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Department of Molecular and Cellular Biology and Dermatology, Houston, Texas, USADepartment of Molecular and Cellular Biology and Program in Developmental Biology, Houston, Texas, USADepartment of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, USA
Multipotent stem cells residing in the bulge region of the hair follicle give rise to cells of different fates including those forming hair follicles, interfollicular epidermis, and associated glands. Stem cell fate determination is regulated by genes involved in both proliferation and differentiation, which are tightly regulated processes. Understanding the molecular mechanisms by which proliferation and differentiation are regulated will provide useful insight into treating human diseases caused by the deregulation of these processes. Two genes involved in regulating proliferation and differentiation are c-Myc and p63, both of which have been found to be deregulated/mutated in several human diseases. Accelerating proliferation leads to neoplastic human diseases and deregulated c-Myc has been implicated in a variety of cancers. Evidence indicates that c-Myc also diverts stem cells to an epidermal and sebaceous gland fate at the expense of the hair follicle fate. Therefore, deregulation of c-Myc has the potential to not only accelerate tumorigenesis, but also influence skin tumor phenotype. In addition, the inhibition of differentiation may also predispose to the development of skin cancer. Recent evidence suggests that the transcription factor p63, is not only responsible for the initiation of an epithelial stratification program during development, but also the maintenance of the proliferative potential of basal keratinocytes in mature epidermis. Mutations in the p63 gene have been shown to cause ectodermal dysplasias and deregulated expression of p63 has been observed in squamous cell carcinomas. In this review, we will discuss recent data implicating a role for both c-Myc and p63 in human skin diseases.
The epidermis is a self-renewing tissue that forms the outer layer of the skin through an intricate balance of cell proliferation and differentiation. Because the skin is readily accessible, it represents an attractive system to analyze the molecular mechanisms responsible for these processes. As a highly regulated organ, the skin maintains strict control of proliferation and differentiation. The balance between cell proliferation and differentiation results in the division of stem cells and the proper entry of daughter cells into differentiation, thus maintaining epidermal homeostasis. Alterations in either proliferation or differentiation have the potential to disrupt normal epidermal homeostasis and lead to disease. Recent studies focusing on c-Myc and p63, both of which are involved in regulating proliferation and differentiation, have provided new insight into the role of these genes in formation of the epidermis during development, the maintenance of stem cells in mature epidermis, and the commitment of multipotent stem cells to different cell fates. In addition, these studies have revealed that skin disorders arise when these genes are mutated or deregulated.
Epidermal Stem Cells
The self-renewing characteristic of the skin and its appendages is supported by stem cells residing within the epidermis and in the bulge region of hair follicles. Stem cells are unique from other cells because they have a high capacity for self-renewal and the ability to produce daughter cells (transit amplifying cells) that undergo terminal differentiation (
). Since cells in the epidermis continually differentiate to replenish cells sloughed from the external surface, the only cells capable of accumulating genetic mutations required for tumorigenesis are epidermal stem cells (
Research over the past few years has expanded our knowledge about epidermal stem cells. Studies utilizing the unique characteristics of stem cells have led to the identification of a population of multipotent stem cells within the epidermis. Label retaining experiments have shown that cells residing in the bulge region of mouse pelage follicles are slow cycling and give rise to both the hair follicle and the epidermis (
). This study established, for the first time, that these stem cells are bipotent. In addition, an elegant study using chimeric hair follicles created from wild-type and Rosa26 mice, which were genetically engineered to express a reporter gene, β-galactosidase, showed that stem cells residing in the bulge region of the hair follicle are multipotent and give rise to hair follicles, sebaceous glands, and interfollicular epidermis (
). Rosa26 cells that were initially present in the bulge region of the chimeric hair follicle were detected migrating downward along the hair follicle towards the hair bulb and upward into the epidermis 4 wk after transplantation. After 6 wk, the Rosa26 cells could be detected in the hair bulb, sebaceous glands, and interfollicular epidermis (
). These studies provide very convincing evidence that a multipotent stem cell population resides in the bulge region of hair follicles.
To date, a unique epidermal stem cell marker has not been identified. But the use of a combination of markers and adhesive properties has allowed the isolation of enriched stem cell populations. Epidermal stem cells express higher levels of β1-integrin compared with transit amplifying cells, allowing the isolation of epidermal stem cells based on their adhesiveness (
), has also been used for the enrichment of epidermal stem cells. An additional approach using a combination of Hoechst and propidium iodide dye to sort cells has led to the isolation of three distinct populations of cells from the basal layer including stem cells, transient amplifying cells, and non-proliferative basal cells (
). The ability to isolate a pure population of epidermal stem cells would be very beneficial for therapeutic applications for a variety of skin disorders.
Role of c-Myc in the Epidermis
Members of the MYC oncoprotein family, c-Myc, N-Myc, and L-Myc, play a role in the pathogenesis of many human neoplastic diseases (
). N-myc is expressed at high levels in pre-B cells, kidney, forebrain, hindbrain, and intestine and continues to be expressed during differentiation (
). This review focuses on c-Myc since it is the predominant member expressed in the epidermis.
The oncoprotein c-Myc plays a role in proliferation, differentiation, and apoptosis. c-Myc is a transcription factor that heterodimerizes with members of the Max/Mad family. To activate transcription of target genes, c-Myc heterodimerizes with Max and binds to E box sequences in the promoters of target genes (
). The transcription factor Mad can bind to both Max and c-Myc, which prevents transcriptional activation of target genes by competing out the association of c-Myc/Max complexes as well as binding E box sequences to block c-Myc/Max binding (
). In the epidermis, c-Myc is expressed at high levels in the basal compartment and as cells differentiate c-Myc levels decline and Mad expression levels increase (
). There are a variety of mechanisms by which c-Myc is tightly regulated including: activation by the WNT signaling pathway, growth factors, and mitogens, as well as inhibition by the TGF-β signaling pathway (
). Previous studies using transgenic mouse models have provided evidence that the WNT pathway regulates stem cell fate determination. Collectively, these studies have found that different levels of active β-catenin influences the specific fate adopted. An increase in β-catenin signaling leads to precocious hair formation (
). β-catenin levels, however, may not be the sole determinate of stem cell fate. c-Myc has been shown to induce differentiation of epidermal stem cells in vitro (
), and recent in vivo studies have found that deregulation of c-Myc expression targeted to epidermal stem cells leads to increased sebaceous gland and epidermal differentiation at the expense of hair follicles (Figure 1) (
). Label retaining cell assays revealed that transgenic mice with deregulated c-Myc expression targeted to epidermal stem cells using a K14 promoter exhibited a depletion of stem cells compared with wild-type littermates (
). These mice also exhibited a decrease in integrin expression that accompanied a defect in wound healing. In addition, similar results were observed in an inducible MycER mouse model, which also exhibited terminal differentiation of keratinocytes into interfollicular epidermis and sebocytes at the expense of hair lineage differentiation (
). Both studies indicate that bypassing the WNT pathway by deregulating c-Myc can also influence stem cell fate, suggesting that c-Myc may play a more pivotal role in stem cell fate determination than previously in realized.
Figure 1Influence of c-Myc on Stem Cell Fate. c-Myc diverts multipotent stem cells residing in the bulge region of the hair follicle to a sebaceous and/or interfollicular epidermis cell fate at the expense of a hair follicle fate
The mechanism by which c-Myc influences stem cell fate is unknown; however, it is possible that c-Myc activates direct targets involved in stem cell fate determination. There have been numerous studies using gene expression profiling to determine potential downstream targets of c-Myc. Studies using an inducible transgenic mouse model have found targets of c-Myc to be involved in proliferation, cell cycle regulation, RNA regulation, protein synthesis and processing, cell adhesion, and regulation of the cytoskeleton (
). Among various downstream targets, this study found α6-integrin, a potential epidermal stem cell marker, to be decreased in expression 2-fold after activation of c-Myc expression. Other studies using in vitro gene expression profiling approaches also provide important insight into the potential downstream targets of c-Myc. For example, studies using an inducible human fibroblast line found an increase in c-Myc expression led to an increase in CD71 expression (
). Smad7 is the inhibitory smad of the TGFβ pathway and overexpression of Smad7 targeted to the epidermal basal compartment has been shown to increase the number of sebaceous glands and induce epidermal hyperplasia (Wang XJ, personal communication), similar to the phenotype exhibited by K14.myc2 mice (
). These results support the ability of c-Myc to divert stem cells from the stem cell compartment to a sebaceous fate at the expense of hair follicles through activating specific targets.
Role of c-Myc and Human Disease
Deregulated c-Myc has been found in several cancer types including breast (
). The role c-Myc plays in cell proliferation is thought to be the key to its involvement in cancer. Studies analyzing c-Myc expression in non-proliferating cells of the suprabasal layer of the epidermis found c-Myc to induce proliferation while inhibiting terminal differentiation (
). c-Myc is involved in the G1 to S phase transition of the cell cycle, which is the time when the DNA is repaired. Premature exit from G1 without proper DNA repair allows mutations to accumulate. Recent studies analyzing targets of c-Myc have found several genes involved in the G1 to S phase transition to be targets of c-Myc. Genes activated by c-Myc include cyclinD2 (
). But if c-Myc induced apoptosis is blocked, then, tumorigenesis could proceed. In fact, studies have looked at the effect of deregulated c-Myc in combination with overexpressing Bcl-xL (
), a member of the bcl-2 family. The anti-apoptotic family members include bcl-2, bcl-xL, mcl-1, and bcl-w and the pro-apoptotic members include bax, bak, bad, bcl-xS, bid, and hrk (
). This family regulates apoptosis by either promoting or inhibiting cell death. Human tumor studies have found Bcl-xL to be highly expressed in human squamous cell carcinoma (SCC) (
). When this apoptosis was suppressed by co-expression of Bcl-xL, c-Myc expression was able to induce tumor progression. Also, data from mammary gland tumors suggest cooperation between Bcl-xL and c-Myc in transformation (
). Cell lines derived from mammary tumors, which arise in MMTV-myc transgenic mice, can be induced to undergo apoptosis by exogenous TGFβ and inhibited by exogenous epidermal growth factor (EGF). In a study to analyze apoptotic pathway genes in these cell lines in the presence or absence of the growth factors, it was found that the expression of Bcl-xL increased with EGF and decreased with TGFβ. The change in Bcl-xL expression was greater than the change in Bcl-2 expression (
). This data suggest that Bcl-xL may be the predominant family member responsible for inhibiting c-Myc induced apoptosis. Although the cooperation between Bcl-xL and c-Myc has not been analyzed in skin carcinogenesis, both have been found to play a role (
). A concern raised with these therapies is that withdrawal of oncogene inactivation may result in tumor regrowth. Recent studies have analyzed the role of inactivation/activation of c-Myc in tumor regression and regrowth. The first study used an inducible mouse model in which c-Myc expression, targeted to the suprabasal layers by the involucrin promoter, could be activated by topical application of 4-hydroxytamoxifen (OHT) (
). Activation of c-Myc in the suprabasal layer resulted in the proliferation of post-mitotic keratinocytes and prolonged activation induced the formation of preneoplastic lesions similar to human epithelial precancerous lesions. Inactivation of c-Myc in the preneoplastic lesions resulted in regression of lesions (
). Additionally, studies using osteogenic sarcoma cells derived from a tetracycline-regulated transgenic mouse model with c-Myc targeted to lymphocytes have found that inactivating c-Myc leads to tumor regression (
). When c-Myc expression was reactivated, the cells underwent apoptosis as opposed to proliferation. This was further analyzed in vivo by implanting osteogenic sarcomas subcutaneously into syngenic mice. Inactivation of c-Myc expression caused tumor regression and subsequent activation of c-Myc resulted in a marked increase of apoptosis in tumor cells (
In summary, c-Myc plays an important role in stem cell fate determination and carcinogenesis. It is tempting to speculate that the effect of c-Myc on stem cell fate determination could influence tumor phenotype. Nevertheless, c-Myc appears to be an important player in stem cell fate determination and the role of c-Myc in both tumor progression and regression indicates the potential use of c-Myc in therapeutic strategies for skin cancer treatment. For tumors with deregulated c-Myc expression, the acute short-term inhibition of c-Myc may lead to tumor-targeted apoptosis with the withdrawal of c-Myc inhibition. This would bypass the potential problems with systemically inhibiting c-Myc and therefore proliferation.
Function and Structure of p63
In addition to deregulated proliferation, alterations affecting proper cell differentiation have also been found to give rise to human disease. Recently p63, a member of the p53 gene family has been implicated in epidermal development and differentiation. The p53 gene family now consists of three genes: p53, p63, and p73. All three genes share sequence homology, although p63 and p73 are more similar to each other than to p53 (
). Each p53 family member contains the three typical domains of a transcription factor: a transactivation domain, a DNA binding domain, and an oligomerization domain (
). It, however, remains to be determined if p63 and p73 regulate p53 target genes under physiological conditions. Despite these similarities between p53, p63, and p73, they differ in several important respects. Unlike p53, p63 and p73 do not represent classical tumor suppressor genes: mice heterozygous for either p63 or p73 are not predisposed to tumor development (
), and germline mutations in p63 (as found in ectodermal dysplasias; see below) are not associated with a cancer-prone phenotype. Rather, p63 and p73 act as key regulators during development. p73 is required for the development of neuronal and pheromonal pathways and p63 for epithelial, limb, and craniofacial development (
). Contrary to p63, the expression pattern of p73 and the phenotype of p73-/- mice do not suggest a role for p73 in skin development or skin cancer susceptibility.
). The use of alternative promoters and transcription start sites gives rise to two classes of p63 transcripts, those encoding proteins with an amino terminal transactivation domain (TA isoforms) and those encoding proteins lacking this domain (ΔN isoforms). ΔNp63 isoforms were shown to be able to inhibit transactivation of a p53 reporter construct by TAp63 isoforms, suggesting that ΔNp63 isoforms have a dominant-negative function (
). In addition to alternative promoter usage, alternative splicing in a part of the sequence that is not present in p53, gives rise to three different carboxy-termini designated α, β, and γ. The α C-terminus of p63 is the longest and is the only C-terminus that contains a SAM (sterile α motif) domain. SAM domains are evolutionary conserved domains that are found in proteins involved in the regulation of developmental processes and were shown to be able to bind to other SAM domains or to SH2 domains (
). The physiological relevance of these interactions, however, is not known. In addition to interaction with lipids, the SAM domain of p63 was shown to interact with apobec-1 binding protein (ABBP1), an RNA processing protein (
). Upon binding to the p63 SAM domain, ABBP1 preferentially splices Fgfr2 into the epithelial-specific Fgfr2-IIIb (K-SAM) isoform. In the absence of this isoform, the epidermis exhibits a reduction in keratinocytes proliferation resulting in severe epidermal hypoplasia (
). Therefore, the binding of the p63 SAM domain to ABBP1 may contribute to the role of p63 in epidermal development and differentiation. Although these are just two examples of interactions with the p63 SAM domain, it is likely that p63 participates in other interactions that mediate its function.
Role of p63 in the Epidermis
In the mature epidermis ΔNp63α is the major p63 isoform expressed and the highest expression levels are observed in the proliferating cells of the basal layer and hair follicles (
The Delta Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay–Wells syndrome-derived mutations.
). These expression data suggest a role for ΔNp63α expression in proliferation of basal keratinocytes. Consistent with this hypothesis, in vitro data using primary mouse keratinocytes demonstrated that ΔNp63α expression can block calcium-induced differentiation of primary keratinocytes, thereby maintaining cells in a proliferative state (
). In zebrafish embryos, ΔNp63 is synthesized prior to epidermal proliferation; however, the nuclear translocation of ΔNp63 during zebrafish development correlates with the time that epidermal proliferation begins. Moreover, in the absence of ΔNp63 expression, epidermal cells in zebrafish embryos fail to proliferate (
). In addition, in the absence of ΔNp63 expression, zebrafish skin does not differentiate resulting in microbial infections and death. Consistent with a role for p63 in maintaining the proliferative potential of epidermal keratinocytes, the observed downregulation of p63 in differentiated layers of vertebrate epidermis was shown to be required for terminal differentiation to take place. In primary keratinocytes, ΔNp63α was shown to interact with the promoters of p21WAF1/Cip1 and 14-3-3σ resulting in transcriptional repression (
The Delta Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay–Wells syndrome-derived mutations.
), this repression may prevent basal keratinocytes from prematurely differentiating. During terminal differentiation, ΔNp63α expression is downregulated resulting in loss of binding of ΔNp63α to the p21WAF1/Cip1 and 14-3-3σ promoters. This may result in the expression of p21WAF1/Cip1 and 14-3-3σ thereby allowing for terminal differentiation to take place. Taken together, these studies suggest that p63 is required for the maintenance of the proliferative potential of basal keratinocytes in the mature epidermis and that p63 expression must be downregulated for terminal differentiation to take place.
In addition to its role in mature epidermis, p63 expression is required for development of the epidermis, as clearly demonstrated by the phenotype of p63-/- mice (
). p63-/- mice fail to form a stratified epidermis resulting in a lack of barrier formation causing dehydration and death within hours after birth. In addition to the failure to develop an epidermis, p63-/- do not develop epithelial appendages such as teeth, hair follicles, and mammary glands. This failure to develop appendages is presumably caused by a failure to participate in epithelial-mesenchymal signalling required for appendage development. In fact, several genes that are induced in the mesenchyme of the limb bud as a result of epithelial-mesenchymal signalling are absent from p63-/- limb buds (
). The single-layered surface epithelium of p63-/- mice does not express keratins K5 and K14. These keratins are the first differentiation markers expressed during normal epidermal development and are markers for epithelia that have committed to initiate an epithelial stratification program (Figure 2). Therefore, although it has been proposed that p63 is required for epithelial stem cell maintenance (
), it is more plausible that p63 is required for the commitment of the originally single-layered surface ectoderm to an epithelial stratification program (
). In fact, we have recently demonstrated that ectopic expression of TAp63α in single-layered lung epithelia results in the development of squamous metaplastic lesions (
). Consistent with these data, it had previously been demonstrated that squamous metaplastic lesions that develop in the lung and uterus express p63, whereas the surrounding single-layered epithelia do not (
). In addition, we have demonstrated that deregulated expression of TAp63α in mature epidermis results in hyperproliferation and a delayed onset of terminal differentiation (
). Taken together, these data support a dual role for p63: initiating epithelial stratification during development and maintaining the proliferative potential of basal keratinocytes in mature epidermis (Figure 3). This hypothesis is further supported by the identification of p63 target genes that are involved in epidermal development and differentiation (
Figure 3Proposed role of p63 in embryonic and mature epidermis. Epithelia that do not express p63 remain single-layered. Upon induction of p63 expression, epithelia commit to initiate a stratification program (A). In the mature epidermis, p63 is expressed in the basal layer, and its expression is downregulated in the differentiated layers. Expression of p63 in the basal layer may maintain the proliferative potential of keratinocytes (B)
Mutations in p63 were shown to underlie a number of human ectodermal dysplasias, which are characterized by abnormalities of the limbs, hair, teeth, nails, sweat glands, and mammary glands (
). Therefore, consistent with the phenotype of p63-/- mice, abnormalities in p63 gene function in humans appear to disrupt the differentiation process of epithelial tissues and their derivatives. All human ectodermal dysplasias caused by p63 mutations are inherited in an autosomal-dominant fashion. Since humans that have a heterozygous deletion of the p63 gene do not develop characteristics of ectodermal dysplasias, it has been suggested that the p63 mutations result in a dominant-negative effect or a gain-of-function. Interestingly, a genotype–phenotype correlation was shown to exist based on the clustering of p63 mutations in different ectodermal dysplasias. For example, patients with ectodermal dysplasia and cleft lip (EEC) harbor missense p63 mutations in the DNA binding domain (
). Interestingly it was found that mutations in the SAM domain abrogate the interaction of p63 with ABBP1, which may partially account for the defects in epithelial development and differentiation observed in these patients (
). Since the SAM domain is only contained in p63α isoforms, this suggests that p63α isoforms are essential for development of ectodermally derived tissues.
Genes that are active during normal development are frequently found to be dysregulated during neoplastic transformation. A number of studies have investigated the role of p63 in neoplastic transformation and tumor progression. Although p63 does not function as a classical tumor suppressor gene (
Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray.
A simple specific pattern of chromosomal aberrations at early stages of head and neck squamous cell carcinomas: PIK3CA but not p63 gene as a likely target of 3q26-qter gains.
). These transgenic mice were more resistant to UV-B induced apoptosis than control littermates, suggesting that ΔNp63α has an oncogenic role. Based on in vitro evidence suggesting that ΔNp63 isoforms have a dominant-negative function towards TAp63 isoforms (
p53 gene family p51(p63)-encoded, secondary transactivator p51B(TAp63alpha) occurs without forming an immunoprecipitable complex with MDM2, but responds to genotoxic stress by accumulation.
), it has been proposed that TAp63 isoforms may have tumor suppressing abilities. But all of these in vitro studies have been performed in cell lines derived from tissues that normally do not express p63. Since ΔNp63α was shown to have cell type-specific functions (
), this may also be the case for TAp63 isoforms. Therefore, like ΔNp63 isoforms, TAp63 isoforms may also have an oncogenic function.
In summary, although the roles of the different p63 isoforms are still elusive, the current data suggest that p63 has a dual role. During development p63 is required for the initiation of an epithelial stratification program, whereas in the mature epidermis p63 is required for the maintenance of the proliferative potential of basal keratinocytes. Deregulation of p63 expression can result in ectodermal dysplasias and SCC. The molecular role of p63 in these disorders, however, remains to be determined and will be further elucidated by the identification of additional interacting proteins and downstream target genes.
c-Myc and p63 have complimentary roles in regulating epidermal homeostasis through the regulation of proliferation and differentiation, respectively. Deregulation of either of these processes leads to human disease. The molecular mechanisms by which these genes regulate epidermal homeostasis are currently being analyzed and future studies will uncover the missing links between these genes and the human diseases in which they are involved.
This work was supported by NIH Grants: AR47898, CA52607, and HD25479 to D.R.R.
References
Andl T.
Reddy S.T.
Gaddapara T.
Millar S.E.
WNT signals are required for the initiation of hair follicle development.
p53 gene family p51(p63)-encoded, secondary transactivator p51B(TAp63alpha) occurs without forming an immunoprecipitable complex with MDM2, but responds to genotoxic stress by accumulation.
A simple specific pattern of chromosomal aberrations at early stages of head and neck squamous cell carcinomas: PIK3CA but not p63 gene as a likely target of 3q26-qter gains.
Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray.
The Delta Np63 alpha phosphoprotein binds the p21 and 14-3-3 sigma promoters in vivo and has transcriptional repressor activity that is reduced by Hay–Wells syndrome-derived mutations.
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