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Importance of alpha-catenin and cell-cell interactions in hair follicle stem cells homeostasis
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Importance of alpha-catenin and cell-cell interactions in hair follicle stem cells homeostasis
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IMPORTANCE OF ALPHA-CATENIN AND CELL-CELL INTERACTIONS IN HAIR FOLLICLE STEM CELLS HOMEOSTASIS by Surbhi A Thesis Presented to the FACULTY OF THE USC VITERBI SCHOOL OF ENGINEERING UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (BIOMEDICAL ENGINEERING) December 2010 Copyright 2010 Surbhi ii __________ Dedication __________ To my mother, father, sister, brother and friends. iii _________________ Acknowledgements _________________ I would like to thank my advisor, Dr. Agnieszka Kobielak, for her guidance and support during my research and study at University of Southern California. I have learned far more beyond the scope of the research project under her supervision. She has been a constant source of motivation and inspiration. Her accessibility and willingness to help her students with their research made the experience very rewarding. Dr. David Z. D‘Argenio and Dr. Zong-Lin Lu deserve a special thanks as my thesis committee members and advisors. In particular, I would like to thank Mischalgrace Diasanta, my graduate advisor, whose timely guidance and assistance de-stressed the process of choosing the right study plan. Many thanks to the members of the Kobielak lab, past and present, especially to Hongjun Zhang, Joseph Kwack and Christine Cao. Deepest gratitude goes to my family and friends for their constant support through the ups and downs in life, and for their unflagging and unconditional love. I cannot be more grateful to my parents who worked industriously to make my life better. I will always remember the caring thoughts of my loving younger sister and brother, who have been my best friends and my stress-busters. I am blessed to have such an amazing family. I would also like to thank all my roommates over the years for their well-cooked food, iv endless chats and motivation. You guys never made me miss home and are my second family. Last but not the least, thanks be to God, for when no one was by my side, he held my hand and walked me though the darkest times. v ________________ Table of Contents ________________ Dedication ii Acknowledgements iii List of Figures vii List of Tables x Abstract xi Chapter 1: Introduction 1 1. Skin 1 2. Hair and Hair Follicles 4 3. Hair Cycle 7 a. Anagen 8 b. Anagen-to-Catagen Transition 8 c. Catagen 8 d. Telogen 9 e. Exogen 9 4 Molecular Mechanisms of Hair Follicle Cycling 9 a. Wnt/ Beta-catenin Pathway 10 b. Shh Signaling 12 c. Bmp Signaling 12 5. Epithelial Stem Cells 14 6. Models of Hair Follicle Stem Cell Activation 17 Chapter 2: Adherence Junctions 19 1. Adherence Junctions 19 2. E-cadherin 21 3. Catenins 22 a. p120 catenin 24 b. Beta-catenin 25 c. Alpha-catenin 26 4. Adherence Junctions and Stem Cell Niche Maintenance 27 Chapter 3: Alpha-catenin 30 1. Alpha-catenin: The Known and the Unknown 30 vi 2. Alpha-catenin and its Role in Tumorigenesis 36 a. Cancer and Cadherin/Catenin Complex 36 b. Consequences of Loss of Alpha-catenin 40 Chapter 4: Methods 42 1. Generation of Transgenic Mice 42 2. Lineage Analysis in vivo (Treatment of Mice with RU486) 43 3. Sampling and Staining 44 4. Fluorescent Imaging 45 5. Keratinocyte Isolation by FACS Analysis 45 Chapter 5: Results & Discussion 46 1. Five Weeks After Treatment 47 2. Five Months After Treatment 49 3. Ten Months After Treatment 56 Chapter 6: Conclusion & Future Directions 63 References 65 Bibliography 72 Appendices: Appendix A 78 Appendix B 79 Annexure C 81 Appendix D 82 Appendix E 85 Appendix F 86 Appendix G 88 Appendix H 89 vii _____________ List of Figures _____________ Figure 1.1: Anatomy of the Skin 1 Figure 1.2: Schematic Drawing Illustrating the Concentric Layers of the Outer Root Sheath (ORS), Inner Root Sheath (IRS) and Shaft in the Bulb 4 Figure 1.3: Development and Cycling of Hair Follicles 6 Figure 1.4: The Hair Cycle 7 Figure 1.5: During Follicle Morphogenesis and Cycling, Skin Stem Cells Change Cadherin Expression in a Fashion Dependent upon Noggin and Wnt Signaling 11 Figure 1.6: Summary of the Signaling Pathways Involved in Hair Follicle Morphogenesis and Regeneration 13 Figure 1.7: Schematic Illustration of the Menagerie of Stem Cells and Their Individual Locations in the Resting (Telogen) Adult Hair Follicle 14 Figure 1.8: Current Model of SC Migration During Skin Homeostasis and Repair 18 Figure 2.1: E-cadherin. 21 Figure 2.2: The Catenin Family at a Glance 23 Figure 2.3: p120-catenin 24 Figure 2.4: Beta–catenin 25 Fig. 2.5: Similar and Distinct Functions of Cadherin–Catenin Proteins in Skin Epidermis 27 Figure 2.6: Multiple Functional Roles of Cell–Cell and Cell–ECM Adhesion in SC Niches 29 viii Figure 3.1: Primary Structure of Alpha–catenin 31 Figure 3.2: Quantification of Fluorescence Recovery after Photobleaching (FRAP) 32 Figure 3.3: Protein–Protein Interactions Between Cadherins, Catenins and the Actin Cytoskeleton 33 Figure 3.4: A Model for Regulation of Cytoskeleton and Membrane Dynamics by the Cadherin–Catenin Complex 34 Figure 3.5: Models of How the Cadherin-Catenin Complex is Linked to and Regulates the Actin Cytoskeleton 35 Figure 3.6: Metastasis is a Multistep Process 37 Figure 4.1: K15-crePR Mice are Mated with Alpha-catenin flox/flox Mice to Obtain Mice with the Ability to Remove Alpha-catenin flox/flox Allele upon Activation of K15-crePR 43 Figure 4.2: Timeline of Treatment of Mice upon the Onset of Second Postnatal Telogen by Steriod RU486 43 Figure 4.3: The Figure Depicts Hair Follicle of Knockout Mice Where the Ablation of Alpha-catenin Takes Place Upon RU486 Treatment 44 Figure 5.1: No Apparent Morphological Change Observed in the KO and WT Mice 5 Weeks After RU 486 Treatment. 48 Figure 5.2: Loss of Hair Shaft But Not Hair Bulb in the KO Mice 5 Months After RU486 Treatment 49 Figure 5.3: Increased Proliferation and Loss of Alpha-catenin in Mosaic Pattern 5 Months After RU 486 Treatment 51 Figure 5.4: No Observable Apoptosis 5 Months After RU 486 Treatment 52 Figure 5.5: No Difference in the Number of Stem Cells Obtained From the KO and WT 54 Figure 5.6: Hair Follicle Differentiating Pathways are Not Affected by Alpha- catenin Ablation, as Observed 5 Months After RU486 Treatment 56 ix Figure 5.7: Loss of Hair Follicle Morphology as a Result of High Proliferation Observed in KO Mice 58 Figure 5.8: Stem Cell Marker Still Expressed in the Regions With Decreased Alpha-catenin Activity in KO Mice 60 Figure 5.9: Lack of Differentiation in Rapidly Proliferating Cells in the KO Mice 62 x _____________ List of Tables _____________ Table 3-1. Alpha-catenin as a Prognostic Marker in Human Cancers 38 Table D-1. PCR Reaction Components and Cycle Information 83 Table D-2. Cre Primers 83 Table D-3. PCR Reaction Components and Cycle Information 84 Table D-4. Lcat-fx Primers 84 xi ________ Abstract ________ In multi-cellular organisms, cell–cell contacts that are mediated by classical cadherins have essential roles in many fundamental processes, such as morphogenesis, maintenance of tissue integrity, wound healing and cell polarity. Furthermore, there is overwhelming evidence that the adherens junctions (AJs) are also an important tumor and/or invasion suppressor. Alpha-catenin is the protein that connects E-cadherin–beta-catenin complexes with the actin cytoskeleton. Although it was previously considered to be a solely structural protein, it has become increasingly clear that alpha-catenin has a central role in both assembling the actin cytoskeleton and regulating its dynamics at cell–cell junctions thus regulating cell polarity. Cell-polarity mechanisms are responsible not only for the diversification of cell shapes but also for regulation of the asymmetric cell divisions of stem cells that are crucial for their correct self-renewal and differentiation. Disruption of cell polarity is a hallmark of cancer. Although null mutations in alpha-E-catenin have been associated with epithelial cancers it has been usually assumed that perturbations in cell–cell adhesion are late rather than early steps in carcinogenesis, and that they are preceded by mutations in cell-cycle-regulated genes that lead to uncontrolled growth. In our previous experiments we have shown that loss of alpha-catenin in the whole xii epidermis during embryonic development results in formation of squamous cell carcinoma. To understand the origin of squamous cell carcinoma as well as focus on the role of cell adhesion and polarity on the maintenance of quiescence stem cell niche and transition toward tumor development, we disrupted in inducible manner the alpha-catenin gene specifically in the hair follicle stem cells using alpha-catenin flox/flox and Keratin15-CrePR mice. The results highlighted that alpha-catenin ablation leads to loss structural tissue integrity in hair follicles, leading to tumor-like cellular populations, which are highly proliferative but lack differentiation. This suggests that knockout of alpha-catenin might lead to asymmetric division in stem cells. This indeed proves that alpha-catenin has an important role to play in the maintenance of adherence junctions. The findings also signify the use of alpha-catenin as a prognostic marker for squamous cell carcinomas. 1 __________ Chapter 1 __________ INTRODUCTION 1.1 Skin Figure 1.1. Anatomy of the skin. [1] Skin is the largest human organ. It is the outer body covering which not only gives us shape and appearance but is also the first line of defense. In addition, the skin 2 also plays other important roles in maintaining body temperature and regulating excessive loss of water. Skin has a number of nerve endings reacting to various sensations like heat, cold, touch etc. The process of formation of skin in mouse starts around day nine of the embryonic stage (E9). The protective covering to seal the body from the external environment is completed day before the animal is born (E18) [2]. There are three primary skin layers, namely, epidermis, dermis and hypodermis (Figure 1.1). Epidermis is the outermost layer of the skin made of stratified squamous epithelium sitting on basal lamina. There are no blood vessels nourishing the cells in the epidermis but diffusion from the capillaries provides the nourishment. Merkel cells, keratinocytes, melanocytes and Langerhans cells are the main type of cells found here. Epidermis is divided into 5 layers viz. Stratum corneum, stratum lucid, stratum granulosum, stratum spinosum and stratum basal, from superficial to deep [3]. 25 to 30 layers of dead cells can be found in the outermost layer [4,5]. The layers of epidermis are formed through the differentiation and keratinization of the cells of the innermost layer. As actively dividing keratinocytes present in the basal layer, withdraw from the cell cycle and undergo terminal differentiation, they remain transcriptionally active and move upward towards the skin. To protect themselves the cytoplasm of these cells is replaced by protein keratin, which provides them with robust strength. Next they produce a barrier by synthesizing and depositing proteins like involucrin, loricrin, SPRR etc. under the plasma membrane. When they reach the granular layer, lipids are packed into lamellar granules, which bunch with keratin to add further strength, and when they reach the corneum, the cells loose nuclei and organelles and are desquamated making them 3 metabolically inert. The keratinized layer helps in regulating water, keeping out pathogens and in protecting from environmental impacts. Differentiating cells that are continuously moving upward in every few weeks regenerates epidermis. Epidermis has the remarkable ability to heal, expand or retract in response to environmental cues [2]. The next layer of skin is the dermis. Dermis consists of connective tissue and is tightly connected to the epidermis through basement membrane. Dermis has many nerve endings and also contains hair follicles, sweat glands, sebaceous glands (SG), apocrine glands, lymphatic vessels and blood vessels. The vascular system in the dermis is responsible for the nourishment and waste removal. The dermis is divided into two regions: papillary region and the reticular region. The papillary region of the dermis bears a number of finger-like projections called papillae, which strengthen the connection between dermis and epidermis. The reticular region consists of irregular connective tissue, which is densely packed. Roots of the hair, sebaceous glands, sweat glands, receptors, nails and blood vessels are found in this region as well. Hypodermis, also known as subcutaneous tissue, is not actually a part of skin. It lies below the dermis and attaches the above layers to the bone and muscle. It contains number of nerves, blood vessels and connective tissue. Main cells found here are fibroblasts, microphages and adipocytes. 50% of the body fat is contained in the hypodermis for insulation and padding of the body [3]. 4 1.2 Hair and Hair Follicles Figure 1.2. Schematic drawing illustrating the concentric layers of the outer root sheath (ORS), inner root sheath (IRS) and shaft in the bulb. The inner root sheath is composed of four layers: Companion layer (CL), Henle‘s layer, Huxley‘s layer, and the inner root sheath cuticle. The companion layer cells are tightly bound to Henle‘s layer, but not to the outer root sheath, thus allowing the companion layer to function as a slippage plane between the stationary outer root sheath and the upwards moving inner root sheath. Further inwards, the inner root sheath cuticle is composed of scales that interlock with the scales of the hair shaft cuticle, anchoring the shaft in the follicle and enabling both layers to jointly move during hair follicle growth. The hair shaft is wrapped by a protective layer of overlapping scales and in mice, but not in humans, shows in its centre regularrows of air spaces believed to play a role in thermal insulation (BM: basal membrane; APM: arrector pili muscle; CTS: connective tissue sheath; DP: dermal papilla; M: matrix; HS: hair shaft, IRS: inner root sheath; ORS: outer root sheath; SG: sebaceous gland) [6] 5 Hairs on the skin have a wide range of functions and are characteristics of mammals. In mouse, hair follicle morphogenesis begins around E13 and progresses in a wave patterns up till birth. Hair follicles produce terminally differentiated keratinocytes, which give rise to the hair shaft. Hair follicle development is a characteristic of fetal and perinatal skin, but is also observed in adult mouse and rabbit skin during de novo wound healing. Studies have also shown that hair follicle formation can be induced in adult human skin. All of the epithelial components of the hair follicles are formed by the ectodermal hair follicle stem cells, activated by specialized dermal cells. As the hair follicle grows down, the inner layers begin to differentiate to form central hair shaft (HS) and the inner root sheath (IRS). The hair shaft is several cell diameters wide and consists of concentric cylinders of cells [6,7]. Concentric layers surrounding the hair shaft form the outer root sheath (ORS) and the inner root sheath (IRS). Inductive mesoderm-derived cells give rise to follicular dermal papilla (DP), which is the permanent part of the follicle base, and the connective tissue. DP is enveloped by hair bulb, which is found at the bottom of the dermis when the follicle is fully mature (Figure 1.2). Keratinocytes are produced as a result of dividing proliferative cells at the base of the follicle. The pigmentary unit of the hair follicle is formed by neural crest-derived melanocyte progenitors [6]. The basal layer and OSR have a number of common biological markers, including Keratin 5, Keratin 14, and 64 integrins. These represent the proliferating compartment of the skin. Mitotically-active cells of SG also express Keratin 5 and Keratin 14. Matrix cells present in the bulb of the follicle and surrounded by DP have the highest proliferation rate. They generate IRS and hair cells (Figure 1.3). Matrix cells express 6 transcriptional factors like Lef1, which are associated in hair follicle differentiation [9,10,11,12]. Figure 1.3. Development and Cycling of Hair Follicles. Selected stages of the morphogenesis of hair follicles and the three stages of follicular cycling (anagen, catagen, and telogen) are shown. The roman numerals indicate morphologic substages of anagen and catagen. The pie chart shows the proportion of time the hair follicle spends in each stage [13]. In postnatal mouse, the ORS of the maturing hair follicles creates a bulge by widening on one end. This specialized area is speculated to contain epithelial stem cells. The adult bulge markers include stem cell CD34 marker and Keratin 15 promoter activity [11,14,15]. 7 1.3 Hair Cycle Hairs are constantly replaced during the lifetime of an animal. To bring about this constant supply, the hair follicles undergo cycles of growth (anagen), apoptotic-mediated regression (catagen) and relative quiescence (telogen) (Figure 1.4). Each hair cycle results in the formation of a new hair shaft and the shedding of the old one. This process, which is actively regulated, is called exogen. Bulge region of the hair follicle are the storehouse of hair-specific epithelial stem cells. Activation of these stem cells leads to the formation of new hair shaft. Figure 1.4. The Hair Cycle [16] 8 1.3.a Anagen In mice, the first growth phase of the hair cycle occurs 4 weeks after birth. During anagen, proliferating matrix cells move upward proliferating intensively and differentiating into the six lineages of IRS and HS [17]. As the cells terminally differentiate, the process of keratinization occurs providing strength and flexibility to the hair shaft. 1.3.b Anagen-to-catagen transition The matrix cells undergo limited divisions before differentiating. This decline in the number of matrix cells and the slow HS and IRS differentiation leads the follicle into the destructive phase called the catagen. The timing of catagen varies slightly among different strains of mice and also significantly varies from one skin region to another [16,17]. 1.3.c Catagen Catagen is the phase of dynamic transition between anagen and telogen. During catagen, matrix, IRS and ORS keratinocytes undergo apoptosis. This leads to rapid regression of the lower two-thirds of the hair follicle. This brings the DP and the bulge close together. The hair follicle stem cells present in the bulge do not undergo apoptosis. The receding lower hair follicle forms a temporary structure known as the epithelial strand, which connects DP and the upper hair follicle. In mice, the old hair shaft stops differentiating and assumes a rounded appearance (club hair) and moves towards the non-cycling upper follicle. By the time DP reaches the club hair, the epithelial strand 9 ceases to exist [17]. 1.3.d Telogen The resting stage of the hair follicle cycling is called telogen in which the follicles lie dormant. For the first telogen, the quiescence phase lasts couple of days, approximately from P19 to P21 where as the second telogen can last more than 2 weeks, beginning around P42. The length of the telogen increases with subsequent cycles. The telogen club hair and the emerging new hair can coexist in the same orifice during several cycles, leading to dense outer coat [17]. 1.3.e Exogen The shedding of the old hair follicle is termed exogen. Exogen is independent of anagen and telogen. The remnant hair follicle can persist even after several hair cycles. This protective mechanism reduces shedding before new fur is available. The shedding usually occurs as a result of a mechanical stimulus [16]. 1.4 Molecular Mechanisms of Hair Follicle Cycling The telogen to anagen transition is brought about by the activation of quiescent stem cells present near DP at the base of the follicle. They undergo rapid proliferation forming new follicles. The mechanism of activation of these epithelial stem cells is obscure, but numbers of studies in the past have determined essential cascades that are crucial for hair cycle stages. 10 The control of hair follicle cycling involves multiple pathways, such as Fibroblast growth factor (Fgf) [18], Transforming growth factor beta (Tgf- ) [19,20,21], Hedgehog (Hh) [22,23,24] and Wnt [25]. 1.4.a Wnt / beta-catenin pathway Wnt signaling pathway is involved in a number of developmental controls both during embryonic and postnatal stages. In the skin, Wnt and beta-catenin play diverse roles in HF morphogenesis, SC maintenance and/ or activation, hair shaft differentiation. Wnt/beta-catenin signaling activation is significant during the HF development whereas its inhibition leads to decline in hair follicles. Studies have revealed that the bulge be deficient in Wnt reporter activity where as the bulge stem cells receive Wnt signals and are necessary for their maintenance. Postnatal studies have revealed that Wnt signaling is involved in hair shaft differentiation [26]. Imbalances in Wnt signaling have been reported to often result in cancers as a result of proliferation and differentiation abnormalities [27]. In the adherence junctions, beta-catenin is completed with E-cadherin and alpha-catenins. The cytoplasmic free beta-catenin is degraded by proteases. Wnt pathway is involved in the stabilization and accumulation of cytoplasmic beta-catenin, which allows beta-catenin to activate TCF/Lef1 transcription factors, which in turn are mandatory for hair follicle morphogenesis and cycling [28]. During morphogenesis and cycling, there is a change in cadherin expression that is dependent upon noggin (controls morphogenesis of ectodermal derivatives) and Wnt signaling. Whenever these pathways are active, there is downregulation of E-cadherin and Lef1 (Lymphoid Enhancer Factor 1, developmental transcription factor required for the inductive formation of several 11 epithelial-derived organs including hair follicles) expression (Figure 1.5) [29,30]. Figure 1.5. During follicle morphogenesis and cycling, skin stem cells change cadherin expression in a fashion dependent upon noggin and Wnt signalling. Wild-type skins from TOPGAL reporter mice at E16.5 (a–d, h, i) or adult (e–g) (left) Skin from a newborn transgenic mouse stained with X-gal in (E) (to test for Wnt signaling) [29] Summing up the role of Wnt signaling in the HF, we can say that the stabilization of beta-catenin props bulge SC activation, proliferation and follicle reproduction. The stabilization of beta-catenin is also responsible for terminal differentiation of matrix cells along hair cell lineage and formation of new HFs. Constantly active beta-catenin expression leads to pilomatricoma hair tumors. In summary, Wnt inhibition leads to SC quiescence and constitutive Wnt expression leads to tumorigenesis. Although the significance of the role of Wnt/beta-catenin signaling in activation of SCs, studies have revealed that the beta-catenin expression does not cause the bulge SCs to lose their quiescent nature. Hence, additional factors along with Wnt signaling are involved in SC activation. 12 1.4.b Shh Signaling Sonic hedgehog (Shh) signaling is similar to Wnt/beta-catenin signaling involved in cell differentiation and proliferation during development [31]. It has been reported that deregulation of the pathway leads to tumorigenesis. Studies have revealed that the pathway is potentially involved in epithelial-mesenchymal cross-talk essential for HF formation and also for HF regeneration during hair cycle. Although Shh is not expressed in the bulge, it signals matrix cell proliferation during the hair cycle. 1.4.c Bmp Signaling Bmp signaling is essential for skin development. Bmp signaling indicates ectodermal cells to differentiate to epidermis [32]. Another function of Bmp appears in the formation of hair placode, dependent on Bmp-inhibitor Noggin. Ablation studies of Bmpr1a gene leads to undifferentiated placode-like cells expressing Lef-1, hence pointing out that Bmp is inhibited during HF morphogenesis. Also, decrease of nuclear beta-catenin was observed in Bmpr1a-lacking matrix cells. These studies and further reports direct us towards the view that Bmp inhibition is required for SC activation to assume their fate as one of the six different layers of a mature HF [33]. In contrast Bmp1a-null ORS continues to multiply and grow downward, forming follicular tumors [34,35]. Bmp signaling also regulates the cyclic initiation of bulge SCs in traveling waves across the mouse body. To summarize, the Bmp pathway controls the nuclear beta- catenin, activation of SCs and HF differentiation. Summary of all the signaling pathways involved in hair follicle morphogenesis is depicted in Figure 1.6. 13 Figure 1.6. Summary of the signaling pathways involved in hair follicle morphogenesis and regeneration. This schematic summarizes many of the signaling pathways involved in hair follicle morphogenesis and hair follicle regeneration. Wnt/beta-catenin signaling acts early in hair follicle specification and quiescent bulge stem cell (SC) activation. The sonic hedgehog (Shh) signaling pathway acts in the second step to promote embryonic and adult hair germ proliferation. Bone morphogenetic protein (Bmp), Notch, and Wnt/beta-catenin signaling pathways act further downstream to allow the normal differentiation of matrix cells into the hair shaft (HS) and its inner root sheath (IRS) envelope [36]. 14 1.5 Epithelial Stem Cells Hair follicles are autonomous mini organs, which are the integral part of the body‘s first layer of defense, the skin. As we have discussed, regenerative capacity of the skin is due to different populations of stem cells present. Although there are considerable differences between the human and the murine skin, research on animals has elaborated our understanding about the various cellular populations of skin. Figure 1.7. Schematic illustration of the menagerie of stem cells and their individual locations in the resting (telogen) adult hair follicle. The stem cell populations are depicted by their distinct gene/protein-expression or promoter-activity: Lgr5 (green, hair germ and bulge), CD34 (orange, bulge), LRC (yellow, bulge), Lgr6 (pink, lower isthmus), Lrig1/MTS24 (blue, isthmus), Blimp1 (violet, sebaceous gland opening) and K15* (a truncated version of the K15 promoter, restricted in its activity to the bulge area). Note that Lgr5-expressing cells and LRCs show minimal overlap. LRC—label retaining cell; IFE—interfollicular epidermis [37]. There are specialized populations of stem cells present in the hair follicles, sebaceous glands and the epidermis. There are two populations of stem cells in the hair follicles- the neural crest-derived melanocyte stem cells contributing to the hair 15 pigmentation and hair follicle stem cells (HFSC‘s). Here we will focus of the populations of stem cells present in the hair follicles- the HFSC‘s. Initially it was believed that HFSC‘s reside in secondary germ. Figure 1. It was assumed that secondary germ moves downwards to the hair bulb during anagen and supplies new cells for hair formation. When anagen ends, secondary hair germ was thought to move upward with DP during catagen to come to rest at the base of the telogen follicle [11]. This cycling of the stem cells was brought into question with the identification of label-retaining cells, presumed to be stem cells in the bulge area of the ORS. HFSC‘s are now found to reside the bulge area of the ORS (Figure 1.7). These cells are responsible for regeneration of the hair follicle and hair shaft during each hair cycle. However, they play addition role during injury and wound healing. These cells migrate upward towards the sebaceous gland and the epidermis and assist in repair. These stem cells are relatively quiescent and hence retain the nucleotide label over time. This remarkable proliferation capacity suggests that bulge cells stay for the lifetime of the animal. There is a lot that still needs to be known regarding these cells- how do they maintain their property of self-renewal and differentiation? Are these cells influenced by their niche microenvironment to behave in a certain way? Or are they fundamentally different from their progeny? There are a number of groups working to reveal the role of these cells and the signaling mechanism involved in the mouse skin. The bulge is thought to be he compartment where epithelial stem cells reside. CD34 and Keratin 15 (K15) promoter 16 activity are the markers of this region in the adult mouse skin. Appearance of bulge corroborates with CD34 initiation at the beginnings of first anagen. Moreover, truncated version of K15 promotor marks the lower part of telogen HF, surrounding the bulge and the parts of the Hair Germ (HG). The overlap between the CD34/LRC- areas and K15+ cells substantiates their stemness. E0pithelial tissue and hair shaft are regenerated as a result of these stem cells. Environment and signaling from the surrounding area plays an important part in the activation of these quiescent stem cells. Bmp signaling in part and unidentified signals from the DP necessitate the reactivation of stem cells at the start of each hair cycle. The Bulge and Hair Germ as a Residence of Hair Follicle Stem Cells The slow cycling of bulge cells protects them from accumulating DNA replication errors. Pulse-chase experiments using titrated thymidine or BrdU (Bromodeoxyuridine) on mouse pups incorporated the label into newly replicated DNA and was found profusely in the basal lamella and ORS. After four to eight weeks of chase, amplifying cells quickly loose the label whereas slow-cycling cells retain the label and are kept in the tissue. Hence, Label-retaining cells (LRCs) have found to be localized in the bulge area of the epidermis in the postnatal epidermis [2]. The use of Histone H2B fused with GFP (Green fluorescent protein) has been successfully used to characterize and isolate the LRCs in the bulge. In the study by Blanpain and colleagues (2004), bulge cells were isolated from the mouse hair follicle, grown in a culture dish and then cells from individual colonies were mixed with newborn dermal cells and grafted onto the back of Nude mice [38]. 17 Strikingly, the skin formed as a result includes epidermis, sebaceous glands and hair follicles, suggesting multipotency and self-renewal properties of the bulge cells. In vivo lineage-tracing experiments using fragment of Keratin 15 promoter- derived Cre recombinase have revealed that all the hair lineages are derived from bulge cells, in the absence of injury or loss of the cell‘s natural environment. Moreover, the ablation of K15+ cells using an inducible gene resulted in failure of hair follicle regeneration. In the absence of these cells, epidermis and sebaceous glands could continue to exist, signifying their sovereignty in the skin lineages. Although there is an irony that is existent between the slow cycling bulge cells and the cell cultures obtained using the bulge cells. Large colonies are obtained when the cells from the bulge are cultured in about two weeks, which contradicts the notion of bulge cells being infrequently dividing. The formation of hair follicles from cultured cells graft suggests that in vitro each colony must have a population of stem cells and the rest could be proliferating stem cell derived transit-amplifying (TA) cells. Despite the paradoxes, it‘s quite certain that the bulge cells do posses attributes of epithelial stem cells. 1.6 Models of Hair Follicle Stem Cell Activation Bulge Activation Hypothesis: This model suggests that the stem cells are activated by unknown signals sent out through direct contact with DP at the conclusion of every hair cycle [2]. This interaction seems to be necessary for the activation of bulge cells, although it is not the only factor as the closeness of bulge and DP has been observed to 18 not always lead to stem cell activation. Cell Migration or the Traffic Light Hypothesis: The colonogenic and morphogenic bulge cells are found to migrate to the base of the bulb in the late catagen and early anagen, and stem cell progeny migrates to DP along ORS, signaling their differentiation pathway (Figure 1.8) [2]. A more dynamic model of stem cell activation has been proposed which is based on maintenance. The highly proliferating HF cells make up the LRCs in the bulge, which are reformed by the maintenance SCs in the hair germ, in the absence of bulge, suggesting the existence of a model of universal authenticity [37]. Figure 1.8. Current model of SC migration during skin homeostasis and repair [39]. 19 __________ Chapter 2 __________ ADHERENCE JUNCTIONS 2.1 Adherence junctions In all aspects of tissue morphogenesis, cell-cell adhesion is involved. Proper adhesion molecules are essential for tissue integrity in multi-cellular organisms. [39]. This is achieved through regulated genetic programs denoting cell-cell and cell-matrix interactions leading to the development of tissues and organs. The tight junctions are involved in controlling paracellular diffusion between cells and the extracellular environment; desmosomes resist mechanical stress and help maintaining epithelium continuum; and adherence junctions (AJs) assemble at initial cell-cell contact site and precede the formation of other junction complexes. The importance of cell adhesion is essential for understanding of tissue formation, wound healing and its dysregulation in many diseases particularly cancers points towards the need to study the molecular basis of cell-cell adherence formation. Tissue morphogenesis necessitates actin cytoskeleton remodeling to achieve cellular profile and 20 dynamics [40,41]. Hence, the understanding of tissue morphogenesis, especially cell adhesion, requires the complete understanding of all the players involved in maintaining homeostasis. AJs are a prominent feature of the stratified epithelia. AJs use homophillic interactions to bind the cytoplasmic epithelial cells to their neighbors. Protein complexes with distinguishing functional structures arbitrate cell-cell adhesion in mammalian epithelium. These include classic cadherins, claudins/occluding, nectin, desmosomal cadherins and catenins [42,43]. Cadherin-family of adhesion receptors facilitate the link between neighboring cells and connect to the actin filaments in the cytoplasm. Cell junctions are abundant in the epithelial tissues and epithelium is often defined by the E-cadherin expression. E- cadherin tail in the cytoplasm binds to a long, twisted coil of 36 short -helices of beta- catenin. Alpha-catenin binds to both beta-catenin and actin filaments. The assembly and maintenance of AJs is under tight transcriptional and posttranscriptional control. Past studies have highlighted other roles for AJ molecules. In vivo studies using conditional gene knockout strategies have revealed that cadherin-catenin complexes are not only involved in cell-cell adhesive contacts, but also play a vital role as signaling centers of various cellular pathways. These new findings suggest that the cadherin- catenin complex may serve as biosensors for the cellular microenvironment [45]. 21 2.2 E-Cadherin Figure 2.1. E-cadherin. Adherens junctions are comprised of the single pass transmembrane protein, E- cadherin. The extracellular domain is proposed to form trans-interactions with E-cadherin on neighboring cells. The intracellular domain has two binding regions; juxtamembrane domain (JMD) and catenin-binding domain (CBD). (TM; Transmembrane) The protein-protein interactions presented are limited to those involved in connections with the actin cytoskeleton. The asterisks represent the region proteins have been shown to bind. Not drawn to scale [46]. E-cadherin belongs to the calcium - dependent adhesion protein family. The structural hallmark of the cadherin family is the presence of 5 extracellular cadherin (EC) repeat domains. Ca 2+ presence is essential for proper conformational organization of the extracellular cadherin domain. Without the presence of Ca 2+ , the domains rotate freely around their linked peptide [47]. Cells with matching cadherins form trans-cadherin interactions, initiating weak cell-cell adhesion and forming Ajs. E-cadherin is an essential component in developing and maintaining cell polarity, sustaining cell survival and controlling cell proliferation. Cytoplasmic tail of E-cadherin binds to different proteins mediating its endocytosis, recycling and degradation, intracellular signaling and gene transcription, and local actin cytoskeleton control. The intracellular tail binds to various cytoplasmic proteins of the armadillo repeat family- p120 catenin (p120), beta-catenin and plakoglobin (also known as -catenin). Cell-cell interactions mediated by cadherins are 22 extremely dynamic. This allows the reorganization of cells during processes like epithelial – to – mesenchymal transition (EMT) in the course of normal development, wound healing and carcinogenesis [48]. Disruption and loss of E-cadherin-mediated adherence junctions as well as down-regulation of E-cadherin is linked to epithelial dedifferentiation, phenotypic alteration of the epithelial cells as well as increased proliferation and invasiveness of epithelial- derived tumors. 2.3 Catenins Although E-cadherin is the core protein associated with adherence junctions, there is also need for localization of a number of other cytoplasmic proteins to aid connections between cadherin complex and actin cytoskeleton, and to mediate numerous signaling pathway. p120, beta-catenin and alpha-catenin are the important members of the catenin family [49]. The catenin family has been well depicted in Figure 2.2 [50]. 23 24 2.3.a p120 catenin Figure 2.3. p120 catenin. Also are mentioned additional proteins known to regulate actin cytoskeleton. Asterisks represent regions known to bind the respective protein. Numbers associated with the asterisks correspond to amino acids flanking the binding site. Not drawn to scale [46]. p120 is a 120 kDa protein (Figure 2.3). The cytoplasmic domain of E-cadherin is divided into two regions. p120 associates with the cadherin juxtamembrane domain (JMD) and is directly responsible for stabilizing cadherin expression at the cell surface, as well as for inducing clustering of cadherins to promote AJs formation. Dynamic phosphorylation of p120 and cadherin is possible to control the interaction of p120 with cadherins. Previous research indicates that increase in adhesiveness of the cells is achieved with p120–E-cadherin interaction. Retention of cadherin complex at plasma membrane increases with p120 where as the loss of E-cadherin stabilization via p120 is connected to tumor progression and invasion. It has been proposed that the binding of p120 to JMD may prevent internalization and degradation of cadherins [51,52,53]. It also results in reutilization of the internalized cadherins back to plasma membrane [54]. It is speculated that in E-cadherin null environment, loss of p120 leads to increased cell-cell adhesion, hinting to p120‘s further potential in influencing cell-cell adhesion. p120 has a well established role as a regulator of cell motility via actin cyctoskeleton by regulating the members of the Rho family of small GTPases. Furthermore, p120 being independent of p120–E-cadherin binding regulates cytoskeletal dynamics and invasiveness by 25 inhibiting RhoA activity. Regulation of transcription of canonical Wnt/beta-catenin target genes is also one of the signaling functions of p120. 2.3.b Beta-catenin Figure 2.4. Beta- catenin. Also are mentioned additional proteins known to regulate actin cytoskeleton. Asterisks represent regions known to bind the respective protein. Numbers associated with the asterisks correspond to amino acids flanking the binding site. Not drawn to scale [46]. Beta-catenin is a 92-kDa protein and it binds to the C-terminal cytoplasmic ―catenin-binding domain‖ (CBD) of cadherins (Figure 2.4). Beta-catenin was recognized in Drosophilla. It was identified as a transcriptional coactivator central to the Wnt signaling pathway that determines cell fate during embryogenesis and tissue renewal. Posttranslational modification of proteins, especially beta-catenin by phosphorylation is instrumental to these interactions. With the study of chimeric E-cadherin/alpha-catenin protein complex expression, the role of phosphorylated beta-catenin in cell-cell adhesion was determined [55]. The modification to the tyrosine residues brings about the E- cadherin/beta-catenin/alpha-catenin adhesion complex dissociation. In the past, it was thought that beta-catenin serves as a direct link cadherins and alpha-catenin bound actin-filaments, such that beta-catenin/alpha-catenin interactions 26 provide physical link between AJs and actin cytoskeleton. However, recent studies have challenged this notion. Most beta-catenin is bound to cadherins, but a second pool is found in the nucleus. Cytoplasmic free beta-catenin enters the nucleus and is important for the regulation of transcription factors of Lef/TCF family and Wnt/beta-catenin signaling pathway directing cell differentiation. The importance of beta-catenin in vasculature development has also been demonstrated using beta-catenin conditional knockout studies in mice endothelial cells. The conditional knockout results in embryonic death between embryonic day (E) 11.5 and 13.5 [56,57]. 2.3.c Alpha-catenin In contrast to other catenins, alpha-catenin was thought to be a non-regulatory component of the cadherin complex in charge for cell-cell adhesion. Since it was known to bind to F-actin, it was believed to bind the cadherin complex to actin filaments, although the E-cadherin/alpha-catenin/actin complex has never been isolated. Recent studies evaluating the functional role of alpha-catenin have demonstrated more complex role of the molecule in tissue organization and morphogenesis. Alpha-catenin is discussed in more details in the next chapter. Figure 2.5 addresses the role of cadherin-catenin complex in the skin. The cadherins are responsible for providing the structural support to the skin whereas catenins take up the role of signaling molecules. 27 Fig. 2.5. Similar and distinct functions of cadherin–catenin proteins in skin epidermis. The schematic diagram shows connections between cadherin–catenin proteins and cellular phenotypes revealed by loss-of- function experiments. While cadherins appear to be primarily responsible for mechanical tissue integrity, catenins also are involved in regulation of multiple signal transduction pathways [57]. 2.4 Adherence Junctions and Stem Cell Niche Maintenance Even at postnatal day two (P2), the proliferative epidermis of the newborn mouse has a population of LRCs almost at the level of potential bulge, suggesting that this specific area of the HF posses explicit structural and molecular determinant forcing cells to quiescence [58]. It has been speculated that the unique properties of the quiescent cells is preserved by their location providing an explicit microenvironment, known as the stem cell ―niches‖ [2,36]. It is thus, an issue of extreme importance to understand how the stem cells are maintained within the niche. Stem cell niches can be classified into two classes: Stromal niches (stem cells having direct membrane contact with niche cells) and 28 Epidermal niches (stem cells having direct contact with extracellular matrix, not with any stromal cells). Discovery of the Drosophila germline stem cell (GSC) niche has validated the stem cell niche theory [59]. It has also provided valuable information pertaining to the adherence molecules and mechanism attaching stem cells to the niche. GSC-niche is localized in the Drosophila ovary where 2 to 3 GSCs are attached to cap cells (a tight group of 5 to 6 stromal cells). The cap cells constitute the GSC-niche as they provide essential maintenance signals for GSCs. Various studies support that GSCs are affixed to the niche through Drosophila E-cadherin-mediated adhesion linking GSCs and cap cells [60]. The removal of Drosophila beta-catenin gene causes the disconnection between GSCs from their niche and leads to differentiation. In a recent report up regulation of E-cadherin expression in a differentiation defective Drosophila ovary has shown to replace normal GSCs in the niche. E-cadherin levels served as ‗a quality control mechanism‘. Differentiated cells were removed and non-differentiated cells were retained in the niche. These studies strongly suggest that the adherence molecules are essential in maintaining the stem cell in their niches and in maintaining their quiescent state. Studies in the epidermis exploring the HFSC-niche have suggested that the interaction of basement membrane and cell adhesion molecules with the basal layer regulates the orientation if the division axis, providing basis for symmetric and asymmetric division (Figure 2.6). Moreover, the targeted deletion of alpha-catenin from the cells of basal layer resulted in arbitrary spindle alignment and disorientation of the cell divisions. The randomization of polarization and loss of spindle control in alpha- 29 catenin knockout (KO) sheds light on the various structural and signaling defects in the alpha-catenin KO epidermis, including the absence of organized columns of stratified cells, the presence of suprabasal mitoses and the formation of internalized masses of disorganized epidermal cells. Figure 2.6. Multiple functional roles of cell–cell and cell–ECM adhesion in SC niches [61]. 30 __________ Chapter 3 __________ ALPHA-CATENIN 3.1 Alpha-catenin: The known and the unknown Alpha-catenin differs in sequence as well as structural organization when compared to other members of the other members of the catenin family, like beta-catenin and p120, which belong to the Armadillo repeat superfamily and share sequence similarity. Alpha-Catenin is a 906-amino-caid protein that is homologous to the membrane- cytoskeletal protein vinculin found in focal adhesion plaques (Figure 3.1). Vinculin and alpha-catenin are distant relatives sharing 20-30% of the sequence. Alpha-catenin binds to actin bundles and has interactions with other actin-binding proteins including vinculin, beta-catenin, spectrin, ZO-1 (zonula occludens-1), afadin, ajuba and actin-assembly regulating proteins like formins [62]. 31 Figure 3.1 Primary structure of alpha-catenin. The figure also shows vinculin-homology regions and binding sites for various partners indicated Numbers are amino acid positions. ZO-1, zonula occludens 1 [63]. In 1984, Vestweber and Kemler discovered alpha-catenin as a 102kDa protein and proposed it being the connecting protein between E-cadherin and actin filaments, even before its amino acid sequence for identified. Binding assays indicate that E-cadherin, beta-catenin and alpha-catenin assembles into a complex with a 1:1:1 stoichiometry [64]. But the interaction of the complex with actin filaments could not be demonstrated. Moreover, FRAP (Fluorescence Recovery After Photobleaching) showed that actin has a very dynamic when present at cell-cell contacts (Figure 3.2). It is highly mobile and has a much higher recovery rate [65]. 32 Figure 3.2. Quantification of fluorescence recovery after photobleaching (FRAP). The numbers of cells (n) quantified are: Ecad-GFP (n = 30), EcadDC-tdDsR (n = 4), GFP-bcat (n = 28), GFP-acat (n = 41), GFP- acatDC (n = 28), GFP-actin (n = 30), and Rhod-actin (n = 16). Error bars show SEM [64,65]. These results contradicted the previous thought role of alpha-catenin as link between cadhenin and actin. However, alpha-catenin exists as a monomer as well as a homodimer. It forms a homodimer in a solution but in the presence of beta- catenin/plakoglobin, a heterodimer of alpha-catenin and beta-catenin/plakoglobin in the ratio 1:1 is formed. The alpha-catenin homodimer has a higher affinity for actin filaments as compared to the monomer, where as the monomer has higher affinity for cadherin- bound beta-catenin. In living cells, it was confirmed that alpha-catenin bound with membrane could be exchanged with the cytosolic pool using FLIP (Fluorescence Loss In 33 Photobleaching). The mutual exclusiveness of these two processes disregarded the earlier hypothesis [65,67]. This suggests that alpha-catenin needs to dissociate from the E- cadherin/beta-catenin complex before it can bind to actin (Figure 3.3). Figure 3.3. Protein–protein interactions between cadherins, catenins and the actin cytoskeleton. Protein interactions formed between cadherin, beta-catenin ( β-cat), alpha-catenin monomers ( α-catM), alpha- catenin dimers ( α-catD) and actin. Differences in the thickness of the arrows represent strengths of protein– protein interactions (i.e. increased thickness shows increased binding). The interaction between cadherin and beta-catenin is regulated by kinases that increase (green box: CK2 and GSK3 β) or decrease (red box: Src, Fer, Abl and EGFR) the binding affinity [67]. Arp2/3 (Actin-related protein 2/3) complex mediates in actin polymerization necessary for lamellipodia (cytoskeletal protein actin projection on the mobile edge of the cell) and cell migration. Additional studies have revealed that alpha-catenin homodimers suppress this process by competing directly with Arp2/3 complex for binding to actin filaments (Figure 3.4). Reports also suggest alpha-catenin‘s role in 34 promoting linear actin polymerization by stimulating formins activity, highlighting the new roles for alpha-catenin. Figure 3.4. A model for regulation of cytoskeleton and membrane dynamics by the cadherin–catenin complex [67]. Studies also suggest alpha-catenin to be an allosteric protein, changing its conformation when one ligand binds leading to change in the affinity of another ligand [63], suggesting the explanation for different binding activities of alpha-catenin. These studies point towards nascent roles of alpha-catenin and provide us with new insight into the complex interactions it has with other proteins. Figure 3.5 point outs the new proposed model for alpha-catenin and its role in cell adhesion. Alpha-catenin concentration at the membrane increases due to the clustering of the cahderin–catenin 35 complex, which is locally exchanged between the membrane and cytoplasmic pool under [66,68]. This is adequate to assemble alpha-catenin into dimers, compete with Arp2/3 complex and bind to actin. This would cease the branched actin polymerization characteristic of lamellipodial protrusion. But alpha-catenin dimers bind to actin and lead to actin reorganization – from branched to bundled arrays. [46, 63,69]. Although studies in the past decade have shed light on the novel functions and roles of alpha-catenin, a lot more needs to be known before we can fully establish alpha- catenin‘s role in cell-cell adhesion and various signaling pathway. Figure 3.5. Models of how the cadherin-catenin complex is linked to and regulates the actin cytoskeleton. A, model based upon the assumption that alpha-catenin binds simultaneously to beta-catenin and actin filaments. B, model showing that alpha-catenin monomers and dimers regulate binding tobeta-catenin and actin filaments, respectively, and that alpha-catenin also inhibits Arp2/3-mediated actin branching and promotes actin bundling [66,68]. 36 3.2 Alpha-catenin and its Role in Tumorigenesis Cancer is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood) [70]. Loss and disruption of cadherin/catenin complex has been a characteristic in cancer. E-cadhenin, being the major cell adhesion protein and beta-catenin, being a mediator of Wnt signaling pathway, have been well studied to establish their role in cancer. On the other hand, alpha-catenin had been ignored in cancer progression studies until recently. 3.2.a Cancer and cadherin/catenin complex E-cadherin role in many human cancers has been extensively studied. Mutations of the somatic or germline origin, epigenetic modifications, transcriptional repression or post-transcriptional regulations may be responsible for changes in cadherin expression in cancer. Most carninomas, cancers of the transformed epithelial cells, show a loss of cell-cell adhesion governed by E-cadherin and go through process of EMT (Epithelial-mesenchymal transition or transformation) [70]. To be able to metastasize, cancer cells must intravasate (invasion of cancer cells through basal membrane into lymph and blood circulatory system), extravasate (exiting the circulatory system to enter 37 surrounding tissues and organs) and establish growth in their new environment (Figure 3.6). Figure 3.6. Metastasis is a multistep process. The major steps of the metastatic process are indicated: local invasion, involving basement membrane destruction and invasion into adjacent tissues; intravasation and survival in the bloodstream; and extravasation into distant organs and proliferation/survival in the new host organ. The specific steps where EMT (epithelial–mesenchymal transition) and the reverse MET (mesenchymal–epithelial transition) process are thought to occur are indicated [71]. Metastasis of tumor is the leading cause of death among cancer patients. Hence the identification of patients prone to develop metastatic lesions is extremely important to formulate the best treatment plan. Current methods of pathological and clinical diagnosis 38 often based on arbitrary lymph sampling and lead to errors in grading cancers. Therefore there arises a need for molecular prognostic markers to work adjunct to current methods to identify metastasis and cancer stage. Table 1 summarizes the use of cadherin/catenin complex as a prognostic marker to establish tumor stage and metastasis. Table 3-1. Alpha-catenin as a prognostic marker in human cancers [64]. Cancer type Loss of alpha-catenin is a statistically significant prognostic factor for the following cancer phenotypes Loss of alpha-catenin is more prognostic than loss of E- cadherin for the following cancer phenotypes Expression of alpha- catenin in cancer metastasis compared to primary tumor Breast Histological type and growth, grade, stage, metastasis, survival Survival, reduced more frequently in diffuse tumors Decreased protein levels, normal/increased protein levels Gynecological Grade, survival d.n.a. Decreased mRNA levels, decreased protein levels, increased protein levels Esophageal/ Laryngeal Differentiation, grade, stage, infiltrative growth, metastasis, survival Survival, metastasis and tumor differentiation Decreased protein levels Colorectal Differentiation, invasion, metastasis, survival Invasion and metastasis to lymph/liver Decreased expression, increased expression Prostate DNA aneuploidy, differentiation, grade, metastasis, survival d.n.a. d.n.a. Thyroid Stage, tumor recurrence metastasis d.n.a. d.n.a. Oral d.n.a. d.n.a. Decreased expression Lung Differentiation, metastasis, survival Metastasis to lymph and survival d.n.a. Liver Survival d.n.a. d.n.a. Pancreatic Grade, stage, metastasis, survival d.n.a. Normal/increased expression] Gastric Differentiation, depth invasion, metastasis Metastasis to lymph and tumor invasion Decreased expression, mixed expression Skin d.n.a. Significantly reduced alpha-catenin levels but not E-cadherin Decreased expression Bladder Grade, stage, cancer progression, invasion, survival Survival d.n.a. Other Tumor growth, size, grade, invasion, survival Survival d.n.a. 39 Beta-catenin is a part of the cadherin/catenin complex as well as plays significant part in the Wnt signaling pathway. Due of these functions, beta-catenin‘s role in cancer as also been investigated. Its involvement in loss of cell-cell adhesion and increase in cancer related gene transcription has been established by expression loss and functional gain mutations. Involvement of alpha-catenin in cancer has been a buzzing field. Numerous studies on human cancerous cell lines have conducted to further explore this area. Since, alpha-catenin was thought to function as a link between E-cadherin and actin filaments, its expression was thought to be down-regulated in cancers due to disruption of cell-cell contact, but now its thought to be due to DNA promoter methylation, histone alteration and post-transcriptional modification. Recent report suggesting that alpha-catenin, independent of cadherin in the cytosol, mediates actin dynamics points. Hence, it keeps a check on cell-cell adhesion and migration by maintaining equilibrium, which is distorted in cancer metastasis [72]. There have been cases where the loss of alpha-catenin serves as a significant prognostic factor than E-cadherin alone for cancer stages. The loss of alpha-catenin and E-cadherin is associated with worse cancer prognosis, than the loss of either one alone. Although cancers in the metastatic intravasation stage have down-regulated alpha-catenin expression, the expression of alpha-catanin is, however, normal or higher at the site of secondary tumor. This has been associated with worse patient survival rate. It is believed 40 that at the new site, alpha-catenin expression is restored to allow adhesion of cancer cells to the surrounding tissue and maintain essential microenvironment. Cellular pathways regulating cell proliferation like Ras/MAPK, NFkB, Hedgehog (Hh) and Wnt, have shown involvement of alpha-catenin. Many reports have also pointed to loss of alpha-catenin leading to increase cell number affecting tissue size and organization; and also potentially leading to cancer. Although there has not been established a clear mechanism by which alpha-catenin affects proliferation, but they do suggest that it functions as a cell density sensor, maintaining cell adhesion and proliferation. Reports of existence of alpha-catenin/beta-catenin heterodimer imply that alpha- catenin may also affect proliferation via Wnt signaling. In the absence of alpha-catenin in cancerous cells, beta-catenin target genes could become up regulated leading to uncontrolled proliferation and cancer. 3.2.b Consequences of loss of alpha-catenin As described above, loss of alpha-catenin in epithelial tumors is a marker of poor prognosis. Further in vivo studies were carried out in mice models to access the role of alpha-catenin. Vasioukhin et al. studied loss of alpha-catenin in animal keratinocytes. The loss of alpha-catenin compromise cell-cell adhesion, altered response to several growth factors including the ones controlling the coupling of alpha-catenin with E-cadherin/beta- 41 catenin complex, dramatic effects in the epithelium similar to that observed with the activation of oncogenes [73, 64]. 42 __________ Chapter 4 __________ METHODS 4.1. Generation of transgenic mice The University of Southern California Institutional Animal Care and Use Committee approved all animal protocols. In order to study the role of alpha-catenin in the maintenance of hair follicle homeostasis, conditional ablation of the alpha-catanin gene was brought about by using the inducible K15CrePR mice model. To be able to conditionally disrupt cell adhesion in stem cells of skin hair follicles we will mate alpha-catenin flox/flox and Keratin15-CrePR mice (Figure 4.1). Keratin 15- CrePR will be activated specifically in stem cells area of mouse skin hair follicle after topical application of RU486. It binds to the modified progesterone (PR) fused to Cre resulting in translocation of Cre to the nucleus where it can act to remove floxed allele from alpha-catenin gene. To identify the transgenic mice, genotypic analysis of the tail samples of the progeny was conducted. The DNA was 43 extracted from the tail biopsy and PCR was carried out using transgene-specific primers. The products were run on a 2% agarose gel to identify the mice of interest. Figure 4.1. K15-crePR mice are mated with alpha-catenin flox/flox mice to obtain mice with the ability to remove alpha-catenin flox/flox allele upon activation of K15-crePR. 4.2 Lineage analysis in vivo (treatment of mice with RU486) Once the identification of the wild type and knockout mice was complete, the treatment with the steroid RU486, which induces Cre recombinase, was started on both the knockout mice as well as the control (flox/+ cre+ mice). The treatment was done topically on mice shaved back skins with 2.5mg/mouse RU486 applied daily for a period of 16d starting in the second post natal telogen (the resting phase of hair follicle). The timeline of the treatment is shown in Figure 4.2. Figure 4.2. Timeline of treatment of mice upon the onset of second postnatal telogen by steriod RU486. 44 The mice were observed for following treatment and samples were obtained at desired intervals. RU486 on K15-CrePR mice results in conditional knockout of alpha- catenin in the HFSCs as depicted in Figure 4.3. Figure 4.3. The figure depicts hair follicle of knockout mice where the ablation of alpha-catenin takes place upon RU486 treatment. The figure is adapted from Ref 36. 4.3 Sampling and Staining After the treatment with the drug is complete and the duration has passed, the mice with desired genotype (flox/flox cre+) mice and the control (flox/+ cre+ mice) were sacrificed to obtain the skin sample. The skin samples were mounted on OCT and thin 10µm samples were transferred on to the slides. To study the effect of the treatment on alpha-catenin, the samples were stained various antibodies. These were further tagged with fluorescent tags. K15-CrePR + RU486 45 4.4 Fluorescent Imaging To verify the absence of alpha-catenin as a result of the treatment with RU486, the skin samples were analyzed using fluorescent microscopy. The drug treatment disrupted the cell adhesion between the cells and hence there was a decrease in the amount of alpha-catenin. This was observable as a decreased expression in the alpha- catenin flox/flox and Keratin15-CrePR mice as compared to the control. Moreover, observable morphological changes differences were observed between the control and the knockout mice. Once the model was verified, immunohistochemistry and specific stem cell markers were used to assess the differences between normal stem cells and stem cells without alpha-catenin at different time points after gene ablation. 4.5 Keratinocyte isolation by FACS analysis FACS (Fluorescent-Activated Cell Sorting) analysis was carried out to isolate different cell populations. Keratinocytes, for further analysis and studies, were harvested from the back skin of the transgenic mice. Subpopulations of cells are separated by tagging the protein of interest with an antibody connected to a fluorescent dye. The cells, being fluorescent upon being excited by the laser, are separated according to the light they scatter as a result. This information allows the computer to sort and to collect the cells. 46 __________ Chapter 5 __________ RESULTS & DISCUSSION Adherence proteins play an extreme importance in maintaining the morphology of the cellular structures. E-cadherin and beta-catenin have known to be essential in cell-cell adherence and their disruption can lead to dramatic morphological changes resembling cancerous growth. Adherence junctions are essential to maintain stem cell niches and their quiescent stages. To evaluate the role of adherence junctions and its crucial component alpha-catenin in the maintenance of the hair follicle stem cells and their niche hair follicle morphology and cycle was evaluated in stem-cells specific alpha-catenin knockout mice and control group. Genotyping was performed using gene amplification by PCR and gel electrophoresis to identify and separate the KO and the control group mice. The identified mice were treated with RU486 which induces Cre recombinase followed by alpha-catenin ablation specifically in hair follicle stem cells. To study the effect of the gene KO, 47 immunofluorescent stainings were performed to evaluate the effect of loss of alpha- catenin. 5.1 Five weeks after treatment: RU486 treatment inhibits normal anagen progression The system that we are using relies on the treatment of the back skin of mice with the modified progesterone in order to activate Cre and ablate the specific gene. Normally this treatment is performed during the telogen stage and after transition to anagen we expected to observe the phenotype in hair follicles due to the alpha-catenin ablation. However we observed that this treatment blocks normal transition of hair follicle from telogen (resting phase) to anagen (growth phase) (Figure 5.1). The mechanism for that is not yet understood but in this case endogenous alpha-catenin appeared to be quite stable on the membrane of the quiescent telogen stem cells. It took longer for these hair follicles to enter new anagen and start to produce new cells with knocked-out alpha-catenin. Therefore initially we haven‘t observed any phenotype. 48 Figure 5.1. No apparent morphological change observed in the KO and WT mice 5 weeks after RU 486 treatment. (a and b) There is no apparent change in the structure of hair follicles as observed by hematoxylein and eosin (H&E) staining. (c, d, e, f, g, and h) There is no observable loss of alpha-catenin or stem cells (CD34 marker) in either the KO or WT mice. (i, j, k, l, m and n) E-cadherin expression and proliferation levels (Ki67 ab) are same in both KO and WT mice samples. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). CD34, stem cell marker; Ecad, E-cadherin; Ki67, proliferating nuclear antigen. 49 5.2 Five months after treatment: Loss of hair shaft but not the hair bulb in the KO mice To further evaluate the role of alpha-catenin, the second set of mice was sacrificed 5 months after the treatment. There was apparent loss of hair on the mice back, belly and there was loss of vibrissae (Figure 5.2 a, b) in the KO mice observed by the naked eye, whereas no loss was observed in the WT mice. The H&E staining of the skin samples confirmed the loss of hair shaft but not the loss of hair bulb in the KO mice (Figure 5.2 c, d). Figure 5.2. Loss of hair shaft but not hair bulb in the KO mice 5 months after RU486 treatment. (a and b) There is loss of hair from the mice back, belly and loss of vibrissae. (c, d) Loss of hair shaft but hair bulb is still present in KO mice (H&E staining). In the KO mice, there was increase in cellular proliferation indicating loss of contact inhibition Alpha-catenin expression was less in KO mouse as compared to the WT, confirming alpha-catenin ablation (Figure 5.3 a). This suggests that loss of alpha-catenin 50 affects the proper differentiation process of hair follicle probably due to the hair follicle cells disorganization after cell-cell junction disruption. Cellular proliferation and anagen-like induction was evident in the KO mice on the basis of proliferation marker Ki67 staining. BrdU (another proliferation marker) and K5 (marker of basal epithelial cells) staining confirmed the presence of proliferating cells (Figure 5.3 b,c). Proliferation was observed along the bulb, suggesting that the observable proliferation might be due to the multiplication of the stem cells. Quiescent stem cell niche in the hair follicle present in the bulb is maintained due to their cellular microenvironment. These stainings suggest that there is a loss of this controlled environmental setting, leading to proliferation possible as a result of loss of adherence. This further highlights the loss of adherence and the loss of contact inhibition in the hair follicle. The loss of contact inhibition leads to uncontrolled growth, often leading to cancerous growths. Absence of this phenomenon is a mark of cancerous cellular populations. The WT mouse samples did not have any evident cellular proliferation and the stainings revealed normal hair follicle morphology. There was no loss of alpha-catenin after the RU486 treatment on WT samples (Figure 5.3 a). 51 Figure 5.3. Increased proliferation and loss of alpha-catenin in mosaic pattern 5 months after RU 486 treatment. (a) There is less alpha-catenin expression in KO mice than WT mice, confirming alpha-catenin ablaton (b) Arrows indicate regions of proliferation in the hair follicle in KO mice. (c) The high proliferation is confirmed by Ki67 proliferation marker. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). BrdU, Bromodeoxyuridine; Ecad, E-cadherin; K5, Keratin 5; Ki67, proliferating nuclear antigen. 52 There is no loss of stem cell marker, no apoptosis but proliferation in KO mice To assess the role of alpha-catenin ablation in apoptosis, Activated caspase (apoptotic marker) staining was performed. There was no apparent difference in the expression of Ac caspase indicating that alpha-catenin ablation doesn‘t lead to cell death (Figure 5.4 a, b). Figure 5.4. No observable apoptosis 5 months after RU 486 treatment. (a, b) Ac Caspase 3 is not expressed at all in the LO or WT mice, indicating that alpha-catenin ablation does not lead to cell apoptosis. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). Ac Casp3, activated caspase, apoptotic marker; Ecad, E-cadherin. 53 Even after the ablation of alpha-catenin, there is no loss of CD34 marker in the KO mouse, indicating that stem cells are still present in the back skin. This was further confirmed using FACS analysis, which did not reveal much difference in the number of cells expressing CD34 (Figure 5.5 a). These findings suggest that initially there is no change in the number of stem cell, despite the proliferation. Hence the proliferating cells do not possess stem cell characteristic and FACS analysis confirms that the number of stem cells do not change either (Figure 5.5 b). 54 Figure 5.5. No difference in the number of stem cells obtained from the KO and WT. (a) Even though there is high proliferation in the KO mice, there is no apparent loss of stem cells in KO in comparison to WT mice. (b) FACS analysis confirmed the findings. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). CD34, stem cell marker. 55 There is no differentiation in the hair follicle cellular population as a result of alpha- catenin ablation Bmp and Wnt pathways are important for maintenance of hair shaft homeostasis and differentiation. P-Smad 1/5/8, marker for Bmp activation, was expressed in same levels in the KO and WT mice (Figure 5.6 a, b). Interestingly the expression of beta- catenin was localized predominately on the membrane but not in the nucleus in alpha- catenin KO hair follicles, suggesting that the Wnt activity was blocked in the KO mouse (Figure 5.6 c). These findings suggest that in the KO mice, Wnt activity mediated by nuclear beta-catenin is affected in the alpha-catenin KO hair follicle resulting in lack of proper differentiation. The activated beta-catenin leads to tumor-like growths as has been observed in previous studies. This suggests that alpha-catenin can influence Wnt signaling by affecting localization of beta-catenin. Although, further work will be required to assess if alpha-catenin plays a direct role in Wnt signaling or if it indirectly alters the pathway via interacting with other pathway regulators like beta-catenin. 56 Figure 5.6. Hair follicle differentiating pathways are not affected by alpha-catenin ablation, as observed 5 months after RU486 treatment. (a) Bmp signaling important for hair shaft differentiation is still present in alpha-catenin KO hair follicles. (b) Beta-catenin localize predominantly on the membranein alpha-catenin KO hair follicles (c) Wnt activity was blocked in the KO mouse. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). -cat, beta-catenin; der, dermis; Ecad, E-cadherin; epid, epidermis; P-Smad 1-5-8, marker for Bmp activation. 5.3 Ten months after treatment: To understand the role of cell-cell junctions and alpha-catenin in hair follicle homeostasis in long term we analyzed the WT and alpha-catenin KO mice skin 10 months after RU486 treatment. There is disruption of hair follicle morphology with loss of the structural identity in alpha-catenin KO mouse. There is evident difference in the KO and WT mice. There is balding and loss of hair shaft in the KO mice where as there is the normal hair coat still present in the WT 57 mouse. H&E staining further reveal the commotion in the hair follicle structure. There is excessive uncontrolled downward growth with the loss of the normal HF structure (Figure 5.7 a). The HF does not represent the defined structure with bulb, hair germ, DP and the hair shaft. The structure represents disoriented cellular mass. High rate of proliferation is observed in the cells once constituting the HF. There is high expression of βeta-4 integrin and Ki67 in the HF and areas of proliferating cells are clearly observable (Figure 5.7 b). This clearly indicates the loss of contact inhibition leading to unorganized cellular structure, similar to tumorous growth. This is also a sign of loss of adherence and further strengthens the notion that alpha- catenin plays an integral role in the maintenance of cell-cell adherence. Also, alpha- catenin might play a part in maintaining the quiescent nature of the stem cells and the signaling pathways that help in sustaining the structural integrity of tissues and organs. 58 Figure 5.7. Loss of hair follicle morphology as a result of high proliferation observed in KO mice (a) Loss of structural morphology of the hair follicles in the KO mice (b) Proliferating cells leading to disruption of the hair follicle morphology similar to cancerous growth. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). 4, beta-4 integrin; Ki67, , proliferating nuclear antigen. 59 Stem cell markers are still present in the KO mice but there is loss of structural integrity in regions deprived of alpha-catenin. CD34 staining reveals the presence of stem cells in the hair follicle niche. Although the stem cell population is still present and not lost as a result of proliferation, there is loss of structural morphology of the hair follicle as a result of loss of alpha- catenin (Figure 5.8). 60 Figure 5.8. Stem cell marker still expressed in the regions with decreased alpha-catenin activity in KO mice. (a-g) Loss of structural morphology of the hair follicles in the KO mice due to the loss of alpha- catenin but CD34 is still expreseed in these regions. (h) Magnified view of the region with CD34 expression with reduced alpha-catenin activity. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). CD34, stem cell marker. 61 These results indicate that that alpha-catenin is indeed the requirement for maintenance of the cellular adherence. Although there is the loss of the HF integrity, the HF stem cells are still present. It is believed that the quiescent nature of the stem cells is the result of their microenvironment. It has been shown that the loss of this microenvironment results in the loss of quiescence and stemness of the stem cells and often leads to differentiation. The salient observation of the presence of stem cells can be explained by the fact that the loss of HF structure doesn‘t make the stem cells to lose their characteristics. Despite the high proliferation rate, the cells are not differentiating to HF cells in the KO mouse back samples. The striking finding is the absence of differentiation in the highly proliferative HF, which was revealed by AE15, hair follicular differentiation marker (Figure 5.9). The absence of differentiation indicates that the cellular masses as a result are undifferentiated, and possess similar characteristics to cancerous growths. This suggests that alpha-catenin interacts with Bmp and Wnt signaling pathways, directly or indirectly. The fact that proliferating cells are not differentiating and the presence of stem cells suggests that the proliferation might be as a result of asymmetric division of stem cells. 62 Figure 5.9. Lack of differentiation in rapidly proliferating cells in the KO mice. (a-d) Absence of hair follicular differentiating marker suggests that the proliferating cells possess cancer-like propertics, and might be the result of asymmetric stem cell division. Color coding of immunostained sections is according to secondary Abs; sections are counter stained with DAPI (blue). AE15, hair follicular differentiating marker; 4, beta-4 integrin. 63 __________ Chapter 6 __________ CONCLUSION & FUTURE DIRECTIONS Alpha-catenin has been shown to play a vital role in the adherence junctions and in the maintenance of cell–cell adhesion. Although, we are aware of these functional properties of alpha-catenin, the way in with it actually interacts with other adherence molecules and signaling pathways is still a mystery. Through the use of conditional ablation of alpha-catenin in mice models, we have found a way to study the role it plays and the extent of its significance. Our study indicates that loss of alpha-catenin is a slow process as a result of gene ablation by RU486 treatment. Despite the fact that the observable changes take long to show, it is evident that the alpha-catenin is essential in maintaining the structural tissue integrity, in this case, of the hair follicle. Although the change is sluggish, the consequences are pretty severe. There is massive proliferation resembling undifferentiated cellular populations found in cancers. The loss of alpha-catenin does not 64 lead to the loss of stem cells or to differentiation. These observations are striking as stem cells require specific microenvironment present in the 'niche' to maintain their quiescent nature. The slow cycling stem cells maintain their stemness when present in the controlled setting. It has been shown that the stem cells, when removed from their niches, lose their stem cell characteristics via symmetric cell division leading to differentiation. The hypothesis that might explain this is that alpha-catenin ablation leads to asymmetric division. This leads to one of the daughter cells to replace the stem cell and the other one to undergo differentiation. The fact that there is lack of differentiation implies that alpha- catenin interacts with Bmp and Wnt signaling, essential for the HF differentiation. In conclusion, further work is essential to exactly determine the cellular interactions of alpha-catenin. There is also a need to evaluate the stem cell potential of the cells expressing CD34 even after the loss of niche architecture. This requires further experiments like chamber graft, to evaluate these characteristics. Chamber grafting will allow us to determine if these cells have the ability to rescue and recreate the hair follicle structure, also establishing their stem cell potential. 65 __________ References __________ 1. Skin Anatomy & Physiology. Digital image. 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Olmeda, David, Gema Moreno-Bueno, David Sarrio, Jose Palacios, and Amparo Cano. "Invasion Program of Normal and Cancer Stem Cells." Cancer Stem Cells: Identification and Targets. Ed. Sharmila Bapat. Hoboken, NJ: Wiley, 2009. 167-196. Print. Paus, R., and Foitzik, K. In search of the ‗‗hair cycle clock‘‘: a guided tour. Differentiation. 2004; 72, 489–511. Paus, Ralf; Cotsarelis, George. "The Biology of Hair Follicles." The New England Journal of Medicine. 1999; 341(7): 491-497. Philpott M.P. and Kealey T. Effects of EGF on the morphology and patterns of DNA synthesis in isolated human hair follicles. J Invest Dermatol. 1994; 102: 186–191. Pokutta S, Drees F, Yamada S, Nelson WJ, Weis WI. Biochemical and structural analysis of alpha-catenin in cell–cell contacts. Biochem Soc Trans . 2008; 36:141–147 Pokutta S, Herrenknecht K, Kemler R, Engel J. Conformational changes of the recombinant extracellular domain of E-cadherin upon calcium binding, Eur. J. Biochem. 1994; 223: 1019–1026. Proksch E, Brandner JM, Jensen JM. The skin: an indispensable barrier. Exp Dermatol. 2008; 17(12):1063-72. Raymond K, Deugnier MA, Faraldo MM, Glukhova MA. Adhesion within the stem cell niches. Curr. Opin. Cell Biol. 2009;21:623–629. 76 Reddy S, Andl T, Bagasra A, Lu MM, Epstein DJ, et al. Characterization of Wnt gene expression in developing and postnatal hair follicles and identification of Wnt5a as a target of Sonic hedgehog in hair follicle morphogenesis. Mech Dev. 2001;107:69–82. Rendl, M., Lewis, L., and Fuchs, E. (2005). Molecular dissection of mesenchymal- epithelial interactions in the hair follicle. PLoS. Biol. 2005; 3, e331. Reya T, Morrison SJ, Clarke MF. et al. Stem cells, cancer, and cancer stem cells. Nature. 2001; 414:105–11. Sato N., Leopold P.L., and Crystal R.G. Induction of the hair growth phase in postnatal mice by localized transient expression of sonic hedgehog. J Clin Invest. 1999; 194: 855– 864. Schneider MR, Schmidt-Ullrich R, Paus R. The hair follicle as a dynamic miniorgan. Curr Biol. 2009;19:R132–142. Shapiro L, Weis WI. Structure and biochemistry of cadherins and catenins. Cold Spring Harb Perspect Biol. 2009;1. Skin Anatomy & Physiology. Digital image. Web. 18 Jan. 2010. <http://www.essentialdayspa.com/images/emerginc/Skin_Anathomy_and_Physiology.gif >. Skin. Wikipedia, the Free Encyclopedia. Web. 18 Jan. 2010. <http://en.wikipedia.org/wiki/Skin>. Soma T., Ogo M., Suzuki J., Takahashi T., and Hibino T. Analysis of apoptotic cell death in human hair follicle in vivo and in vitro. J Invest Dermatol. 1998; 111: 948–954. Song X, Zhu CH, Doan C, Xie T. Germline stem cells anchored by adherens junctions in the Drosophila ovary niches. Science 2002; 296:1855-7. St-Jacques B., Dassule H., Karavanova I., Botchkarev V., and Li J. Sonic hedgehog signaling is essential for hair development. Curr Biol. 1998; 8: 1058–1068. Stenn, K.S., and Paus, R. Controls of hair follicle cycling. Physiol Rev. 2001; 81, 449– 494. Taipale J, Beachy PA. The hedgehog and wnt signalling pathways in cancer. Nature. 2001; 411:349–354. Takai, Y. and Nakanishi, H. Nectin and afadin: novel organizers of intercellular junctions. J. Cell Sci. 2003; 116, 17–27. 77 Takeichi, M. Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 1995; 7: 619–627. Trempus C, Morris R, Bortner C, Cotsarelis G, Faircloth R, Reece J et al. Enrichment for living murine keratinocytes from the hair follicle bulge with the cell surface marker CD34. J Invest Dermatol. 2003; 120:501–11 Tumbar, Tudorita, and Elaine Fuchs. "Epithelial Hair Follicle Stem Cells." Essentials of Stem Cell Biology. By Robert Paul Lanza. Elsevier, 2009. 189-97. Print. Vasioukhin V, Bauer C, Degenstein L, Wise B, Fuchs E. Hyperproliferation and defects in epithelial polarity upon conditional ablation of alpha-catenin in skin. Cell. 2001;104:605–617. Wang LC, Liu Z-Y, Gambardella L, Delacour A, Shapiro R, et al. Conditional disruption of hedgehog signaling pathway defines its critical role in hair development and regeneration. J Invest Dermatol. 2000;114:901–908. Weis WI, Nelson WJ. Re-solving the cadherin-catenin-actin conundrum. J Biol Chem. 2006;281:35593–35597. Xi R.Anchoring stem cells in the niche by cell adhesion molecules. Cell Adh Migr. 2009 Oct;3(4):396-401. Yamada S, Pokutta S, Drees F, Weis WI, Nelson WJ. Deconstructing the cadherin- catenin-actin complex. Cell. 2005;123(5):889–901. Yamada, S. and Nelson, W.J. Synapses: sites of cell recognition, adhesion and functional specification. Annu. Rev. Biochem. 2007; 76: 267–294. 78 ___________ Appendix A ___________ Polyformaldehyde (PFA) fixation method for immunofluorescence 1. Thaw slides for 10-20 minutes at room temperature. 2. Fix in 4% PFA (in 1xPBS) for 10 minutes at room temperature. 3. Wash 3x in 1x PBS for 5 minutes. 4. Permeabilize sections in 0.1% TritonX-100 (in 1x PBS) for 10 minues. 5. Wash 3x in 1x PBS for 5 minutes. 6. Block in 2.5% NGS and 2.5% NDS in PBS-T + 0.1% BSA for 1 hour at room temperature. 7. Incubate in primary antibody diluted in PBS + 0.1% BSA for minimum 1 hour at room temperature or overnight at 4 o C. 8. Wash 3x in 1x PBS for 5 minutes. 9. Incubate in secondary antibody diluted in blocking solution for 1 hour at room temperature. 10. Wash 3x in 1x PBS for 5 minutes. 11. Counterstain with DAPI (in 1x PBS) for 2 minutes. 12. Wash with 1x PBS. 13. Mount with fluorescence mounting medium and add coverslip. 14. Store on -20 o C. 79 ___________ Appendix B ___________ H & E Staining Slide Preparation Frozen Sections 1. Thaw slide for 10-20 minutes at room temperature. 2. Fix in 4% PFA (in 1xPBS) for 10 minutes at room temperature. 3. Wash 2x in 1x PBS for 5 minutes. Paraffin Sections 1. Deparaffinize sections in xylene or Citrus Clearing Solution 2x for 5 minutes. 2. Hydrate 2x in 100% ethanol for 5 minutes. 3. Hydrate in 95% ethanol for 5 minutes. 4. Hydrate in 80% ethanol for 5 minutes. 5. Rinse in distilled water. 6. Wash 2x in 1x PBS for 2 minutes. Staining 1. Incubate in hematoxylin for 4 minutes. 80 2. Rinse 2x with 1x PBS. 3. Wash with 1x PBS for 2 minutes. 4. Wash 5x in water for 2 minutes. 5. Incubate in 0.1% NH 4 OH (in H 2 O) for 10 seconds. 6. Wash 2x in water for 2 minutes. 7. Incubate in freshly prepared eosin mix for 3 minutes. 0.5 mL of 1% eosin stock 0.5 mL of 100% ethanol 5 L of acetic acid, glacial 8. Wash 2x in 95% ethanol for 1 minute. 9. Mount with 80% glycerol. 81 ___________ Appendix C ___________ RU 486 (Mifepristone) Treatment- Tropical Application to Mouse Back Skin RU 486 has been classified as a contragestive due to its ability to interrupt an early pregnancy. RU 486 binds to progesterone receptor. Hence, it‘s recommended to always wear gloves with handling it. Stock Solution 0.5g of RU 486 (Mifepristone) was suspended in 20ml 100% ethanol (25mg/ml solution). The solution was wrapped in foil and stored at -20 o C. Treatment 200 L of RU 486 Stock Solution (25mg/ml) was applied to the shaved back daily (5mg per treatment) for 16 days. 82 ___________ Appendix D ___________ Isolation of Genomic DNA and Genotyping 1. The tail sample from the mouse is added to a mixture of 200 l of TNES and 10 l of Proteinase K. 2. Incubate the test tubes overnight at 55 o C. 3. Add 60 l 6M NaCl and shake for 30 seconds. 4. Spin the tube for 5 minutes at full speed in a centrifuge. 5. Transfer the supernatant into a microfuge tube. 6. Add equal volume (approximately 270 l) of 100% ethanol and mix by inversion. 7. Spin at room temperature for 5 minutes at full speed. 8. Decant supernatant and wash with 200 l of 70% ethanol. 9. Air dry the pellet and re-suspend it in 100 l of H2O. 10. Incubate at 65 o C for 20 minutes in the thermocycler. 11. Keep in the fridge for 1-2 hours before using for PCR. 83 Cre Products Cre + = 600 bp Table D-1. PCR reaction components and cycle information. Reaction Components Vol/Rxn H20 13.25 10X Buffer 2.5 25 mM MgCl2 3 10 mM each dNTP 0.75 Cre-F 10microM 1.5 Cre-R 10microM 1.5 5 U/µl Taq Pol. 0.5 DNA (2 µl per Rxn) from 200 dilution 2 Total 25 Cycling Reaction A Step Temp Time Note 1 94 °C 5 min 2 94 °C 30 sec 3 62 °C 45 min 4 72 °C 60 sec Go to step 2, 35 times 5 72 °C 5 min 6 4 °C Separated by gel electrophoresis on a 1.5% agarose gel. Table D-2. Cre primers. Cre-F 5'- TGCTGTTTCACTGGTTATGCGG Cre-R 5'- TTGCCCCTGTTTCACTATCCAG 84 Catna1 tm1Efu (alpha-catenin flox) Jackson strain 004604 Products +/+ = 100 bp +/- = 100 bp and ~350 bp -/- = ~350 bp Table D-3. PCR reaction components and cycle information. Reaction Components Vol/Rxn H20 8.25 10X Buffer 2.5 Qsolution 5 25 mM MgCl2 3 10 mM dNTP 0.75 10 µM Lcat-Fx-F 1.5 10 µM Lcat-Fx-R 1.5 5 U/µl Taq Pol. 0.5 DNA (2 µl per Rxn) from 200 dilution 2 Total 25 Cycling Reaction A Step Temp Time Note 1 94 °C 5 min 2 94 °C 30 sec 3 62 °C 45 min 4 72 °C 60 sec Go to step 2, 35 times 5 72 °C 5 min 6 4 °C Separated by gel electrophoresis on a 1.5% agarose gel. Table D-4. Lcat-fx primers. Lcat-fx-F 5'- CAT TTC TgT CAC CCC CAA AgA CAC -3' Tm = °C 24-mer A=7, C=10, G=2, T=5 Amplifies a 100 bp product from the wild type allele and a ~350 bp product from the floxed allele. Lcat-fx-R 5'- gCA AAA TgA TCC AgC gTC CTg gg -3' Tm = °C 23-mer A=6, C=6, G=7, T=4 85 ___________ Appendix E ___________ Agarose Gel Electrophoresis 1. Add 2 grams of agarose in 100 ml TAE buffer to make 2% agarose gel. 2. Heat solution in a microwave until agarose is completely dissolved. 3. Allow it to cool till 50-55 o C. 4. Prepare gel-casting tray by using the appropriate casting system. Place appropriate number of combs in gel tray. 5. Once cool, add 10 l of SYBR Green in the gel. 6. Pour the gel into the gel tray and allow it to cool for 15-30 minutes at room temperature. 7. Remove comb(s), place in electrophoresis chamber and cover with TAE buffer. 8. Add loading buffer to samples. As a guideline, add 1.5 l of 10x Loading Buffer to a 20-25 l PCR/DNA solution. 9. Load DNA and standard (Ladder) onto gel. 10. Electrophorese at 100V for 1 hour. 11. Visualize DNA bands using gel-imaging system. 86 ___________ Appendix F ___________ Cryostat Sectioning 1. Thaw the samples a bit so as to be removed from the cryomolds. 2. Apply OCT on the detached stage covering an area large enough to accommodate the sample. 3. Attach the specimen to the prepared base of OCT. Attachment should be made when OCT is partially frozen, but not yet hardened. Slowly add OCT around specimen until covered, and leave to harden. 4. When the specimen is ready, attach the stage to the cryostat with consideration given to the angle desired for positioning the sections on each slide. The position of the stage with specimen can be adjusted clockwise and counter-clockwise approximately 5 degrees in either direction. Tilt can also be adjusted. Refer to manual for directions. 5. Place the blade into its holder in the cryostat. Examined the blade for nicks. If the area of the blade that will be cutting the specimen is compromised, the blade can be moved in either direction. Replacement of the blade may be necessary. 6. ‗Trim‘ the sample till the desired sectioning depth is achieved. 87 7. Set the thickness to 10 microns the large wheel on the right hand side of the cryostat anti-clockwise. 8. Place the roll-bar onto the blade and turn the handle in a smooth, steady motion. 9. Once a section has been cut, lift the roll bar to expose the section and gently place a slide on top of it. The section should then stick to the slide. 10. Leave the slides to air-dry before use. Slides can be stored at -20 0 C until required. 88 ___________ Appendix G ___________ Collecting Samples from the Mouse Back Skin 1. Euthanize the desired mice from which the samples are to be collected. 2. Shave the back skin of the mouse. 3. Pin down the mouse on a board. 4. Carefully cut out a rectangular piece of back skin preferably covering the treated area. 5. Remove fatty tissue and blood vessels from the skin. 6. Cut the skin sample to fit in the cryomold. 7. Fill the cryomold with OCT and place the skin samples in the cryomold perpendicular to the table. 8. Keep the cryomolds on dry ice to harden the OCT. Keep the samples in place while hardening. 9. Label and store the samples at -20 o C. 89 ___________ Appendix H ___________ Isolation Stem Cells from adult mouse skin 1. Shave and then remove back skin from the adult mouse. (Take the sample for OCT and /or PFA for staining). 2. Remove fat and veins from dermis (by scalpel scraping). 3. Incubate skin in 0.25% trypsin at 4C O/N (at least 8h) (skin facing epidermis up floating in trypsin) in 10cm dish. 4. Scrape epidermis in trypsin, mix them by pipetting several times (- 30ml) then add 3vol. of (E low Ca media 0.05mM) and filtrate through 0.7um filter 5. Spin down the cells at 300g (250g optional) (1129rpm Allegra Beckman Coulter centrifuge) at 4C 10min (20min). 6. Remove supernatant and resuspend cells in 20ml 1xPBS (or * E low Ca media 0.05mM) then filtrate through 0.4um. 7. Resuspend in 1%FBS Chelex then ready for staining: *alternatively - wash in 20ml 1xPBS (1x) (if E low Ca was added in step 6) then resuspend in 1% FBS Chelex (no calcium) – cells are ready for staining: Alternatively split the cells – freeze unused one to 20% (or 90%) FBS Chelex and 10%DMSO) 90 8. Count the cells (usually from 1 adult mouse back skin you can isolate approx. 4- 6mln cells). Staining for Stem Cells in skin for FACS: Controls for settings - tubes 1-4 use unstained cells (usually if you resuspend your sample cells in 520ul 1%FBS Chelex. Use 30ul for each control + 230ul more of 1% FBS Chelex (tubes 1-4). Use remaining 400 ul as for sample. 1. Unstained 30 +270ul 2. L6 - 30 +270ul + 2ul CD49f-conjugated PE 30‘ at 4C 3. CD34 - 30 +270ul + 2ul CD34 Alexa 700 conjugated 30‘ at 4C 4. DAPI (1:10000) before FACS Samples: 1. staining I Ab – 30‘ 4C: 400ul 1xPBS 1%FBS Chelex (no Ca) + 8ul L6-PE (1:50 for 2-10mln cells) + 8ul CD34-Alexa 700 (1:50 for 2-10mln cells) -Wash – add 3ml 1xPBS spin down at 300g (10min) then
Abstract (if available)
Abstract
In multi-cellular organisms, cell–cell contacts that are mediated by classical cadherins have essential roles in many fundamental processes, such as morphogenesis, maintenance of tissue integrity, wound healing and cell polarity. Furthermore, there is overwhelming evidence that the adherens junctions (AJs) are also an important tumor and/or invasion suppressor. Alpha-catenin is the protein that connects E-cadherin–beta-catenin complexes with the actin cytoskeleton. Although it was previously considered to be a solely structural protein, it has become increasingly clear that alpha-catenin has a central role in both assembling the actin cytoskeleton and regulating its dynamics at cell–cell junctions thus regulating cell polarity. Cell-polarity mechanisms are responsible not only for the diversification of cell shapes but also for regulation of the asymmetric cell divisions of stem cells that are crucial for their correct self-renewal and differentiation. Disruption of cell polarity is a hallmark of cancer. Although null mutations in alpha-E-catenin have been associated with epithelial cancers it has been usually assumed that perturbations in cell–cell adhesion are late rather than early steps in carcinogenesis, and that they are preceded by mutations in cell-cycle-regulated genes that lead to uncontrolled growth. In our previous experiments we have shown that loss of alpha-catenin in the whole epidermis during embryonic development results in formation of squamous cell carcinoma.
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Surbhi
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Core Title
Importance of alpha-catenin and cell-cell interactions in hair follicle stem cells homeostasis
School
Viterbi School of Engineering
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Master of Science
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Biomedical Engineering
Publication Date
10/08/2010
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University of Southern California
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alpha-catenin,hair follicle,homeostasis,OAI-PMH Harvest,Squamous Cell Carcinoma,stem cell
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Kobielak, Agnieszka (
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rajsurbhi@gmail.com,surbhi@usc.edu
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alpha-catenin
hair follicle
homeostasis
stem cell