of Smad2 overexpression on multiple mouse tissues such as the skin, palate, and teeth (Cui et al., 2005; Hosokawa, Urata, Ito, Bringas, & Chai, 2005; Ito et al., 2001; Owens, Han, Li, & Wang, 2008), but none of those studies have reported the role of Smad2 overexpression on the progression of periodontal disease. The following list provides an overview of the rationale for examining the effect of Smad2 overexpression on the progression of periodontal disease.  1) TGF-β increases apoptosis and reduces the mitotic activity of epithelial cells.  2) Apoptosis and mitotic activity play a significant role in the progression of periodontal disease by reducing the number of cells available for the barrier function of the junctional epithelium.  3) Inflammatory cytokines are up-regulated by TGF-β through the SMAD2-mediated intracellular signalling pathway. These previous findings have led to the following hypothesis for this proposal: Smad2 overexpression alters apoptosis, cell proliferation, and inflammatory cytokine secretions in the junctional epithelium, which are associated with periodontal attachment loss. This hypothesis will be tested by three specific aims developed to examine the attachment loss in the K14-Smad2 transgenic mice that   2 overexpress Smad2. The first aim is to determine if Smad2 overexpression causes alveolar bone loss, as alveolar bone loss is an important feature in periodontal disease. The next aim is to analyze the role of Smad2 overexpression on the junctional epithelium that represents the first line of defence against periodontal disease. The final aim is to examine the secretion of molecules from the junctional epithelium cells of K14-Smad2 overexpression mice and determine how they are associated with alveolar bone resorption. 1.2 Anatomy and Development of the Junctional Epithelium The junctional epithelium (JE) is the part of the dento-gingival unit that is attached to the tooth surface. The JE develops from the reduced enamel epithelium and it has been proposed that over time it is replaced by the basal cells of the oral gingival epithelium (Salonen, Kautsky, & Dale, 1989). The JE extends coronally to the base of the oral sulcular epithelium and apically to the connective tissue attachment at the cementoenamel junction (Fig. 1). The JE forms the lining of the interdental col (Gargiulo & Wentz, 1961).    3 Figure 1: The Dento-Gingival Unit  Represents the different parts of the dento-gingival unit from a 12 months WT mouse. OGE=oral gingival epithelium. E=enamel. D=dentin. CEJ=cementoenamel junction. A=alveolar bone. CT=connective tissue. JE=junctional epithelium.  The biological width is the dimensions of soft tissues that are attached to the tooth up to the level of the alveolar bone and in average it is 2.04 mm (JE+ connective tissue attachment) (Gargiulo & Wentz, 1961). The JE represents an average of 0.97 mm from the total biological width. The remaining is the oral sulcus 0.69 mm and the connective tissue attachment 1.07 mm (Gargiulo & Wentz, 1961). The biological width is not constant and varies both from tooth to tooth and between individuals; in particular the JE demonstrates variability between 0.9 and 1.14 mm (Vacek, Gher, Assad, Richardson, & Giambarresi, 1994). The clinical significance of the biological width lies in that any violation to the width during restorative dental treatment of teeth can result in gingival inflammation, gingival recession, and alveolar bone resorption (Newcomb, 1974; Tal, Soldinger, Dreiangel, & Pitaru, 1989). The JE has two basal laminas (Schroeder, 1969). The first basal lamina faces the tooth surface (internal basal lamina) and the second basal lamina faces the connective tissue (external basal lamina) (Bosshardt & Lang, 2005). The external basal lamina matrix has the same structures as other basement membranes such as collagen types IV and VII, laminin, heparan sulfate proteoglycan, fibronectin, nidogen (entactin), and the proteoglycan perlecan (Bosshardt & Lang, 2005). The internal basal lamina is not considered a true basal lamina and differs from the external basal lamina in that it lacks collagen types IV and VII, most laminin isoforms, perlecan, and a lamina fibroreticularis (Hormia, Owaribe, & Virtanen, 2001; Kogaya, Haruna, Vojinovic, Iwayama, & Akisaka, 1989; Salonen & Santti, 1985). The internal basal lamina is distinguishable from the   4 external basal lamina in that it contains laminin V (Oksonen, Sorokin, Virtanen, & Hormia, 2001).  1.3 The Role of the JE in Maintaining Health The JE is the first line of defence against periodontal disease; it provides an important barrier activity by contacting the tooth surface with cells directly attached (DAT cells) through hemidesmosomes ( Salonen, Kautsky,& Dale, 1989; Listgarten, 1966). The JE maintains its integrity by expressing multiple molecules that interact in a cell-cell or cell-surface manner. Integrins are cell surface receptors that allow cells to interact with ECM; also integrins play a role in cell-cell interactions (Danen & Sonnenberg, 2003). The JE expresses multiple integrins, namely α6β4 (Gürses, Thorup, Reibel, Carter, & Holmstrup, 1999), αvβ6 (Ghannad et al., 2008), α2β1, α3β1, and α6β1 (Del Castillo et al., 1996). Each integrin has specific function for example α6β4 is responsible for the formation and stabilization of hemidesmosomes (Wilhelmsen , Litjens , & Sonnenberg , 2006) and α2β1 is the major collagen (collagen type I) binding integrin (Arlinghaus , & Eble , 2013).   Any change of the expression of the integrins might alter the types of integrins present and play a role in periodontal disease progression (Bosshardt & Lang, 2005). Indeed the absence of αvβ6 integrin resulted in a severe periodontal disease in mice (Ghannad et al., 2008). The JE expresses molecules that are related to cell-cell contact such as E-cadherin and carcinoembryonic Ag-related cell adhesion molecule (CEACAM1) (Ye, Chapple, Kumar, & Hunter, 2000). In general, E-cadherin and CEACAM1 provide the cohesion function of the JE cell (Heymann, Wroblewski, Terling, Midtvedt, & Öbrink, 2005). For that reason, the loss of these   5 molecules could reduce the JE integrity and induce periodontal disease (Bosshardt & Lang, 2005). The JE cells play an active role in the synthesis of a variety of molecules that are part of the defence against bacterial invasion. In health the JE contains PMNs as they keep and maintain defence against continuous bacterial challenge from the oral cavity (Tonetti, Imboden, & Lang, 2012). In addition to maintaining the JE integrity, CEACAM1 directs and guides PMNs through the JE (Heymann et al., 2005). Another important molecule in the PMNs guidance within the JE is the intercellular adhesion molecule-1 (ICAM-1). ICAM-1 acts as a ligand for the leucocytes integrin β2 (Crawford & Hopp, 1990). Leucocytes bind to ICAM-1 and migrate to protect against bacterial invasions (Tonetti et al., 2012). A molecule of a particular interest in the host response against periodontal disease is IL-8 cytokine, which has a chemotactic ability. The IL-8 has the ability to select the best path for PMNs as it directs them to bacterial challenge (Tonetti, Gerber, & Lang, 1994). The JE has the ability to secrete antimicrobial molecules such as α- and β-defensins, cathelicidin family members, and calprotectin (Dale, 2002).These antimicrobial molecules have the ability to kill invading microorganisms and maintain periodontal health (Dale, 2002). The JE expresses a variety of growth factors that help in cell growth, proliferation, differentiation, and wound healing (Nordlund, Hormia, Saxén, & Thesleff, 1991). Epidermal growth factor (EGF) has the ability to induce proliferation and differentiation of epithelial cells. EGF is expressed in JE in both health and periodontal disease, which can help the JE during the healing process (Tajima et al., 1992).    6 The JE maintains health through a physical barrier connection with teeth and the secretion of multiple molecules that regulate immunity, kill bacterial invasion, and promote growth (Bosshardt & Lang, 2005). Therefore any disturbance of the JE’s integrity or the ability of secretion protection could reduce the defensive properties of the JE, leading to periodontal disease progression (Overman & Salonen, 1994).   Damage to the JE cells could induce periodontal disease. Clinical diagnosis of periodontal disease has been identified as attachment loss of either the soft tissues or alveolar bone and in many cases both are damaged (Armitage, 1999). During routine dental examination dentists evaluate the levels of soft tissues clinically and bone loss radiographically (Armitage, 1996). Any change in the level of attachments is considered periodontal disease and the underlying cause should be investigated (Armitage, 1999). 1.4 Periodontal Disease and Genetics  Genetics play a major role in the progression of periodontal disease as almost 50% of periodontal disease is attributed to genetic and hereditary factors (Michalowicz et al., 2000). The main role of genetic aspects of periodontal disease is that genetically inherited properties make the host response susceptible to periodontal disease progression (Hassell & Harris, 1995; Marazita et al., 1994). Multiple syndromes have been identified with gene mutations that have been linked to the causation of periodontal disease (Genco & Borgnakke, 2013). An example of gene mutations is the mutation in cathepsin-C gene, which is expressed at high levels in immune cells such as polymorphonuclear leucocytes, and macrophages (Hart et al., 1999). Cathepsin-C gene mutation causes a syndrome called Papillon–Lefèvre syndrome (PLS) (Hart et al., 1999). PLS is characterized by severe periodontal disease and Palmoplantar keratosis (Hart et al., 1999).   7 Another important point is the expression of specific genes that might increase the progression of periodontal disease (Genco & Borgnakke, 2013). The overexpression of IL-1α in oral epithelium has been found to increase the susceptibility to and progression of periodontal disease (Dayan, Stashenko, Niederman, & Kupper, 2004). On the other hand, a knockout of a single gene can cause severe periodontal disease as exemplified by the experimental knockout of the αvβ6 integrin (Ghannad et al., 2008). Theoretically any change in periodontal tissue-specific gene expression could increase the susceptibility and progression of periodontal disease (Taba, Souza, & Mariguela, 2012). Thousands of up- and down-regulated genes are involved in periodontal disease but the diagnosis of periodontal disease cannot be based only on patterns of gene expression (Demmer et al., 2008). Periodontal disease is a complex multi-factorial disease therefore it is very difficult to establish a cause and effect relationship for any one specific causative factor. Due to the complexity of periodontal disease the hallmark of periodontal disease diagnosis is attachment loss (Armitage, 1999).  1.5 The Role of Apoptosis on the Progression of Periodontal Disease  Apoptosis or programmed cell death is present in the JE epithelium in health and disease, especially the coronal portion of the JE epithelium that normally has high numbers of apoptotic cells when compared with other locations (Jarnbring, Somogyi, Dalton, Gustafsson, & Klinge, 2002). The mitotic activity of the JE is also high to replace the cells that are lost through apoptosis. This high turnover rate is presumed to eliminate diseased and injured cells to establish an equilibrium between microorganisms and epithelial cells (Watanabe et al., 2004). The JE is unique in that the basal cells of both the   8 internal and external basement membranes have the ability to proliferate (Bosshardt & Lang, 2005). The ratio of apoptotic JE cells to the total number of JE cells differs between health and disease (Ruissen, Van De Kerkhof, & Schalkwijk, 1995). With chronic periodontitis, the number of apoptotic cells increases within the base of the JE, which gives an indication of the linkage of JE apoptosis with the initiation of periodontal disease (Tonetti, Cortellini, & Lang, 1998). Bacterial invasion also increases the apoptosis ratio in the JE (Stathopoulou et al., 2009). Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Streptococcus gordonii, and Porphyromonas gingivalis have all been shown to increase the apoptosis of oral epithelial cells, providing additional evidence that apoptosis of epithelial cells is an important component in the progression of periodontal disease (Dickinson et al., 2011; Stathopoulou et al., 2009). 1.6 TGF-β  Superfamily The TGF-β superfamily consists of Cripto, Nodal, TGF-β, Activin, and bone morphogenetic protein (BMP) (Kitisin, Saha, Blake, & Golestaneh, 2007). These growth factors can activate both SMAD and non-SMAD signalling pathways (Attisano & Wrana, 2002). In general the SMAD pathway is activated when TGF-β binds to TGF-β receptor type II (Kitisin et al., 2007). The interaction of TGF-β with TGF-β receptor type II phosphorylates the glycine-serine (GS)-rich region of TGF-β receptor type I (Kitisin et al., 2007). In turn TGF-β receptor type I phosphorylates the receptor SMADs (SMAD2 and SMAD3) (Attisano & Wrana, 2002). Receptor SMADs together with the co-mediator SMAD4 form a complex that enters the nucleus with the potential to alter patterns of gene expression (Massagué & Chen, 2000) (Fig. 2). The non-SMAD pathway occurs   9 when the TGF-β superfamily activates GTPases, MAP kinase, and Phosphatidylinositol 3-kinases (PI3K) pathways (Mu, Gudey, & Landström, 2012; Weiss & Attisano, 2013; Zhang, 2009). The multiple ability of the TGF-β superfamily to control multiple signalling pathways justifies the broad role that it plays in many cellular functions (Gordon & Blobe, 2008). The TGF-β superfamily has different effects on cells within different organs. An example is the role of TGF-β in osteoclast differentiation (Fiorelli et al., 1994), as some studies have shown that it induces osteoclast differentiation (Fiorelli et al., 1994) while other studies have shown that TGF-β reduces osteoclast differentiation through the reduction of the secretion of RANKL by osteoblasts (Quinn et al., 2001). For that reason studying a single path and selected molecules of the TGF-β superfamily would be appropriate to detect the effect of each growth factor on multiple tissues.    10    Figure 2: TGFβ  Signalling Through SMAD2 TGF-β binds to TGF−β receptor type II that in turn phosphorylates TGF-β receptor type I. TGF-β receptor type I phosphorylates SMAD2. SMAD2 together with the co-mediator SMAD4 form a complex that enters the nucleus with the potential to alter patterns of gene expression.  1.7 TGF-β  Activation The pro TGF-β is cleaved by enzymes called furin-like enzymes within cells leading to the formation of the active form of the TGF-β (Dubois, Laprise, Blanchette, Gentry, & Leduc, 1995; Massagué & Chen, 2000). Small latent complex (SLC) is formed by the binding of the mature TGF-β to the latency-associated protein (LAP) (Dallas et al., 1994). Large latent complex (LLC) is generated by the binding of SLC to the latent TGF-β-binding proteins (LTBP) (Saharinen & Keski-Oja, 2000). Almost all cells secrete TGF-  11 β in the form of LLC in high amounts, as it is used as a reservoir (Miyazono, Olofsson, Colosetti, & Heldin, 1991; Todorovic et al., 2005). TGF-β has to be separated from the LAP to be biologically active and bind to its cell surface receptors to start the TGF-β signalling process (Annes, Munger, & Rifkin, 2003).  1.8 The Role of TGF-β  in Wound Healing The literature shows conflicting results when looking at TGF-β in wound healing. TGF-β1 enhanced wound healing by stimulating cell migration (Santibáñez, Iglesias, Frontelo, Martínez, & Quintanilla, 2000), increase wound contraction by inducing the α-smooth muscle actin (Desmoulière, Geinoz, Gabbiani, & Gabbiani, 1993), and increased the production of ECM molecules (Leask & Abraham, 2003). On the other hand, TGF-β1 was found to reduce the growth and the migration of gingival keratinocytes in wound healing, which will reduce the re-epithelialization process (Glick et al., 1993). The overexpression of TGF-β1 has shown delayed re-epithelialization of burn wounds in the epidermis of transgenic mice. The opposite is also true as the deletion of TGF-β1 in transgenic mice has showed increased re-epithelialization when compared to WT mice (Koch et al., 2000). TGF-β1 has been shown to stimulate the formation of ECM (Powell et al., 1999). The formation of ECM is beneficial during periodontal disease healing, however TGF-β1’s ability to form ECM will lead to scar formation in the skin and gingiva (Powell et al., 1999), which might not be aesthetically pleasing to patients (Arx, Salvi, Janner, & Jensen, 2008). TGF-β has the ability to control the expression of collagen type I genes, as it influences the COL1A2 gene by a TGF-β response element (Inagaki Y, Truter S, & Ramirez F, 1994). The overexpression of SMAD2 affects both   12 healing and oral gingival cells. The multiple effects of SMAD overexpression are discussed in Table1. Table 1: The Effects of SMAD2 Overexpression on Healing and Gingival Cells  Authors Effects of SMAD2 overexpression on healing  Effects of SMAD2 overexpression on gingival cells  (Tomikawa K etal.,2012)   (Shimoe M etal.,2014)    (Hosokawa R etal., 2005)     (Gregory LG etal.,2010)     (Meng XM etal.,2010)    Reduce the re-epithelialization of the gingiva during wound healing.      Delay wound healing in the oral gingival and skin wounds.     Increased epithelial airway hyper reactivity after allergen challenge by up-regulating IL-25 and activin A. Reduction in TGFβ1-SMAD3 activation, which results in a reduction of collagen I expression in tubular epithelial cells. Inhibition of cytokeratin 16 which reduced the migration of keratinocyte. Reduces the proliferation of oral epithelial cells through the down regulation of P15 and P21.  Defects in the migration of the basal keratinocytes, which results in the migration of the suprabasal layer in the wound.      13 1.9 The JE and TGF-β  Receptors Expression in Health and Disease TGF-β1 is expressed in JE cells in health (H. Lu, Mackenzie, & Levine, 1997). There is a difference in the expression of TGF-β receptors in health, as TGF-β type II receptor (TβRII) is present at a higher level than TGF-β type I receptor (TβRI) (H. Lu et al., 1997). The levels of TGF-β1 have been shown to increase in chronic periodontitis patients (Skaleric, Kramar, Petelin, Pavlica, & Wahl, 1997), and TβRI is up-regulated in advanced periodontal disease (J.-P. Lu et al., 2003), which gives an indication of the active role of TGF-β signalling in active periodontal disease. Due to the variability of TGF-β  receptors in the JE it would be preferable to focus downstream of the receptors on the SMAD intracellular signalling pathway. The overexpression of Smad2 in the K14-Smad2 mouse model is independent of the presence or absence of the TGF-β  receptors (Ito et al., 2001). The overexpression of Smad2 would result in a situation analogous to continuous TGF-β signalling and what role it might have on periodontal disease and health. 1.10 The Role of TGF-β  on Apoptosis and Mitotic Activity TGF-β signalling has been found to cause apoptosis in epithelial cells through activation of the intracellular SMAD proteins (Schuster & Krieglstein, 2002). There are 8 SMADs that can be classified into 3 different groups. SMADs 1, 2, 3, 5, and 8 are called receptor-activated SMADs; SMAD4 has been called the common mediator SMAD. SMAD7 is involved in the inhibition of the intracellular signalling process (Brown, Pietenpol, & Moses, 2007). When TGF-β and activin bind to a specific growth receptor they activate the receptor complex, resulting in the phosphorylation of the intracellular   14 SMAD2 (Derynck & Zhang, 2003). Phosphorylated SMAD2 binds with SMAD4 to form a cytoplasmic complex that will enter the nucleus to function as a transcription factor leading to changes in DNA transcription (Massagué, Seoane, & Wotton, 2005). One result of this transcriptional activation is to increase caspase transcription, specifically caspase3, which plays a critical role in the apoptosis pathway (Wyllie, 2010). Another pathway of SMAD2 related apoptosis is through the activation of the BCL-2 family (van der Heide, van Dinther, Moustakas, & Dijke, 2011). BCL-2 family has both pro-apoptotic and anti-apoptotic molecules (Brenner & Mak, 2009). The increased expression of the pro-apoptotic molecules such as BAX, BAD, BIK, and BIM will increase the release of cytochrome c from the mitochondria and lead to programmed cell death (Lindsay, Esposti, & Gilmore, 2011). On the other hand the increased anti-apoptotic molecules, namely BCL-2, BCL-XL, and XIAP, will protect from apoptosis (Lindsay et al., 2011). TGF-β signalling has been reported to reduce the mitotic activity of epithelial cells (Coffey & Moses, 1989). TGF-β induces p21Cip1 and 2150p15Ink4b, which are cyclin-dependent kinase inhibitors, and at the same time TGF-β down-regulates transcription factors involved in proliferation, for example, MYC, ID1 and ID2 (Ijichi et al., 2004; Seoane, 2006; Yagi et al., 2002). SMAD2 overexpression could increase apoptosis and reduce mitotic activity of the JE, a combination of changes that would reduce the protective ability of the JE cells (Overman & Salonen, 1994). 1.11 The Role of Inflammatory Cytokines/Chemokines in the Progression of Bone Loss The overexpression of Smad2 will induce GADD45β expression leading to the activation of P38 MAPK (Takekawa et al., 2002). As P38 becomes activated, it will up-regulate pro-inflammatory cytokines, namely TNF-α and IL1β (X.-L. Chen, Xia, Ben,   15 Wang, & Wei, 2003). Another path that might activate TNF-α and IL1β is apoptosis, as cleaved caspase 3 has been shown to activate inflammatory cytokines (Joshi, Kalvakolanu, & Cross, 2003). Both TNF-α and IL1β cytokines have been linked to bone loss as they play a central role in osteoclastogenesis by inducing stromal cells, T-lymphocytes, and osteoblasts to secrete RANKL, which will bind to the RANK receptors of the osteoclast precursors leading to an activation of the IkB kinase (T. A. Silva, Garlet, Fukada, Silva, & Cunha, 2007). IkB kinase phosphorylates the inhibitor of kappa B leading to the release of nuclear kappa B that will act as a transcriptional factor resulting in increased osteoclastogenesis (D. Drugarin, Drugarin, Negru, & Cioaca, 2003). In addition, TNF-α will induce a higher apoptosis ratio of the osteoblasts through TNF-related apoptosis-inducing ligand (TRAIL) (Mori, Brunetti, Colucci, & Ciccolella, 2006). The activation of TRAIL will activate caspase-8 and caspase-3 leading to a higher apoptosis ratio of osteoblasts (Mori et al., 2009). By increasing both osteoclastogenesis and the increased apoptotic ratio of osteoblasts the balance of bone resorption and bone deposition will tip more towards bone resorption leading to bone loss, which is considered the hallmark of the progression of periodontal disease (Graves, Li, & Cochran, 2011). 1.12 K14-Smad2 Mice Phenotype Overexpressing Smad2 in mice driven by the cytokeratin 14 promoter resulted in multiple phenotypical characteristics (Ito et al., 2001). The macroscopic phenotypes of K14-Smad2 mice were smaller body size until the age of three months, thicker skin, shorter tail, less hair on the ventral skin surface, fragile chalky white incisors and the skin of the ear’s pinna was retarded when compared to their wild type WT counterparts (Ito et   16 al., 2001). The microscopic phenotypes were the tail’s ill-defined basement membrane with reduced laminin content, increased proliferation rate in the keratinocyte layer of the epidermis, disorganized ameloblasts, and the amelogenin was not confined in the enamel matrix but also in between the disorganized ameloblasts (Ito et al., 2001). K14-Smad2 mice showed impaired wound healing (Tomikawa et al., 2012). The basal keratinocytes of the K14-Smad2 mice had a lower migration rate during wound healing when compared to WT mice due to the inhibition of keratin 16 expression by SMAD2 (Hosokawa et al., 2005).  The overexpression of Smad2 rescued the cleft palate in TGFβ3 -/- mice (Cui et al., 2005). The TGFβ3 -/- mice were mated with K14-Smad2 mice and the offspring that were TGFβ3 null but K14-Smad2 positive exhibited a rescue of the cleft palate defect (Cui et al., 2005). The exact mechanism of the rescue is not fully understood, but clearly restoration of the intracellular SMAD2 signalling pathway was sufficient to complete the process of palatogenesis. The studies showed that SMAD2 plays a significant role in the palatal shelf medial edge epithelium during palatal development. Studying the effect of the knockout of Smad2 currently is not possible in vivo as the knockout of Smad2 resulted in multiple severe defects during the three weeks of embryonic development and the absence of lower jaws or eyes (Nomura & Li, 1998).  None of the previous studies (Cui et al., 2005; Hosokawa et al., 2005; Ito et al., 2001; Owens et al., 2008) have examined the role of Smad2 overexpression in JE cells on the progression of periodontal disease. The K14-Smad2 mouse model is unique and specific as Smad2 is overexpressed only in tissues that express cytokeratin 14 (Ito et al., 2001). Cytokeratin 14 is expressed in the JE cells but not in connective tissue, bone,   17 cementum, and PDL cells, therefore it is specific to study the role of Smad2 overexpression in JE cells and what effect that might have on the periodontal tissues.  1.13 Hypothesis Smad2 overexpression alters apoptosis, cell proliferation, and inflammatory cytokine secretions in the junctional epithelium, which are associated with periodontal attachment loss.  1.14 Aims and Objectives Aim 1: To detect and quantify alveolar bone loss that occurs as a result of Smad2 overexpression (Chapter 4). Objective 1  To investigate in vivo the effect of Smad2 overexpression in JE cells on alveolar bone loss by determining bone loss, bone density, and bone volume for K14-Smad2 mice and compare them with their WT counterparts. Objective 2  To quantify the secreted molecules that cause bone loss (TNF-α, IL1-β, IF-γ, OPG, and RANKL) in K14-Smad2 mice compared to WT mice and to link them to periodontal disease progression. Objective 3  To quantify the number of osteoclasts in K14-Smad2 mice and compare them to WT mice. Aim 2: To analyze the effect of Smad2 overexpression on the viability and proliferation of JE (Chapters 2 and 3).  Objective 1  To assess the JE surface area of K14-Smad2 mice and compare them to WT mice.   18 Objective 2  To quantify the apoptotic rate of the JE of K14-Smad2 mice and compare them to WT mice. Objective 3  To evaluate the role of Smad2 overexpression in apoptosis of JE cells by quantifying pro-apoptotic molecules (cleaved caspase 3, BAX, BAD, BIK and BIM) and anti-apoptotic molecules (BCL-2, BCL-XL, and XIAP) in K14-Smad2 mice and comparing them to WT mice.  Objective 4 To determine the mitotic activity of the JE cells of K14-Smad2 and WT mice. Objective 5 To test the role of Smad2 overexpression on the proliferation rate of the JE cell of K1-Smad2 mice and WT mice by determining effects of molecules that control mitosis (P21, P15, P27, c-MYC, and pRB).    19 Chapter 2: SMAD2 is Involved in the Apoptosis of Murine Gingival Junctional Epithelium Associated with Inhibition of BCL-2 2.1 Overview 2.1.1 Objective Gingival junctional epithelium (JE) actively contributes to the homeostasis of the periodontium. Altered activation of TGF-β signalling is implicated in the epithelium from chronic periodontitis. However, little is known about the effects of TGF-β signalling on the JE. In this study, we investigated the relationship between SMAD2, which plays an important role in mediating TGF-β signal, and induction of apoptosis in the JE. 2.1.2 Methods K14-Smad2 transgenic mice were used to observe the effect of overexpression of Smad2 driven by CK14 promoter in the JE. We performed TUNEL technique to evaluate the epithelial apoptosis. Expression of apoptosis-related genes were examined using real-time PCR and immunofluorescence. 2.1.3 Results K14-Smad2 mice showed an increased number of phospho-SMAD2 positive JE cells associated with an increase in TGF-β1 expression. K14-Smad2 mice have a significantly higher percentage of TUNEL positive cells in the JE. Immunofluorescence double labelling revealed that TUNEL positive cells showed immunoreactivity to phospho-SMAD2. Real-time PCR analysis of apoptosis-related gene expression provided evidence of lower expression of BCL-2 in the gingival tissue from K14-Smad2 mice. There was a strong positive reaction for BCL-2 protein in the junctional epithelium of   20 wild type mice, while the gingival tissue of K14-Smad2 transgenic mice had only a faint signal for BCL-2. 2.1.4 Conclusions The present study provided evidence that SMAD2 plays a crucial role in the induction of apoptosis in gingival JE through inhibition of BCL-2. 2.2 Introduction Gingival junctional epithelium (JE) represents the first line of defence against microbial plaque in the dento-gingival complex (Bosshardt & Lang, 2005).The epithelium can serve as part of the local immune system, providing not only a fundamental structure as a physical barrier but function crucial for the host response against bacterial infection via the expression of a large variety of cytokines and antimicrobial peptides (Beagrie & Skougaard, 1962; Dale, 2002; Dickinson et al., 2011; Miyauchi et al., 2001). The structural and functional integrity of the JE therefore contributes to the homeostasis of periodontal tissue. TGF-β is a pleiotropic cytokine that controls homeostasis in the adult tissue. Aberrant activation of TGF-β signalling has been linked to various diseases such as fibrosis and cancer (Bierie & Moses, 2006; Pohlers et al., 2009). Previous studies showed distinct expression patterns of TGF-β ligands and their receptors in the JE as chronic periodontitis progressed (H. Lu, Mackenzie, & Levine, 1997; Ye et al., 2003), which indicated aberrant activation of TGF-β signalling  could be linked with the disease. However, little is known about the effects of TGF-β signalling in the JE. Therefore, it is of interest to investigate whether uncontrolled persistent activation of TGF-β signalling has the potential to alter the response of the JE.   21 TGF-β is known to exert its biological effects by activating a diverse range of intracellular signal transduction pathways. To dissect the molecular mechanism of TGF-β dependent effects on gingival epithelial tissue, we took advantage of a mouse model system that induces overexpression of Smad2, which is one of the crucial downstream effectors for TGF-β (Feng & Derynck, 2005; Massagué, Seoane, & Wotton, 2005) under the control of the cytokeratin 14 promoter. SMAD2 has been demonstrated to induce apoptosis that is observed in epithelial cells such as gastric (Ohgushi et al., 2005) and prostate epithelial cells (Seoane, 2006; Yang, Wahdan-Alaswad, & Danielpour, 2009). Activation of SMAD2 by autocrine and paracrine actions of TGF-β may regulate apoptosis in the JE during periodontal inflammation. In this study, we clarified the linkage of canonical SMAD pathway and induction of apoptosis in the gingival JE using K14-Smad2 transgenic mice. Secondly, we investigated the underlying mechanism by which overexpression of Smad2 induced apoptosis in the JE. Our result revealed that overexpression of Smad2 causes apoptosis of the gingival JE associated with regulation of BCL-2. 2.3 Materials and Methods  2.3.1 Animals and Genotyping All animal procedures complied with guidelines of, and were approved by, the Animal Care Committee of The University of British Columbia. K14-Smad2 transgenic mice were originally provided by Dr. Yang Chai (Ito et al., 2001). The genotype of the mice was determined by PCR using genomic DNA extracted from ear biopsies. Primer sets to detect the K14-Smad2 transgene were designed to be specific for the cytokeratin 14 promoter region (Ito et al., 2001).   22 2.3.2 Real-time PCR A total of five eight-week-old male mice were used in this study. Gingival tissue was dissected and total RNA was extracted using RNeasy Mini Kit (Qiagen, CA, USA) and quantified by spectrometry at 260 and 280 nm. First standard cDNA synthesis was performed with 1 µg of total RNA extract in a total volume of 20 µl using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, CA, USA). SYBR-Green based real-time PCR was performed with a Roter-Gene RG3000 using SsoFast EvaGreen Supermix (Bio-Rad). Primers used in this study were obtained from Primer Bank (Tgf-β1: ID6755775a1, Tgf-β2: ID15029686a1, Tgf-β3: ID13529608a1, Bcl-xl: ID118129881b1, Xiap: ID157951673b1, Bax: ID133778943b1, Bad: ID133892666b1, Bim: ID90093352b2, Bik: ID#13277643a1 and Gapdh: ID#126012538b1) except Bcl-2 (forward primer: 5′-CTGGCATCTTCTCCTTCCAG-3′ and reverse primer: 5′-GACGGTAGCGACGAGAGAAG-3′) (Spandidos, Wang, Wang, & Seed, 2010). Gapdh expression levels were used as a reference for normalization. 2.3.3 Immunohistochemistry Eight-week-old male mice were used in this study. Tissue samples were collected from the left and right molar regions and fixed with 4% paraformaldehyde solution. They were then decalcified in a 20% sodium citrate and formic acid solution (2:1) for seven days at 4 °C. The decalcified tissue blocks were dehydrated through graded ethanol and embedded in paraffin. Sections (5 µm thick) of the frontal plane parallel to the long axis of the maxillary teeth, including the root apex, were cut and collected on glass slides. After deparaffinization and antigen retrieval, sections were blocked with 1% BSA/0.1% Triton-X/PBS blocking solution at room temperature for 30 min and incubated with the   23 primary antibody against cytokeratin 14 (1:100, Santa Cruz Biotechnology, CA, USA), phospho-SMAD2 (1:20, Cell Signaling Technology, MA, USA), BCL-2 (1:100, Cell Signaling Technology) and E-cadherin (1:100, BD Transduction Laboratories) for 2 h at room temperature followed by a fluorescent labelled secondary antibody (1:100, Invitrogen, CA, USA) for 1 h at room temperature, washed with PBS, and cover-slipped with mounting media including DAPI. All sections were examined with a Nikon Laser Scanning Confocal microscope (C1) that was equipped with an argon (488 nm) and two He–Ne lasers (543 nm and 633 nm). Sections were scanned with a Plan Fluor 40X NA0.75 lens and a Plan Apo VC 60X NA1.4 oil lens. For quantification of the number of phospho-SMAD2 positive cells, total (DAPI) and phospho-SMAD2 positive cells were counted in JE and the percentage of positive cells was determined. Eight sections with 100 µm distance each obtained from first and second molars in the maxilla were analyzed, and average values were obtained for three biological replicates in each group. These mean values were used for the Student t-tests that compared phospho-SMAD2 positive cells in the control and the K14-Smad2 mice groups. 2.3.4 Analysis of Junctional Epithelial Apoptosis Rate Apoptotic cells were detected in paraffin sections by the TUNEL technique using an In Situ Cell Death Detection Kit (Roche Applied Science, Basel, Switzerland). Slides were cover-slipped with mounting medium including DAPI. Total (DAPI) and apoptotic (TUNEL positive) cell counts were made in JE that was shown as the area surrounded by the dotted line in Fig. 4A. The percentage of apoptotic JE cells was calculated as follows: apoptotic JE cells (%) = number of TUNEL positive staining cells/number of total DAPI positive cells × 100. Eight sections with 100 µm distance each obtained from first and   24 second molars in the maxilla were analyzed, and average values were obtained for three biological replicates in each group. These mean values and the standard deviations were used for the Student t-tests that compared apoptotic cells in the control and the K14-Smad2 mice groups.  2.4 Results 2.4.1 Activation of Overexpressed SMAD2 Was Associated With an Increase in Endogenous Tgf-β1 Expression in Gingival Epithelial Tissue  We performed real-time PCR to confirm overexpression of Smad2 mRNA in the K14-Smad2 transgenic mice. Gingiva from K14-Smad2 mice had 5-fold higher expression of Smad2 mRNA than that isolated from wild type mice (Fig. 3A). We performed immunofluorescence to determine the level of SMAD2 protein under the control of the cytokeratin 14 promoter in wild type and K14-Smad2 transgenic mice. We confirmed cytokeratin 14 expression in gingival JE, sulcus epithelium and oral epithelium. The strongest signals were observed in JE (Fig. 3B). Gingival tissue from K14-Smad2 mice had high levels of phospho-SMAD2, which is the active form. Positive cells were dominant in JE and basal cell layer of oral epithelium, which corresponded to the localization of cytokeratin 14 (Fig. 3B). K14-Smad2 mice had an 11-fold higher percentage of phospho-SMAD2 positive JE cells compared to wild type mice (Fig. 3C). We also evaluated the expression level of endogenous TGF-βs in the gingival tissue to determine whether activation of overexpressed SMAD2 was mediated by TGF-βs. K14-Smad2 mice had a 2-fold increase in the endogenous Tgf-β1 compared to the wild type control (Fig. 3D). Tgf-β2 was not detectable in either wild type or K14-Smad2 mice. There was no significant difference in Tgf-β3 between the experimental and control   25 samples (Fig. 3D).  Figure 3: Activation of Overexpressed SMAD2 was Associated with an Increase in Endogenous Tgf-β1 Expression in Gingival Epithelial Tissue (A) Quantitative real-time PCR analysis of Smad2 mRNA in gingival tissue. **Differs significantly (P < 0.05). (B) Immunolocalization of phospho-SMAD2 (red) and cytokeratin 14 (green) in gingival tissue from wild type and K14-Smad2 mice. Original magnification (600×). (C) Quantification of phospho-SMAD2 positive cells expressed as percent of total nuclei in the JE. **P < 0.01. (D) Quantitative real-time PCR analysis of TGF-βs mRNA in gingival tissue. *P < 0.05. Error bars represent the standard deviations. The white scale bar represents a 100µm 2.4.2 Increased JE Apoptosis in K14-Smad2 Mice TUNEL assay was conducted to examine whether overexpression of Smad2 can   26 induce apoptosis in JE. Sections treated with DNase I were used as positive control of apoptosis. No TUNEL positive nuclei were observed in negative control sections (Fig. 4A). K14-Smad2 mice exhibited a 12-fold increase in TUNEL positive JE cells (36%) compared to the wild type (3%) (Fig. 4B). Immunofluorescence double staining revealed TUNEL positive JE cells showed immunoreactivity to phospho-SMAD2 (Fig. 4C).      27 Figure 4: Increased JE Apoptosis in K14-Smad2 Mice (A) Representative image of TUNEL staining (green) from wild type and K14-Smad2 mice, counterstained with DAPI (blue). The area surrounded by dotted line represents JE, in which we counted apoptotic cells. Original magnification (600×). (B) Quantification of apoptotic JE cells on the basis of TUNEL-positive staining expressed as percent of total nuclei in the JE. **P < 0.01. (C) Immunofluorescence double staining of phospho-SMAD2 (red) and TUNEL (green), counterstained with DAPI (blue). Arrowheads indicate double positive JE cells. Original magnification (1200×). Error bars represent the standard deviations. The white scale bar represents a 100µm   2.4.3 Reduction in BCL-2 Expression in K14-Smad2 Mice We performed real-time PCR to investigate the underlying mechanism by which overexpression of Smad2 induced apoptosis in the JE. Figure 5 provides the evidence that more than a 2-fold lower expression of anti-apoptotic Bcl-2 occurs, but no significant difference in the expression level of anti-apoptotic Bcl-2 family genes; Bcl-xl and Xiap, pro-apoptotic Bcl-2 family genes; Bax, Bad, Bim, and Bik in the gingival tissue from K14-Smad2 mice when compared to wild type mice. At the protein level, immunofluorescence analysis, presented in Figure 6, demonstrated a strong positive signal for BCL-2 in wild type mice, especially in the JE. On the other hand, the gingival tissue of K14-Smad2 transgenic mice had only a very faint signal for BCL-2.     28   Figure 5: Reduction in Bcl-2 mRNA Expression in K14-Smad2 Mice (A) Quantitative real-time PCR analysis of gene expression encoding anti-apoptotic Bcl-2 family proteins, Bcl-2, Bcl-xL and Xiap in gingival tissue from wild type and K14-Smad2 mice. **P < 0.01. (B) Quantitative real-time PCR analysis of gene expression encoding pro-apoptotic Bcl-2 family proteins, Bax, Bad, Bim, and Bik in gingival tissue from wild type and K14-Smad2 mice. Error bars represent the standard deviations       29        A                                                       B  Figure 6: Reduction in BCL-2 Protein Expression in K14-Smad2 Mice (A) Immunolocalization of BCL-2 (red) and E-cadherin (green) in gingival tissue from wild type and K14-Smad2 mice. Original magnification (400×). (B) Higher magnification of boxed area on (A). Original magnification (600×). The white scale bar represents a 100µm 2.5 Discussion TGF-βs have important roles in the control of tissue homeostasis. Little is known about the effects of TGF-β signalling in the JE during periodontitis in which aberrant TGF-β activation was implicated (H. Lu et al., 1997; Ye, Chapple, Kumar, & Hunter, 2000). Here we demonstrate that the SMAD2 activation induces apoptosis in gingival JE by regulating the anti-apoptotic BCL-2 family member, Bcl-2.   30 2.5.1 Mouse Model System We used K14-Smad2 transgenic mice to dissect the molecular mechanism of TGF-β dependent effects in the JE. Overexpression of Smad2 was confirmed to be expressed in only gingival epithelial cells with cytokeratin 14 expression. SMAD2 becomes activated by receptor-mediated phosphorylation to form a complex with SMAD4 and regulate the expression of TGF-β targeted genes (Feng & Derynck, 2005; Massagué et al., 2005). Detection of a higher level of phosphorylated SMAD2 in K14-Smad2 mice provided evidence that overexpression of Smad2 using a cytokeratin 14 promoter system functions successfully in mediating signals in the JE. We also showed that overexpressed Smad2 increased expression of Tgf-β1, thereby generating a positive feedback loop leading to phosphorylation of the overexpressed SMAD2, which is consistent with a previous study (Ito et al., 2001). This mouse model is a powerful tool to evaluating TGF-β/SMAD pathway dependent effects in gingival epithelial tissue. 2.5.2 Overexpression of Smad2 Induces Apoptosis in JE TGF-β is a pleiotropic cytokine that regulates diverse cellular processes such as cell growth, apoptosis, and epithelial–mesenchymal transition (Heldin, Landström, & Moustakas, 2009). With respect to the apoptotic response, the canonical SMAD2 pathway plays an important role in mediating the signals as well as a p38 dependent pathway, which appears to be cell type-dependent (Moustakas & Heldin, 2005; Schuster & Krieglstein, 2002).We evaluated whether overexpressed Smad2 can induce apoptosis in the JE using the TUNEL technique. The results demonstrated a higher apoptotic ratio in K14-Smad2 mice compared to controls. This indicates that the SMAD2 signalling pathway played a role in inducing TGF-β dependent apoptosis in JE. Colocalization of p-  31 SMAD2 and TUNEL signals provide strong evidence to support the relationship between activation of SMAD2 and induction of JE apoptosis. In gingival tissue, apoptosis was detected in both the superficial layers of the JE in clinically healthy teeth (Tonetti, Cortellini, & Lang, 1998) and pocket epithelium in chronic periodontitis (Vitkov, Krautgartner, & Hannig, 2005; 2009). However, JE cells collected from patients with chronic periodontitis had a higher apoptotic ratio than those from healthy teeth (Zhang & Li, 2009). Maintenance of the balance between cell proliferation and cell death is a prerequisite for the proper function of JE to control the constant microbiological challenge. Altered JE cell turnover with increased numbers of apoptotic cells may be induced by the TGF-β/SMAD2 pathway during periodontitis, which may contribute to the progression of periodontitis due to loss of junctional epithelial integrity. 2.5.3 Underlying Mechanism by Which Overexpression of Smad2 Induces Apoptosis in JE TGF-β has been described to induce cell death by controlling the balance between anti-apoptotic factors such as BCL-2 and BCL-XL and pro-apoptotic BCL-2 family members such as BAX, BIM, and BIK depending on the cellular context (Q. Lu, Patel, Harrington, & Rounds, 2009: Motyl et al., 1998; Ohgushi et al., 2005; Spender et al., 2009; Yu et al., 2008). In models of mesothelial and pulmonary endothelial cell apoptosis induced by TGF-β1, SMAD2 was correlated with BCL-2 repression (Q. Lu et al., 2009; Lv et al., 2012).In gingival JE, activation of SMAD2 alters the expression of the anti-apoptotic BCL-2 family member, BCL-2, but does not affect the pro-apoptotic ones. A previous study reported that no BCL-2-positive cells were detected in the JE with strong TUNEL positive signals (Tonetti et al., 1998). We also demonstrated an inverse correlation between the apoptotic cell ratio and Bcl-2 expression level in the JE. Our   32 present data therefore suggest that BCL-2 plays a pivotal role in gingival JE cell homeostasis, and reduction in the expression level of Bcl-2 shifts a life-death balance towards cell death in the JE. Although SMAD2 was not involved in the alteration of gene expression in the pro-apoptotic Bcl-2 family members, we cannot exclude their involvement in JE apoptosis during chronic periodontitis since the p38 dependent pathway activated by TGF-β may regulate their transcriptional expression and augment the apoptotic response in the JE. 2.6 Conclusion In conclusion, this study demonstrates that overexpression of Smad2 results in increased apoptosis of the gingival JE cells. This is associated with reduction in Bcl-2 expression in the JE. The TGF-β/SMAD signalling may play an important role in the progression of periodontitis by regulating JE cell apoptosis.   33 Chapter 3: Smad2 Overexpression Reduces the Proliferation of the Junctional Epithelium  3.1 Overview 3.1.1 Objective The overexpression of the intracellular signalling molecule of the transforming growth factor–beta family (TGF-β) Smad2 was found to induce apoptosis and inhibit the proliferation rate of oral epithelial cells. Therefore, the aim of this study was to investigate in vivo the effect of Smad2 overexpression on the proliferation rate of the junctional epithelium (JE).  3.1.2 Methods  Smad2 overexpression was driven by the cytokeratin 14 promoter (K14-Smad2) in transgenic mice. The K14-Smad2 mice were compared with wild type (WT) mice selected as the control group. Tissue samples were stained with hematoxylin and eosin and analyzed by image analysis. Immunohistochemistry was conducted for proliferating cell nuclear antigen (PCNA) and c-MYC as markers of cell proliferation. The expression of cyclin-dependent kinase inhibitors (P15, P21, and P27) was determined by real-time polymerase chain-reaction (RT-PCR). The quantity of phosphorylated retinoblastoma (pRB) was determined with Western blots. 3.1.3 Results  The overexpression of Smad2 altered the area of the junctional epithelial cells in one-year-old K14-Smad2 mice. The area was 32,768 (± 3,473) µm2 for the WT and 24,937.25   34 (± 1,965) µm2 for the K14-Smad2 mice. There was a significant difference in the proliferation rates of the JE (PCNA-positive cells) between the WT and K14-Smad2 mice, 20.7% (± 1.1) and 2.1% (± 0.5), respectively. A significant difference in c-MYC expression occurred between experimental and control samples. The K14-Smad2 mice had a mean of 2.3% (± 0.6), and the WT mice had a mean of 20.1% (± 3.6). Smad2 overexpression up-regulated the mRNA expression of P15 by 2.3-fold and that of P27 by 5.5-fold in the K14-Smad2 mice. Finally, the pRB protein showed a 2.3 (± 0.5)-fold increase in K14-Smad2 mice when compared with WT mice.  3.1.4 Conclusions Smad2 overexpression inhibits the proliferation of JE cells by down-regulating c-MYC and up-regulating P15 and P27, which resulted in an increase in pRB, leading to cell-cycle arrest. 3.2 Introduction The junctional epithelium (JE) is the part of the dento-gingival unit that is attached to the tooth surface. The junctional epithelium develops from the reduced enamel epithelium and over time it is replaced by the basal cells of the oral gingival epithelium (Salonen, Kautsky, & Dale, 1989).The junctional epithelium extends coronally to the base of the oral sulcular epithelium and apically to the connective tissue attachment at the cementoenamel junction and forms the lining of the interdental col (Gargiulo & Wentz, 1961). The junctional epithelium is the first line of defence against periodontal disease; it provides an important barrier activity by contacting the tooth surface with cells directly attached to the tooth (DAT cells) through hemidesmosomes (Listgarten, 1966).The junctional epithelial cells play an active role in the synthesis of a   35 variety of molecules that are part of the defence against bacterial invasion such as the carcinoembryonic Ag-related cell adhesion molecule (1CEACAM1), which directs and guides PMNs through the junctional epithelium, and IL-8 cytokine, which has a chemotactic ability (Heymann, Wroblewski, Terling, Midtvedt, & Öbrink, 2005; Tonetti, Imboden, & Lang, 2012). Therefore the balance between cell death and mitotic activity is critical to maintain the number of junctional epithelial cells, which affects the defence properties of the junctional epithelium and eventually disease progression (Bosshardt & Lang, 2005; Overman & Salonen, 1994). Transforming growth factor beta (TGF-β) is a potent cytokine that is involved in both development and disease (Chang, Brown, & Matzuk, 2013). TGF-β signalling occurs through the binding of TGF-β to the type II receptor, which in turn phosphorylates the TGFβ type I receptor activating the intracellular kinase. The activation of type I results in the phosphorylation of the intracellular transcription molecule SMAD2. SMAD2 and SMAD4 bind together and enter the nucleus to start the transcription of TGFβ dependent genes that mediate multiple processes including apoptosis, cell proliferation, and the secretion of inflammatory cytokines (Wyllie, 2010). To study the effect of Smad2 overexpression on the JE cells we used a mouse model (K14-Smad2) that overexpresses Smad2 specifically in epithelium using a cytokeratin14 promoter (K14) (Ito et al., 2001). The transgenic mouse model presents an advantage as it induces an overexpression of Smad2 controlled by the K14 promoter specifically in JE cells, which makes the model suitable to investigate the role of Smad2 overexpression in the JE cells (Fujita et al., 2012). Our lab recently published data showing an increased apoptotic index of the JE   36 cells in Smad2 overexpression mice. Smad2 overexpression induced apoptosis of the junctional epithelial cells through the down-regulation of the anti-apoptotic molecule BCL2 (Fujita et al. 2012). The JE cells ordinarily have a high proliferation rate, which could overcome the increase in apoptosis thus maintaining the homeostasis of the JE (Watanabe et al., 2004). Previous studies have shown that TGF-β inhibits the proliferation rate of cells through the up-regulation of the cyclin-dependent kinase inhibitor and the repression of the c-MYC. c-MYC is a transcription protein that represses cyclin-dependent kinase inhibitors (p15, and p21) leading to an inhibition of the cell cycle progression from G1 to S phase (Seoane, 2006). The hypothesis of this study is that Smad2 overexpression will reduce the proliferation rate of JE cells through the up-regulation of the cyclin-dependent kinase inhibitors secondary to the down-regulation of c-Myc. The availability of the K14-Smad2 transgenic mice permits the role of Smad2 overexpression to be examined in vivo in the JE cells, thus the aim of the current study was to investigate the role of Smad2 overexpression on the proliferation rate of the JE cells in vivo. 3.3 Materials and Methods 3.3.1 Animals and Genotyping K14-Smad2 mice that have Smad2 overexpression through a K14 promoter were selected to represent the model of this study. Dr. Yang Chai, University of Southern California Center for Craniofacial Molecular Biology, generously provided these mice. All methods were within the guidelines of and approved by the Animal Care Committee of The University of British Columbia. The genotype of the mice was detected using a primer set that detected the K14-Smad2 transgene through the cytokeratin14 promoter   37 region. Mice that overexpressed Smad2 were analyzed at 3 and 12 months of age were the test group and compared to age-matched wild type controls. The total sample size was 40 mice. Sample size was divided as shown in Figure 7. The sample size was based on power analysis (G*power software) that was conducted for all methods, which gave a power of 0.8 for each.  3.3.2 Histology and Immunohistochemistry 3.3.2.1 Decalcification and Paraffin Embedding of the Samples Hemisections of mice maxillae were dissected under the microscope, and the samples were decalcified using Ethylenediaminetetraacetic acid (EDTA) for four to six weeks at room temperature. The decalcified samples were embedded in paraffin blocks and stored at -50 C. 7µm sections were obtained for subsequent analysis.  3.3.2.2 Hematoxylin and Eosin Slides were stained with Harris’s hematoxylin for 2.5 minutes and then transferred to running tap water for 20 minutes; thereafter the slides were immersed in eosin for 40 seconds, dehydrated, and cleared through a series of ethanol and Xylene respectively. Finally, slides were cover-slipped using mounting media and viewed under light microscopy (Wazen, Moffatt, Zalzal, Yamada, & Nanci, 2009). 3.3.2.3 Immunohistochemistry EDTA antigen retrieval was done and then the sections were blocked with 2% goat serum for 30 minutes at room temperature. 2µl primary antibody against E-cadherin (Cell Signaling Inc, mouse polyclonal), PCNA (Cell Signaling Inc, mouse polyclonal) and c-MYC (Abcam Inc, rabbit polyclonal) were incubated with the sections for 2 hours and then rinsed with PBS three times for 5 minutes. Secondary antibody goat anti-mouse   38 (Alexa Fluor 488 IgG, Invitrogen, Inc) and goat anti-rabbit (Alexa Fluor 568, IgG, Invitrogen, Inc) were incubated with the slides for 1 hour at room temperature, followed by PBS rinse three times for 4 minutes. Finally, slides were cover-slipped with mounting media, which included DAPI to identify the cell nuclei. Nikon Laser Scanning Confocal microscopy (C1) was used to examine the slides (Watanabe et al., 2004). Liver and small intestine tissues were used as positive control samples for PCNA and c-MYC proteins respectively. Negative control samples were obtained by omitting the primary antibody. 3.3.3 Real-time PCR The samples were collected from the buccal and palatal attached gingiva of the first and second molar teeth of the K14-Smad2 and wild type mice. Tissues were homogenized using a mortar and pestle, and the RNA was purified using RNeasy Mini Kit (Qiagen). The total RNA was measured, and equal amounts of the RNA were used for cDNA synthesis using Iscript Kit (Bio-Rad). Nucleotide sequences for the PCR primers were obtained from the National Center for Biomedical Information as shown in Table 2.  Table 2: Primer Sequences for p15, p21, p27, and Gapdh  Primer  Forward Reverse P15  P21  P27  Gapdh  5’-CCCTGCCACCCTTACCAGA-3’  5’-CCTGGTGATGTCCGACCTG-3’  5’-TCAAACGTCAGAGTGTCTAACG -3’  5’-GGTCCTCAGTGTAGCCCAAG-3’ 5’-CAGATACCTCGCAATGTCACG -3’  5’-CCATGAGCGCATCGCAATC -3’  5’-CCGGGCCGAAGAGATTTCTG -3’  5-‘AATGTGTCCGTCGTGGATCT-3’    39 cDNA samples were added to a PCR amplification mixture containing forward and reverse primers and SsoFast EvaGreen Supermix PCR master mixture (Bio-Rad Laboratories, California, USA). Then samples were subjected to a denaturation reaction for 5 minutes at 94C. Finally, annealing and DNA synthesis were done for 60 seconds at 60 C. The data were normalized against Gapdh and calculated by the CT method (Hart, Shaffer et al. 2004). 3.3.4 Western Blots The attached gingiva from the first and second molar of the maxilla of K14-Smad2 transgenic mice and wild type mice were collected under the microscope. The tissue samples were homogenized using 500-µL lysis buffer NP-40 and 10 µL of the proteinase inhibitor cocktail on ice. The samples were placed in dry ice for 20 minutes, then the tubes were centrifuged at 12000 rpm for 15 minutes at -4C. The protein concentrations were determined through the bovine serum assay standard and then equal protein samples were subjected to 5% SDS-PAGE buffer and electrophoresed for 45 minutes at 160V at room temperature. The samples were transferred from the SDS-PAGE gels to a membrane (BioRad trans blot pure nitrocellulose) at 60V for 2 hours. Then the membranes were washed with PBS for 10 minutes on a shaker. Blocking solution Odyssey (Li-Cor) was applied for 1 hour at room temperature. The primary antibody for phosphorylated retinoblastoma protein (Rabbit polyclonal, Abcam) was applied at a concentration of 0.2 µg/ml for 1 hour. The membrane was washed in PBS 5 times for 5 minutes. The samples were incubated with a fluorescent secondary anti-rabbit IgG antibody for 1 hour. Then the membrane was washed in PBS 5 times for 5 minutes. Finally, the membrane was scanned and the bands intensities were quantified using the   40 Image tool program (Lohinai et al., 2001). 3.3.5 Statistical Analysis The data were interpreted using One-way ANOVA and student t test. 3.4 Results 3.4.1 Smad2 Overexpression Altered the Surface Area of the JE Cells The JE surface area was measured using image tool at 3 and 12 months of age for both K14-Smad2 and wild type mice. Connecting the internal basement membrane to the first horizontal cell in the external basement membrane outlined the JE surface area. As shown in Figure 7 the mean JE surface area of the K14-Smad2 remained almost unchanged from 23036.5(±3754) µM2 at 3 months to 24937.25(±1965) µM2 at the 12 months time point. On the other hand the mean surface area of the wild type mice was 21728(±4724) µM2 at 3 months and increased significantly to 32768(±3473) µM2 at the 12 months time point (Fig. 7).   41  Figure 7: Smad2 Overexpression Altered the JE Surface Area (A) H&E image showing the JE of three months WT mice. (B) H&E image of K14-Smad2 JE surface area at three months time point. (C) H&E image showing the JE of 12 months K14-Smad2 mice. (D) H&E image showing the JE of 12 months WT mice. (E) Quantification of the JE surface area of the three months and the one-year samples. *(P<0.05). Error bars represent the standard deviations. Scale bars represent a 100µm     42 3.4.2 Smad2 Overexpression Reduces the Proliferation Rate of the JE Cells Proliferating cell nuclear antigen (PCNA) positive cells were selected as representative of proliferating cells. The positive cells (PCNA+DAPI) were counted and divided by the total number (DAPI) of JE cells to identify the percentage of proliferating cells. The 3-month-old K14-Smad2 mice had a significantly lower proliferation rate than their wild type counterparts the mean proliferation of the JE cells was 20.79% in the wild type mice and 1.31% in the K14-Smad2 mice (Fig. 8).      43  Figure 8: Smad2 Overexpression Reduced the Proliferation Rate of JE Cells (A) Representative image of PCNA positive cells (Green) and DAPI (Blue) from WT mice at 3 months time point. (B) PCNA positive cells and DAPI from K14-Smad2 mice at 3 months time point. (C) PCNA window of WT 3 months mice showing multiple positive cells. (D) PCNA window of K14-Smad2 mice showing limited positive cells. The dotted line represents the JE. Original magnification (x600). (E) A x1200 Magnification of WT mice showing multiple colocalizations between PCNA and DAPI. (F) A x1200 Magnification of K14-Smad2 mice showing limited colocalizations between PCNA and DAPI. (G) Positive control small intestine tissue showing multiple PCNA positive cells. (H) Negative control of JE cells showing no PCNA signal. (I) Quantification of the PCNA positive cells represented by PCNA +DAPI positive cells. **(P<0.001). The white bar represents 100mm. Error bars represent the standard deviations     44 3.4.3 Increased SMAD2 Up-regulates c-MYC  The mean c-MYC positive cells in the JE were counted and the percentage of c-MYC positive cells calculated. Increased SMAD2 reduced the number of c-MYC positive cells in the JE in K14-Smad2 experimental mice (2.3%) compared to the wild type controls (20.1%) (Fig. 9).      45 Figure 9: Smad2 Overexpression Inhibits C-MYC (A) Representative image of C-MYC (Red) expression in the JE with E-cadherin (Green) from WT mice at 3 months time point. (B) c-MYC positive cells and E-cadherin from K14-Smad2 mice at 3 months time point. (C) c-MYC window showing positive cells of WT mice. (D) C-MYC window no positive cells of K14-Smad2 mice. The dotted line represents the JE. Original magnification (x600). (E) A x1200 Magnification of WT mice showing multiple colocalizations between c-MYC and DAPI. (F) A x1200 Magnification of K14-Smad2 mice showing limited colocalizations between c-MYC and DAPI. (G) Positive control liver tissue showing multiple c-MYC positive cells. (H) Negative control of JE cells showing no c-MYC signal. (I) Quantification % of C-MYC positive cells **(P<0.001). The white bar represents 100mm. Error bars represent the standard deviations   3.4.4 Smad2 Overexpression Increased Both P15 and pRB to Inhibit JE Cell Proliferation  SMAD2 increased the expression of the mRNA of the cyclin-dependent kinase inhibitor p15 by 5.56-fold when compared to wild type mice. P21 gene expression did not show any statistically significant difference between K14-Smad2 and wild type mice. Smad2 overexpression increased (2.3-fold) the pRB protein in the JE cells of K14-Smad2 mice when compared to wild type mice (Fig. 10).     46  Figure 10: Smad2 Overexpression Up-regulates P15, P27 and Increases the Protein Level of p-RB (A) Western blots of K14-Smad2 and WT mice representing P-RB and GAPDH. (B) Quantification of the P-RB western blot results **(P<0.001). (C) Real-time PCR expression of P15, P21, and P27. **(P<0.001). Error bars represent the standard deviations  3.5 Discussion The junctional epithelium has an important protective role providing the barrier ability and the secretion of defence molecules (Bosshardt & Lang, 2005; Heymann et al., 2005; Listgarten, 1966; Overman & Salonen, 1994; Tonetti et al., 2012). In our lab we previously published data showing that Smad2 overexpression causes an increased apoptotic index in the JE cells through the inhibition of BCL2 (Fujita, Alotaibi et al., 2012). The present study expanded on the original findings to determine if increased levels of SMAD2 also altered the JE proliferation and thus the combined effects of increased apoptosis and decreased proliferation would negatively impact JE homeostasis. In the current study the JE of K14-Smad2 mice showed a reduced surface area when   47 compared to wild type mice at 12 months, which indicated that the JE does not have the same size/integrity as the wild type control tissue. The change in JE area at 12 months can be explained by a combination of increased apoptosis and a reduced proliferation rate that decreases the replacement of the dead cells. A limitation of the current methodology to collect the JE tissue samples for the Western blots and the real-time PCR was a potential confounding variable. The JE cells were isolated by doing Modified Widman incisions, which included oral and JE epithelium for both test and control groups. To understand the underlying mechanism of the role of Smad2 overexpression on the reduction of the proliferation rate we examined molecules that control the proliferation rate such as c-MYC, cyclin-dependent kinase inhibitors (P15, P21, and P27) and pRB (Warner, Blain, Seoane, & Massagué, 1999). It was reported in vitro that Smad2 overexpression induces p21 and p15, cyclin-dependent kinase inhibitors that inhibit cell cycle progression from G1 to the S phase and thus prevent proliferation (Shimoe et al., 2014). Our findings have the same outcomes as the previous in vitro study since increased levels of SMAD2 result in an up-regulation of the expression of both P15 and P27. Some differences in specific cyclin-dependent kinase inhibitors can be explained by the specific epithelial cells and tissues examined. The in vitro study used oral epithelial cells and in our study we specifically investigated the JE epithelial cells. It has been reported that TGF-β down-regulates transcription factors involved in proliferation such as c-MYC (Warner et al., 1999). Previous studies have shown that c-MYC was a repressor of P27 (Amendola et al., 2009) and P15 (Staller et al., 2001), and down-regulation of c-MYC would up-regulate these cyclin-dependent kinase inhibitors . Current results supported by the current in vivo analysis of increased intracellular signalling in the TGFβ   48 pathway reinforce this mechanism to reduce cell proliferation. Studies have shown that the activation of cyclin-dependent kinase inhibitors would lead to increased levels of pRB, again consistent with the findings in the present study. The increased levels of pRB in turn release E2F a transcriptional factor that causes cell cycle arrest (Ravitz and Wenner 1997).  From the results presented above we can concluded that Smad2 overexpression in reduces the proliferation rate of the JE cells. Smad2 overexpression reduces the surface area of the JE cells by inhibiting c-MYC, which in turn up-regulates the cyclin-dependent kinase inhibitors (P15, and p27) leading to excess phosphorylation of the retinoblastoma protein.   49 Chapter 4: Smad2 Overexpression Induces Alveolar Bone Loss by Up-regulating TNF-α  4.1 Overview 4.1.1 Background Previous studies found that Smad2 overexpression reduced the surface area of the junctional epithelium (JE) through increased apoptosis and a reduced proliferation rate. The aim of the current study was to investigate whether Smad2 overexpression in JE cells induced alveolar bone loss, and to understand the mechanisms regulating the bone loss. 4.1.2 Methods A mouse line was created that used a cytokeratin 14 (K14) promoter to overexpress Smad2 in the epithelium of the transgenic mice (K14-Smad2)(Ito et al., 2001). Hemi maxilla samples were stained with Van Gieson’s and Ponceau S solutions and photographed. Micro CT radiographs (µCT) were used to assess bone loss, bone volume, and bone density. The expression of Tnfα, Il1-β, Ifγ, Rankl, and Opg were assessed by RT-PCR. Western blots were used to detect the protein levels of TNF-α and IL1-β. Immunohistochemistry was done for Tartrate-resistant acid phosphatase (TRAP) as a marker for osteoclasts. Wild type (WT) mice were used as controls in all steps of the current study.  4.1.3 Results Smad2 overexpression induced alveolar bone loss at 12 months of age in the K14-Smad2 mice. The µCT analysis provided evidence that K14-Smad2 mice had 52.5% (±4.2) root   50 exposed compared to 32.4%(±3.2) in the WT mice. There was a significant difference in alveolar bone volume in the K14-Smad2 mice when compared to WT mice 2.65mm3 (±0.3) and 4.3 mm3 (±0.35) respectively. K14-Smad2 mice also had reduced bone density 696.8 mg/cc (±70) at 12 months when compared to WT mice 845.9 mg/cc(±10). The mRNA levels of Tnfα and Rankl increased by 3.26- and 2.5-fold respectively in the K14-Smad2 mice when compared to controls. The protein level of TNF-α was also significantly increased to 2.8-fold in K14-Smad2 mice when compared to WT mice. Smad2 overexpression increased the total numbers of osteoclasts in K14-Smad2 mice (3.4 ±0.2)-fold when compared to WT mice.  4.1.4 Conclusion Smad2 overexpression increased the osteoclastogenesis through the up-regulation of TNF-α and RANKL to induce alveolar bone loss.  4.2 Introduction The JE develops from both the reduced enamel epithelium and the oral epithelium (Salonen, Kautsky, & Dale, 1989). The JE provides protection to the underlying periodontium by two mechanisms. The first mechanism is through cells directly attached to the tooth (DAT cells) by hemidesmosomes. DAT cells provide a physical barrier that prevents bacterial invasion (Listgarten, 1966), and the JE cells have high apoptotic and proliferation rates that allow microorganisms to be pushed into the gingival cervical fluid away from the periodontal tissues. The second mechanism is through the secretion of multiple molecules that help in the protection and development of the periodontium, such as Intercellular adhesion molecule-1 (ICAM-1 or CD54), Lymphocyte function antigen-3 (LFA-3), Interleukin-8 (IL-8), Epidermal growth factor (EGF), and defensins (Crawford,   51 1992; Dale, 2002; Tajima et al., 1992; Tonetti, Imboden, & Lang, 2012). Any disturbance to the JE cells could reduce the protective ability of the JE cells and lead to periodontal disease progression (Bosshardt & Lang, 2005). Transforming growth factor beta 1 (TGFβ1) is a cytokine that belongs to the transforming growth factor beta superfamily. TGFβ1 was found to be involved in health maintenance and disease progression through the regulation of multiple cellular functions such as proliferation, differentiation, and apoptosis (Ehinger, Bergh, Johnsson, Gullberg, & Olsson, 1997; Granerus, Schofield, Bierke, & Engström, 1995; Takizawa et al., 2001). When TGFβ1 binds to the TGFβ receptor type II (TβRII) it activates Type I receptor (TβRI), which in turn phosphorylates SMAD2(Massagué, Seoane, & Wotton, 2005). SMAD2 is a transcription factor of the TGFβ signalling pathway (Kitisin, Saha, Blake, & Golestaneh, 2007). SMAD2 activation leads to the binding of SMAD2 and SMAD4 (Nakao et al., 1997). The SMAD2- SMAD4 complex enters the cell’s nucleus to activate the transcription of multiple genes that ultimately alters the cell’s function (Massagué & Chen, 2000). One result of this transcriptional activation is an increase in caspase3 expression that is linked to apoptosis (Wyllie, 2010). TGF-β has been reported to reduce the mitotic activity of epithelial cells (Seoane, 2006). TGF-β induces P21Cip1 and 2150 P15Ink4b, which are cyclin-dependent kinase inhibitors (Tomikawa et al., 2012). TGF-β down-regulates transcription factors involved in proliferation, for example MYC, ID1 and ID2 (Seoane, 2006). Our laboratory used a mouse model that overexpresses Smad2 driven by a cytokeratin 14 promoter (K14) (Fujita et al., 2012). The K14 promoter resulted in the overexpression of Smad2 in the epithelial cells especially the JE cells (JE) (Fujita et al., 2012). Interestingly, it was shown that the overexpression of Smad2   52 increased the apoptotic index and at the same time reduced the proliferation rate of the JE, consequently reducing the JE surface area (Alotaibi, Kitase, & Shuler, 2014). Apoptosis has been linked to the activation of inflammatory cytokines, such as TNF-α and IL1β (Joshi, Kalvakolanu, & Cross, 2003). It has been shown that Smad2 overexpression increases the secretion of TNF-α through a cross-talk up-regulation of the P38 MAP kinases (X.-L. Chen, Xia, Ben, Wang, & Wei, 2003; Takekawa et al., 2002). Both TNF-α and IL1β cytokines have been shown to up-regulate RANKL leading to increased binding to RANK (Garlet, Martins, Fonseca, Ferreira, & Silva, 2004; Steeve, Marc, Sandrine, Dominique, & Yannick, 2004). The RANKL-RANK complex will activate nuclear factor kappa β (T. A. Silva, Garlet, Fukada, Silva, & Cunha, 2007), which will up-regulate the osteoclastogenesis process (D. Drugarin, Drugarin, Negru, & Cioaca, 2003). Periodontal disease is a complex disease process with multiple etiologic factors. A clinical diagnosis of periodontal disease has been identified as attachment loss of either the soft tissues or alveolar bone and in many cases both are damaged (Armitage, 1999). During routine dental examination dentists evaluate the levels of soft tissues clinically and bone loss radiographically (Armitage, 1996). Any change in the level of attachments is considered periodontal disease and the underlying cause should be investigated (Armitage, 1999). We hypothesized that Smad2 overexpression alters the expression of inflammatory cytokines in JE cells. This overexpression of the cytokines is associated with bone loss and periodontal disease progression. The specific aims of the current study were: 1) to investigate in vivo the effect of Smad2 overexpression on alveolar bone loss;   53 and 2) to investigate secreted molecules that control bone metabolism and link them to periodontal disease progression. 4.3 Materials and Methods 4.3.1 Animals and Genotyping We thank Dr. Yang Chai for providing the K14-Smad 2 mice. Methods within this study followed the guidelines of the Animal Care Committee of The University of British Columbia. To identify the K14-Smad2 transgene a PCR primer set was used to detect the cytokeratin 14 promoter region. Three age time points were selected (3,6, and 12 months) to analyze the K14-Smad2 mice and compared with their age-matched wild type counterparts as a control group. The total sample size was 40 mice divided into groups, with 5 mice in each of the groups analyzed. G*power software (Heinrich-Heine-University, Düsseldorf, Germany) was used to calculate the sample size as it gave a power of 0.9 for all groups in each methodology.  4.3.2 Photographs Mice hemi maxilla were defleshed using 2%KOH then the samples were stained with Van Gieson’s solution for 30 seconds followed by Ponceau S solution for 5 minutes. The combination of Van Gieson’s and Ponceau S solutions allowed for identification of collagen that stains the roots and outlines the cemento-enamel junction (Ghannad et al., 2008; Leach, 1946). To record the staining of the tissue samples the specimens were mounted and photographed (Canon REBEL T2i, Macro lens 100mm, with ring flash, Japan) (Barczyk, Olsen, & da Franca, 2009).   54 4.3.3 Micro CT Analysis  Mice Hemi maxillae from 6 and 12 months of age K14-Smad2 and WT mice were scanned for their entire length at 45 kV, for 12.5 min (VivaCT, Scanco, Brüttisellen, Switzerland). The settings of the Micro CT were 55 kvp and 140uA. Calibration was done with a phantom. The nominal pixel size was 10um. The frame matrix was 4096 x4096 pixels. Exposure was 316 ms. 3D images were reconstructed and Micro CT analysis performed to quantify the height of alveolar bone, bone density, and bone volume (Um, Jung, Kim, & Bak, 2010; Wilensky, Gabet, Yumoto, Houri-Haddad, & Shapira, 2005). 4.3.4 Histology and Immunohistochemistry 4.3.4.1 Decalcification and Paraffin Embedding of the Samples Hemisection of mice maxillae were collected using a dissecting microscope. The samples were decalcified with Ethylenediaminetetraacetic acid (EDTA) at room temperature for a period of from 4 to 6 weeks that was governed by the thickness of the samples. The samples were embedded in paraffin blocks. The specimens were sectioned at 7-mm thickness, sections placed on slides, then deparaffinized and rehydrated.  4.3.4.2 Hematoxylin and Eosin Harris’s hematoxylin (2.5 minutes) was used to stain the slides. The slides were washed extensively with tap water for 20 minutes and then stained with eosin for 40 seconds. Dehydration and clearing of the slides were done by a series of graded ethanol then xylene. A cover slip was placed on the slides with the use of a mounting media. The slides were viewed by light microscopy (Wazen, Moffatt, Zalzal, Yamada, & Nanci, 2009).   55 4.3.5 Immunohistochemistry Prior to the application of the primary antibody, the sections were blocked with goat 2% serum blocking solution for 30 minutes at room temperature. The primary TRAP antibody (1:100, Goat polyclonal, Santa Cruz Biotechnology Inc, CA, USA) was incubated with slides for 2 hours at room temperature. The slides were rinsed with PBS for 5 minutes three times. The slides were incubated with the secondary antibody, rabbit-anti mouse (1:100, Rabbit polyclonal, Invitrogen Corporation, CA, USA). Slides were rinsed with PBS for 5 minutes three times. The slides were incubated for 10 minutes in streptavidin (50mL) for enzyme conjugation (HRP- streptavidin Invitrogen corporation, CA, USA). Slides were washed 3 times in PBS for 2 minutes. The slides were incubated with AEC Chromogen (100mL) for 10 minutes (AEC Chromogen, Invitrogen Corporation, CA, USA). The slides were rinsed well in distilled water for 10 minutes. The slides were counter-stained with hematoxylin for 60 seconds. The slides were placed under running tap water for 20 minutes then covered with a cover slip with a mounting medium. The slides were viewed under light microscopy (Garcia et al., 2011).  4.3.6 Real-time PCR Two sets of samples were collected. The first set represented the buccal and palatal attached gingiva of the first and second molar teeth that were collected from 5 K14-Smad2 and wild type mice. The second set of samples were bone hemi-sections of mouse maxillae from the 1st and 2nd molar regions. The total sample size for the second set was 10 mice representing 5 mice in each group.  The bone samples were placed in a Spex 6700 bone-milling machine. The Spex 6700 was filled with liquid nitrogen then the bone samples were milled for 5 minutes or until the entire sample produced a   56 homogenous bone powder. The tissues from both the first and second sets of samples were homogenized using a mortar and pestle, and the RNA was purified using RNeasy Mini Kit (Qiagen). A spectrophotometer was used to measure the total RNA, and only good quality RNA was used as the A260/A280 ratios for all RNA samples were above 2.0. Equal amounts of RNA from each sample were used for cDNA synthesis using Iscript Kit (Bio-Rad). Nucleotide sequences for the PCR primers were obtained from the National Center for Biomedical Information for Tnf-α , Il1-β,  Ifγ, Rankl, and Opg (Table 3). PCR amplification was done by the addition of a mixture containing forward and reverse primers and SsoFast EvaGreen Supermix PCR master mixture (Bio-Rad Laboratories, California, USA) to cDNA samples. Then samples were denatured for 5 minutes at 94C. The next step was annealing for 60 seconds at 60 C then DNA synthesis for 60 seconds at 60C. The data were normalized against Gapdh and calculated by the CT method (Hart et al., 2004). Table 3: Primer Sequences for Tnf-α  , Il1-β ,   Ifγ , Rankl, Opg and Gapdh  Primer  Forward Reverse Tnf-α   Il1-β  Ifγ   Opg   Rankl   Gapdh  5’-CAGGCGGTGCCTATGTCTC-3’   5’-GAAATGCCATTTGACAGTG-3’   5’-ATGAACGCTACACACTGCATC-3’   5’-ACC CAGAAACTG GTC ATC AGC-3’  5’-CAGCATCGCTCTGTTCCTGTA-3’  5’-GGTCCTCAGTGTAGCCCAAG-3’  5’-CGATCACCCCGAAGTTCAG-3’  5’-CTGGATGCTCTCACTAGGACA-3’  5’-TCTAGGCTTTCAATGACTGTG-3’  5’-CTGCAATACACACACTCATCACT-3’   5’-CTGCGTTTTCATGGAGTCTCA-‘3  5‘-AATGTGTCCGTCGTGGATCT-3’    57 4.3.7 Western Blots Tissue collection from the attached gingiva was done under the dissecting microscope. The tissue samples represent the attached buccal and lingual gingiva from the maxilla of first and second molar of K14-Smad2 transgenic mice and wild type control mice. Homogenizations of tissues were done using a 500-µL NP-40 lysis buffer and 10 µL of the proteinase inhibitor cocktail on ice. Samples were stored on dry ice for 20 minutes then centrifuged at 12000 rpm for 15 minutes at -4C. Bovine serum albumin standard was used to determine protein concentrations. Equal quantities (15 mg) of the protein samples were mixed with 5% SDS-PAGE buffer and electrophoresed for 45 minutes at 160V at room temperature. The proteins were transferred to a membrane (BioRad transblot pure nitrocellulose) at 60V for 2 hours. Membranes were washed with PBS for 10 minutes. Blocking was done by the application of Odyssey (Li-Cor) blocking solution for 1 hour at room temperature. This was followed by application of the primary antibody: TNF-α (Rabbit polyclonal, Abcam Inc, MA, USA) or IL1-β (Rabbit polyclonal, Santa Cruz Biotechnology Inc, CA, USA), used at a concentration of 0.2 µg/ml for 1 hour. The membrane was washed in PBS 5 times for 5 minutes. A secondary antibody, anti-Rabbit IgG, conjugated to a fluorescent dye was incubated with membranes for 1 hour. Membranes were washed in PBS 5 times for 5 minutes. The membranes were transferred then scanned using a Licor Odyssey scanner (Lincoln, NE, USA). The bands’ intensities were quantified using the Image tool program (Lohinai et al., 2001). 4.3.8 Statistical Analysis The data were interpreted using One-way ANOVA and Student’s t test.   58 4.4 Results 4.4.1 Smad2 Overexpression Results in Severe Alveolar Bone Loss  The overexpression of Smad2 resulted in severe bone loss. As shown in Figure 11 µCT linear bone measurement showed that samples from 12 months old K14-Smad2 had 52.5% (± 4.2) root exposed compared to 32.4%(±3.2) for the WT mice. At 6 months of age there was no statistically significant difference between K14-Smad2 and WT mice with 22.4% (±3.6) and 20.2%(±3.6) bone loss respectively (Fig. 11). The total sample size for the linear bone measurements was 20 mice representing 5 mice in each group.    59  Figure 11: Smad2 Overexpression Induces Chronic Alveolar Bone Loss (A) Photograph of 6 months WT mice with mild loss. (B) Photograph of 6 months K14-Smad2 mice with mild loss. (C) A representative photograph of a hemi maxilla stained with Van Gieson’s and Ponceau S solutions of WT 12 month mice with mild bone loss. (D) Photographs of hemi maxilla stained with Van Gieson’s and Ponceau S solutions of K14-Smad2 12 month mice with severe bone loss. (E) µCT image of 6 months WT mice with mild bone loss. (F) µCT image of 6 months K14-Smad2 mice with mild bone loss. (G) µCT representative image of 12 months WT mice with mild bone loss. (H) µCT image of 12 months K14-Smad2 mice with severe bone loss. (I) Quantification of the linear bone loss (exposed roots %) of 6 months and 12 months time points of both K14-Smad2 and WT mice*(P<0.05). Error bars represent the standard deviations  The 1st and 2nd maxillary molars were digitally extracted using µCT viewer program to remove the effect of teeth on bone measurements. The images of bone   60 without teeth were interpreted for bone volume and density. The bone volume measurements at the 1st and 2nd maxillary molars revealed that there was a significant difference between 12-month-old K14-Smad2 and WT mice with 2.65 mm3 (±0.3) and 4.3mm3 (±0.35) bone volume respectively. There was a statistically significant difference in the bone density at 12 months of age with K14-Smad2 696.8 mg/cc (±70) and WT 845.9 mg/cc (±10) (Fig. 12).       61 Figure 12: Smad2 Overexpression Reduces Both Bone Volume and Bone Density (A) Representative µCT image showing the alveolar bone of 12 months WT mice without the 1st and 2nd molars. (B) An µCT image showing the alveolar bone of 12 months K14-Smad2 mice without the 1st and 2nd molars. (C) Quantification of bone volume of K14-Smad2 and WT 12 month mice *(P<0.05). (D) Quantification of bone density of both K14-Smad2 and WT 12 month mice *(P<0.05). Error bars represent the standard deviations  4.4.2 Increased SMAD2 Up-regulates Tnf-α and Rankl  Smad2 overexpression increased the mRNA levels of 6 months K14-Smad2 Tnf-α to 3.26(±0.6)-fold when compared to WT mice. Also, SMAD2 increased the protein level (2.8 ±0.7-fold increase) of TNF-α in 6 months K14-Smad2 when compared to their WT controls. However there were no statistical differences with Il1-β and Ifγ mRNA levels in either group. The overexpression of Smad2 up-regulated 6 months K14-Smad2 Rankl levels to a 2.5(±1.2)-fold increase when compared with WT mice. There were no differences in the levels of expression of Opg between WT and K14-Smad2 mice (Fig. 13).    62   Figure 13: Smad2 Overexpression Up-regulates Tnf-α and Rankl (A) Real-time PCR expression of Tnf-α  , Il-1β, and Ifγ representing the 6 months time point of WT and K14-Smad2. *(P<0.005). (B) Real-time PCR expression of Opg, and Rankl representing the 6 months WT and K14-Smad2. *(P<0.005). (C) Western blots of K14-Smad2 and WT mice representing TNF-α and GAPDH. (D) Quantification of the TNF-α western blot results *(P<0.005).  4.4.3 The Overexpression of Smad2 Increased the Number of Osteoclasts  TRAP positive cells were selected to represent osteoclasts. The numbers of osteoclasts (TRAP positive cells) were counted over a surface area of 1mm2. An area from the base of the JE cells until mid-alveolar bone was included in the evaluation. Both the buccal and lingual alveolar bones were evaluated for TRAP positive cells. The mean TRAP positive cells were calculated from 5 mice in each group. A total of 3 slides (12 sections per slide) per mouse were included to represent the sample size. Smad2   63 overexpression increased the numbers of osteoclasts in 6 months K14-Smad2 to 3.4 (±02) fold when compared to WT mice (Fig. 14).   Figure 14: Smad2 Overexpression Increased the Number of Osteoclasts in K14-Smad2 Mice TRAP positive cells (brown stain and red arrows) were counted and the mean cell counts were calculated over a surface area if 1mm2. (A) TRAP stain image of WT mice showing no osteoclasts. (B) TRAP stain image of K14-Smad2 mice showing multiple osteoclasts on the buccal alveolar bone. Original magnification 600X. (C) Quantification of the TRAP stain for both WT and K14-Smad2 mice *(P<0.005). Error bars represent the standard deviations  4.5 Discussion An increase in of TGF-β in JE cells has been shown to be correlated with periodontal disease (Steinsvoll, Halstensen, & Schenck, 1999). TGF-β has to bind to its receptors to start the process that results in the phosphorylation of the intracellular transcription factor SMAD2 (Nakao et al., 1997). Phosphorylated SMAD2 combined with SMAD4 enters the nucleus with subsequent alteration in the patterns of gene   64 expression process, which ultimately results in changes in multiple cellular functions (Massagué & Chen, 2000). Interestingly, the expression of TGF-β receptors in JE cells has been shown to be different in health and disease (Lu, Mackenzie, & Levine, 1997). Therefore to study the impact of the overexpression of the TGF-β signalling pathway the focus should move from the cell surface receptors to the intracellular levels of the phosphorylated SMAD2 transcription factor (Alotaibi et al., 2014; Fujita et al., 2012; Ito et al., 2001). A positive advantage of the K14-Smad2 mouse model is that the overexpression of Smad2 and subsequent phosphorylation of SMAD2 is independent of the presence or absence of TGF-β receptors and TGF-β growth factors. Thus, SMAD2 intracellular signalling is independent of both the growth factors and the cell surface receptors (Ito et al., 2001). Another important point is that due to the K14 promoter the overexpression of Smad2 only occurs in tissues of ectodermal origin. Smad2 is only overexpressed in epithelial cells and not in bone, connective tissue, and PDL cells (Ito et al., 2001). Thus, the K14-Smad2 mouse model is more specific to study the role of Smad2 overexpression in JE cells and what effect that might have on alveolar bone loss without trying to manipulate TGFβ ligand and receptors. It has been reported that there is a genetic component in the etiology that can be attributed to 50% of periodontal disease cases (Michalowicz et al., 2000). Indeed, gene mutations (Hart et al., 1999), the knockout of a single gene (Ghannad et al., 2008) and/or the overexpression of another gene (Dayan, Stashenko, Niederman, & Kupper, 2004) have been shown to cause severe periodontal disease. It is interesting that gingivitis may stay stable for long periods of time and might not progress to periodontitis. The progression of gingivitis to periodontitis is controlled by multiple factors such as the   65 presence of invasive microorganisms, a susceptible host, and a conducive environment that aids periodontal disease progression (Genco, 1996). The findings of the current study support the premise that focal changes in gene expression can alter the host response to periodontal disease and increase the susceptibility to periodontal disease progression (Genco,1992). It is clearly presented in the current study that Smad2 overexpression in JE cells resulted in severe periodontal disease when compared to WT mice. It is interesting for future studies to further investigate different levels of gene expression and the magnitude of periodontal disease, which might help clinicians to accurately classify and treat periodontal disease. It is well documented that the JE has a protective role in that it secretes defence molecules and has barrier ability (Bosshardt & Lang, 2005). Our lab has published data highlighting the effect of Smad2 overexpression on JE cells. The overexpression of Smad2 increased the apoptotic rate of JE cells by down-regulating Bcl2 (Fujita et al., 2012). Other studies have shown that Smad2 overexpression reduced the JE cell’s proliferation rate by down-regulating c-MYC the repressor of cyclin-dependent kinases P15 and P21 (Alotaibi et al., 2014). The combined effect of increased apoptosis and reduced proliferation rate of the JE cells in K14-Smad2 resulted in a decrease in the amount of JE in K14-Smad2 mice when compared with their WT counterparts (Alotaibi et al., 2014). The current study advances the knowledge of those original findings, as it shows that Smad2 overexpression in JE cells induces alveolar bone resorption by up-regulation of inflammatory cytokines associated with bone loss. Bone resorption is considered a hallmark in periodontal disease that eventually causes the loss of the bony support for teeth and consequently leads to tooth loss   66 (Armitage, 1999). The current study shows clearly that the overexpression of Smad2 in JE cells causes severe bone loss. To overcome the limitation of photographic assessment of bone loss, µCT radiographs were performed to accurately analyze the mineralized tissue to detect the level of alveolar bone and relate it to the length of the roots. In other studies, µCT radiographs provide data about bone density and bone volume characteristics that are also affected by periodontal disease (Um et al., 2010; Wilensky et al., 2005). The results of the current study show that the overexpression of Smad2 in JE cells resulted in severe bone loss in K14-Smad2 mice when evaluating the crown:root ratio levels. Occlusal wear should be considered when evaluating the levels of the crown:root ratio. With occlusal wear, roots might over-erupt giving undesirable crown root ratio (Steedle & Proffit, 1985). Another point to consider is cementum deposition, which might change the results of the study by increasing the vertical dimensions of roots. It has been shown that cementum deposition is increased in occlusal overload (hypercementosis) (Comuzzie & Steele, 1989). However this is not the case in the current study, as there was no statistical difference in root length between K14-Smad2 and WT mice when measured by µCT from the CEJ to the total root length, which include dentine and cementum (data not presented). The literature linking bone density and periodontal disease presents conflicting results (Genco & Löe, 1993). Some studies concluded that bone density did not have an effect on periodontal disease progression (Ward & Manson, 1973). While others show the opposite concluding that lower bone density could enhance bone loss and increase the progression of periodontal disease (Esfahanian, Shamami, & Shamami, 2012). Bone density and volume have been considered as risk modifiers of periodontal bone loss, as   67 subjects with lower bone density had more bone loss when compared to healthy individuals (Tezal et al., 2000). Increased inflammatory cytokines such as TNF-α are linked to periodontal disease and other diseases associated with lower bone density such as osteoporosis (Manolagas & Jilka, 1995). The current investigation agrees with these findings as K14-Smad2 mice had a higher TNF-α level combined with lower density and volume of bone when compared to the WT mice. To investigate the mechanism of Smad2 overexpression and alveolar bone loss we examined molecules that are involved in bone metabolism such as TNF-α, IL1-β, IFγ, RANKL, and OPG (Koide et al., 2013; T. A. Silva et al., 2007). The current study showed an increase in TNF-α, a potent inflammatory cytokine that has the ability to induce alveolar bone loss both in vivo and in vitro (Abu-Amer, Ross, Edwards, & Teitelbaum, 1997). Bone loss in the current study can be explained by the up-regulation of the inflammatory cytokine TNF-α, which has been shown to increase the number of osteoclasts by up-regulating RANKL, which is consistent with the observations in our study. RANKL will bind to the RANK receptors of the osteoclast precursors leading to an activation of the Ikb kinase and ultimately more osteoclasts will be formed (D. Drugarin et al., 2003; T. A. Silva et al., 2007). With more osteoclasts formed the balance of bone resorption and bone deposition will be directed toward resorption leading to periodontal bone loss (Graves, Li, & Cochran, 2011). From the results above we can conclude that Smad2 overexpression induces alveolar bone loss by up-regulating TNF-α and RANKL, leading to increased numbers of osteoclast and osteoclastic activity.   68 Chapter 5: General Discussion, Conclusions, and Future Directions 5.1 General Discussion 5.1.1 JE to Maintain Health  The JE maintains health for the periodontium, as it secretes protective molecules and growth factors, and aids with the guidance of inflammatory cells to bacterial insult (Tonetti, Imboden, & Lang, 2012; Ye, Chapple, Kumar, & Hunter, 2000). Any alteration to the JE integrity will reduce the protection of the underlying periodontal tissues as seen in our results. The activation of SMAD2 during periodontal disease was reported in the literature, which indicated that Aggregatibacter actinomycetemcomitans had the ability to activate phosphorylation of SMAD2 during periodontal disease. This linkage of bacterial insults and TGF-β signalling in periodontal disease progression provides a clear foundation for our studies (Yoshimoto et al., 2014). We demonstrated that the overexpression of Smad2 in JE cells resulted in severe periodontal bone loss that had a pattern of chronic progression. Our results showed that Smad2 overexpression resulted in a reduction of the JE surface area at 12 months of age (Alotaibi, Kitase, & Shuler, 2014). The reduction of the JE surface area was attributed to an increase in the apoptotic ratio combined with a reduction of the proliferation rate of JE cells (Alotaibi et al., 2014; Fujita et al., 2012). Our results showed that Smad2 overexpression in JE cells reduced the anti-apoptotic molecule BCL2, which contributed to JE cells’ increased apoptosis (Fujita et al., 2012). Smad2 overexpression resulted in the increase of cyclin-dependent kinase inhibitors P21 and P15 by down-regulating their repressor, c-MYC (Alotaibi et al., 2014). The reduced JE surface area has a clinical significance because after supportive periodontal therapy or surgical procedures JE heals as a long JE that has been shown to   69 be stable over time (Beaumont, O'Leary, & Kafrawy, 1984; Yukna, 1976). Thus, the reduced JE surface area might not be stable over time and could aid in the progression of periodontal disease.  5.1.2 Recombinant TGFβ1 Mediated Periodontal Regeneration  Our results could explain the different results in the literature regarding TGFβ1 effects on periodontal regeneration. Some data in the literature show positive results with TGFβ1 mediated regeneration while others show negative or no results in relation to regeneration with the TGFβ1 (Lind et al., 1996; Wikesjö et al., 1998). A shortcoming of all of those studies was that they focused on the TGFβ ligand-receptor interaction, as they increased the levels of TGFβ1 without investigating whether the TGFβ1 intracellular signalling pathway was initiated or not (Lind et al., 1996; Wikesjö et al., 1998). It has been reported that the receptors of TGFβ1 differ between health and disease (Lu, Mackenzie, & Levine, 1997; Ye et al., 2003), and for that reason alone it is understandable that the literature reports conflicting results. A main advantage of using the K14-Smad2 mouse model was the ability to focus on the intracellular signalling transcription factor Smad2 irrespective of the presence or absence of TGFβ1 ligand or receptors (Ito et al., 2001). The constitutive phosphorylation of SMAD2 made it possible to examine the downstream effects of TGFβ1 signalling (Fujita et al., 2012). It is interesting to note that although Smad2 overexpression induced severe bone loss in the mice in our studies, other studies have reported that inhibiting TGFβ1 also resulted in severe bone loss, which gives an indication that there is likely an equilibrium state of activation of TGFβ1 signalling that is required in maintaining the health of the   70 periodontium.  5.1.3 Smad2 and Bone Loss  Periodontal disease is an inflammatory disease associated with increased numbers of inflammatory cells, inflammatory cytokines, and increased osteoclastic activity (Sterrett, 1986). Our results showed that the overexpression of Smad2 up-regulated TNF-α, a potent inflammatory cytokine that is involved in periodontal disease progression. Smad2 overexpression in JE cells also up-regulated RANKL, a protein involved in osteoclastogenesis. When RANKL binds to RANK on the osteoclast precursor cells the intracellular nuclear kappa B will be released to alter gene expression resulting in more osteoclast formation (T. A. Silva, Garlet, Fukada, Silva, & Cunha, 2007). Our results clearly show that SMAD2 up-regulates TNF-α and RANKL, and that an increased number of osteoclasts were found in the K14-Smad2 mice. Periodontal disease progress can be characterized as either chronic or aggressive (Smith, Seymour, & Cullinan, 2010). The difference between the two models of disease progression is the amount of destruction within a period of time (Smith et al., 2010). With aggressive periodontitis periodontal tissues are broken down in a short period of time (Lang et al., 1999). Chronic periodontitis represents periodontal tissue destruction over a longer period and according to the literature the tissue destruction follows either a linear pattern of destruction or a random burst pattern of tissue destruction (Socransky, Haffajee, Goodson, & Lindhe, 1984). Our results show that the bone destruction in the K14-Smad2 mouse model has a chronic pattern of disease, as the bone destruction was significant only at the 12 months of age time point. In other words, the continuous Smad2 overexpression in JE releases TNF-α over time leading to a chronic bone destruction   71 pattern that is detected at 12 months of age.   5.2 Conclusion The conclusions generated by the research in this dissertation (Fig. 15) are as follows:  1- Smad2 overexpression increased the apoptosis rate in JE cells by inhibiting BCL2, the anti-apoptotic molecule. 2- The overexpression of Smad2 in JE cells reduced the proliferation rate of the JE cells by down-regulating c-MYC, the repressor of the cyclin-dependent kinase inhibitor. 3-  Smad2 overexpression up-regulated the cyclin-dependent kinase inhibitors P21 and P15.  4- The combined effect of increased apoptosis and reduced proliferation rate induced by Smad2 overexpression resulted in a reduction of the surface area of the JE. 5- Smad2 overexpression in JE cells up-regulated TNF-α and RANKL. 6- The overexpression of Smad2 increased the numbers of osteoclasts. 7- The overexpression of Smad2 in JE cells resulted in severe alveolar bone loss that follows a chronic pattern of destruction.   72    Figure 15: Smad2 Overexpression and the Progression of Periodontal Disease (A) Smad2 overexpression in JE cells (purple color) reduced the surface area of the JE by: (1) increasing the apoptosis rate in JE cells (by inhibiting BCL2, the anti-apoptotic molecule); (2) reducing the proliferation rate of the JE cells (SMAD2 down-regulated c-MYC, the repressor of the cyclin-dependent kinase inhibitor, which resulted in the up-regulation of cyclin-dependent kinase inhibitors P21 and P15). (B) Smad2 overexpression in JE cells resulted in severe alveolar bone (pink and orange colors) loss by: (1) up-regulating TNF-α and RANKL; and (2) increasing the numbers of osteoclasts.  5.3 Future Directions  1- We have shown clearly that Smad2 overexpression upregulates TNF-α. It would be interesting for future studies to show the actual mechanism by which Smad2 activates TNF-α. Future studies should detect TNF-α by immunohistochemistry   73 and in situ hybridization to detect which cells secrete TNF-α. A recommended pathway for the activation of TNF-α is P38 MAPK as some studies have shown that TGFβ can active TNF-α through this pathway. 2- Detecting the level of SMAD2 that causes disease is an important factor that should be examined in the future. Our study shows clearly that the overexpression of Smad2 causes severe periodontal disease in mice. Interestingly, inhibiting TGFβ1 resulted in severe periodontal disease in mice. Future studies should focus on examining tissues from healthy patients and patients with severe periodontal disease to determine the levels of SMAD2, TGFβ1 and RANKL by immunohistochemistry and Western blots, as it would give an indication about which levels are linked to periodontal disease.  3- The K14-Smad2 mice displayed an up-regulation of RANKL, which resulted in more osteoclasts. Theoretically in clinical situations Smad2 overexpression should not respond to supportive periodontal therapy or surgery because the cause of periodontal disease in this situation is an overexpression of a gene that results in more osteoclasts. It would be interesting for a therapeutic purpose to inhibit the function of osteoclast with osteoclast inhibitors such as cathepsin K inhibitor. However, this approach should be done in mice, as the cathepsin K inhibitors are still under investigation for their clinical safety in humans.   74 References  Abu-Amer, Y., Ross, F. P., Edwards, J., & Teitelbaum, S. 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