A dominant negative form of p63 inhibits apoptosis in a p53-independent manner

Stem cells are a source of differentiated cells in multiple tissues. If genetic alterations occur in stem cells, the problem persists and malignant cancers may arise. DeltaNp63alpha-a homologue of the tumor suppressor p53-is exclusively expressed in proliferating undifferentiated epithelial cells and cancer cells of epidermal origin. Here, we show that DeltaNp63alpha antagonizes DNA damage-induced apoptosis in a p53-independent manner. We found that upon cellular injury, DeltaNp63alpha must be downregulated before apoptotic program can be activated. The 5637 cell line has abundant levels of DeltaNp63alpha and mutant p53, and it is resistant to DNA damage-induced apoptosis. The knockdown of DeltaNp63alpha by RNA interference sensitized these cells to apoptosis upon genotoxic insult. This suggests that DeltaNp63alpha plays an anti-apoptotic role regardless of the p53 status. Considering the frequent mutations of p53 in tumor cells, our results provide important implications for the treatment of cancers in which p63 is amplified.     

Embryonic stem cells as a cellular model for neuroectodermal commitment and skin formation

Embryonic stem (ES) cells can be differentiated into many cell types in vitro, thus providing a potential unlimited supply of cells for cognitive in vitro studies and cell-based therapy. We recently reported the efficient derivation of ectodermal and epidermal cells from murine ES cells. These differentiated ES cells were able to form, in culture, a multilayered epidermis coupled with an underlying dermal compartment, similar to native skin. We clarified the function of BMP-4 in the binary neuroectodermal choice by stimulating sox-1(+) neural precursors to undergo specific apoptosis while inducing epidermal differentiation through DeltaNp63 gene activation. We further demonstrated that DeltaNp63 enhances ES-derived ectodermal cell proliferation and is necessary for epidermal commitment. This unique cellular model further provides a powerful tool for identifying the molecular mechanisms controlling normal skin development and for investigating p63-ectodermal dysplasia human congenital pathologies.     

ATM kinase is a master switch for the ΔNp63α phosphorylation/degradation in human head and neck squamous cell carcinoma cells upon DNA damage

We previously found that the pro-apoptotic DNA damaging agent, cisplatin, mediated the proteasome-dependent degradation of DeltaNp63alpha associated with its increased phosphorylated status. Since DeltaNp63alpha usually plays an opposite role to p53 and TAp63 in human cancers, we tested the notion that phosphorylation events induced by DNA damage would affect the protein degradation of DeltaNp63alpha in HNSCC cells upon cisplatin exposure. We found that DeltaNp63alpha is phosphorylated in the time-dependent fashion at the following positions: S385, T397, and S466, which were surrounded by recognition motifs for ATM, CDK2 and p70s6K kinases, respectively. We showed that chemical agents or siRNA inhibiting the activity of ATM, CDK2 and p70s6K kinases blocked degradation of DeltaNp63alpha in HNSCC cells after cisplatin exposure. Site-specific mutagenesis of DeltaNp63alpha residues targeted for phosphorylation by ATM, CDK2 or p70s6k led to dramatic modulation of DeltaNp63alpha degradation. Finally, we demonstrated that the DeltaNp63alpha protein is a target for direct in vitro phosphorylation by ATM, CDK2 or p70s6K. Our results implicate specific kinases, and target phosphorylation sites in the degradation of DeltaNp63alpha following DNA damage.     

p63 expression during normal cutaneous wound healing in humans

p63, a recently identified member of the p53 family, was shown to play a role in morphogenesis and, probably, in tumors of keratinocyte origin. Because p63 seems to be a marker of keratinocytes with a high proliferative potential, the expression of this protein was studied along with another marker of cell proliferation, Ki67, during normal epidermal regeneration in humans. Serial biopsies of human skin healing by a secondary intention were taken at various time intervals (between days 2 and 21 after the injury) and were studied immunohistochemically with the use of a 4A4 monoclonal antibody against the DeltaNp63 variant and MM1 monoclonal antibody against the Ki67 antigen. In the normal and injured skin, the expression of the DeltaNp63 protein was restricted to the epidermal keratinocytes and hair follicle keratinocytes. In the first days of the healing process, there was a dramatic down-regulation of both DeltaNp63 and Ki67 expression in the area of the epidermal tongue invading under the crust. Five days after the injury, induction of DeltaNp63 in the basal keratinocytes could be detected, followed by a gradual increase of its expression in subsequent days. Several days after complete wound closure, DeltaNp63 was still strongly expressed not only in the basal keratinocytes but also in the entire spinous layer, whereas the Ki67 expression was restricted to single cells in the basal layer. The results indicate that DeltaNp63 could be involved in the control of physiologic processes, such as cell proliferation and migration, related to epidermal repair during healing of normal skin in humans.     

A dominant-negative form of p63 is required for epidermal proliferation in zebrafish

Epidermal stem cells play a critical role in producing the multilayered vertebrate skin. Products of the p63 gene not only mark the epidermal stem cells, but also are absolutely required for the formation of mammalian epidermis. We find that early zebrafish embryos express a dominant-negative form of p63 (DeltaNp63), which accumulates in the nucleus just as epidermal growth begins. Using antisense morpholino oligonucleotides, we show that DeltaNp63 is needed for epidermal growth and limb development and is specifically required for the proliferation of epidermal cells by inhibiting p53 activity. While the structure of fish epidermis is very different from that of higher vertebrates, our study shows that DeltaNp63 has essential and ancient role in the development of skin.     

Glycogen synthase kinase-3beta regulates DeltaNp63 gene transcription through the beta-catenin signaling pathway

Overexpression of DeltaNp63 has been observed in a number of human cancers, suggesting a role for DeltaNp63 in carcinogenesis. In the present study, we show that inhibition of glycogen synthase kinase-3beta (GSK-3beta) by lithium chloride (LiCl) elicited a stimulatory effect on DeltaNp63 promoter activity in HEK 293T cells. Exposure to LiCl induced DeltaNp63 promoter activation as well as DeltaNp63 protein expression in the cells. The effect of GSK-3beta on DeltaNp63 expression was further confirmed by the use of two highly specific GSK-3beta inhibitors, SB216763 and SB415286. Further study showed the presence of a putative beta-catenin responsive element (beta-catenin-RE) in the DeltaNp63 promoter region, and the stimulation of DeltaNp63 promoter activity by GSK-3beta inhibitor is markedly abolished by mutation or deletion of the putative beta-catenin-RE. Data are also presented to show that beta-catenin acts together with Lef-1 to influence DeltaNp63 promoter activity and protein expression.     

Tumor necrosis factor alpha-induced apoptosis requires p73 and c-ABL activation downstream of RB degradation

The retinoblastoma protein (RB) suppresses cell proliferation and apoptosis. We have previously shown that RB degradation is required for tumor necrosis factor alpha (TNF-alpha) to induce apoptosis. We show here the identification of two apoptotic effectors, i.e., c-ABL tyrosine kinase and p73, which are activated by TNF-alpha following RB degradation. In cells expressing a degradation-resistant RB protein (RB-MI), TNF-alpha does not activate c-ABL. RB-MI also inhibits TNF-alpha-mediated activation of p73. Genetic deletion and pharmacological inhibition of c-ABL or p73 diminish the apoptotic response to TNF-alpha in human cell lines and mouse fibroblasts. Thymocytes isolated from Rb(MI/MI), Abl(-/-), or p73(-/-) mice are resistant to TNF-alpha-induced apoptosis compared to their wild-type counterparts. This is in contrast to p53(-/-) thymocytes, which exhibit a wild-type level of apoptosis in response to TNF-alpha. Thus, c-ABL and p73 contribute to apoptosis induced by TNF-alpha, in addition to their role in promoting DNA damage-associated cell death.     

DeltaNp63 is essential for epidermal commitment of embryonic stem cells

In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.     

p63/p51-induced onset of keratinocyte differentiation via the c-Jun N-terminal kinase pathway is counteracted by keratinocyte growth factor

p63/p51, a homolog of the tumor suppressor protein p53, is chiefly expressed in epithelial tissues, including the epidermis. p63 affects cell death similar to p53, and also plays important roles in the development of epithelial tissues and the maintenance of epithelial stem cells. Because it remains unclear how p63 regulates epithelial cell differentiation, we examined the function(s) of p63 in keratinocyte differentiation through the use of a keratinocyte culture system. DeltaNp63alpha (DeltaNp51B), a p63 isoform specifically expressed in basal keratinocytes, suppressed the differentiation of specific late-stage proteins, such as filaggrin and loricrin. In contrast, DeltaNp63alpha induced keratin 1 (K1), which is expressed at the start of differentiation, via c-Jun N-terminal kinase (JNK)/AP-1 activation. However, p63 did not induce K1 expression in the basal layer in vivo, although basal keratinocytes had high levels of p63. This discrepancy was explained by the suppression of K1 expression by dermis-secreted keratinocyte growth factor. This suppression occurred via extracellular signal-related kinase (ERK) signaling, and counteracted the p63-mediated induction of K1. Thus, a precise balance between p63 and keratinocyte growth factor mediates the onset of epithelial cell differentiation, through JNK and ERK signaling. These data may provide mechanistic explanations for the pathological features of skin diseases, including psoriasis.     

Porcine epidermal stem cells as a biomedical model for wound healing and normal/malignant epithelial cell propagation

This article summarizes research using cells derived from epidermis of the miniature pigs for use as a cell therapy for skin repair and as a model for squamous carcinoma of the head and neck. Stem cells are an important "tool" for biomedical research. Adult stem cells are defined functionally, as cells that have the capacity to self-renew as well as the ability to generate differentiated cells. They are present in defined tissue microenvironments called niches. Asymmetric mitosis allows them to produce one daughter cell with the properties of stem cells (self-renewal) and a second cell with characteristics of progenitor cells, or transit amplifying cells, which proliferate quickly but with a limited number of mitotic divisions. Porcine epidermal stem cells, located in the bulge region of the outer root sheath of hair follicles, migrate in vitro from hair sheaths and because they are resistant to anoikis (detachment induced apoptosis), survive in non-adhesive conditions to form spheroids. These cells express keratins, galectin-1 and their nuclei are rich in DeltaNp63alpha. Interestingly, the multiple phenotype analysis of the human tumor cells in squamous carcinoma of head and neck revealed similarities with epidermal stem cells. These cancer stem cells are usually located on the periphery of the tumor where the invasive front of the tumor responsible for its aggressive behavior is located. In contrast, extensive expression of markers of terminal differentiation such as expression of glycoligands reactive for the endogenous lectin, galectin-3, indicates better tumor prognosis.