Differential expression of p63 isoforms in female reproductive organs

p63 is the identity switch for uterine/vaginal epithelial cell fate, and disruption of p63 expression by diethylstilbestrol (DES) induces cervical/vaginal adenosis in mice. In this article, we report the expression patterns of p63 isoforms (TA, DeltaN, alpha, beta and gamma) in mice, focusing on the reproductive tract. We also present the reproductive tract phenotype of female p63-/- mice. Finally, to better evaluate the potential role of p63 in human development of DES-induced cervical/vaginal adenosis, we describe the ontogeny of p63 in human female fetuses. In adult mice, the DeltaN isoforms of p63 were expressed only in squamous/basal/myoepithelial cells of epithelial tissues, while TA isoforms of p63 were highly expressed in germ cells of the ovary and testis. In fetal mice, the DeltaN and alpha forms of p63 were expressed in the cloacal and urogenital sinus epithelia. In the female p63-/- mice, the sinus vagina developed, but p63-/- sinus vaginal epithelium failed to undergo squamous differentiation confirming an essential role of p63 in squamous epithelial differentiation. Although TAp63 was highly expressed in developing primordial germ cells/oocytes, p63-/- ovaries and oocytes developed normally. The ontogeny of p63 in female reproductive organs was essentially identical in mouse and human. In the human fetus at the susceptible stage for DES-induced cervical/vaginal adenosis, most cervical/vaginal epithelial cells were columnar and negative for p63. Therefore, inhibition of p63 expression by DES should change the cell fate of human Müllerian duct epithelial cells and cause cervical/vaginal adenosis as previously demonstrated in mouse.     

The development of cervical and vaginal adenosis as a result of diethylstilbestrol exposure in utero

Exposure to exogenous hormones during development can result in permanent health problems. In utero exposure to diethylstilbestrol (DES) is probably the most well documented case in human history. DES, an orally active synthetic estrogen, was believed to prevent adverse pregnancy outcome and thus was routinely given to selected pregnant women from the 1940s to the 1960s. It has been estimated that 5 million pregnant women worldwide were prescribed DES during this period. In the early 1970s, vaginal clear cell adenocarcinomas (CCACs) were diagnosed in daughters whose mother took DES during pregnancy (known as DES daughters). Follow-up studies demonstrated that exposure to DES in utero causes a spectrum of congenital anomalies in female reproductive tracts and CCACs. Among those, cervical and vaginal adenoses are most commonly found, which are believed to be the precursors of CCACs. Transformation related protein 63 (TRP63/p63) marks the cell fate decision of Müllerian duct epithelium (MDE) to become squamous epithelium in the cervix and vagina. DES disrupts the TRP63 expression in mice and induces adenosis lesions in the cervix and vagina. This review describes mouse models that can be used to study the development of DES-induced anomalies, focusing on cervical and vaginal adenoses, and discusses their molecular pathogenesis.     

Involvement of activin signaling in abnormalities of mouse vagina exposed neonatally to diethylstilbestrol

Perinatal exposure to a synthetic estrogen, diethylstilbestrol (DES), causes cervicovaginal adenosis and permanent hyperplastic cornified vaginal epithelium with keratinization in mice. To investigate the mechanisms of the induction of vaginal abnormalities by DES, we have focused on activin A signaling. We have found that the βA-subunit mRNA is mainly expressed in the neonatal vaginal stroma, whereas activin A receptor type IB is localized in the neonatal vaginal epithelium. SMAD2, the intracellular signaling protein, is phosphorylated in the neonatal vagina. Cell proliferation in the vaginal epithelium grown in vitro is reduced by DES treatment or by activin signaling suppression through inhibin treatment. Thus, activin A (a homodimer of the βA-subunit) in the stroma stimulates epithelial cell proliferation in the neonatal vagina. DES treatment decreases the expression of the βA-subunit and activin receptor IIB but increases the expression of the βB-subunit and inhibin receptor. Neonatal DES treatment inhibits the phosphorylation of SMAD2 in the vaginal epithelium, indicating the inhibition of activin A signaling in the vaginal epithelium by neonatal DES treatment. Treatment with DES or inhibin, a native antagonist of activin, induces adenosis-like structures and keratinization in the vagina grown in vitro. These data suggest that the suppression of activin A signaling by DES is involved in the induction of cervicovaginal adenosis and keratinization in the neonatal mouse vaginal epithelium.     

Roles of p63 in the diethylstilbestrol-induced cervicovaginal adenosis

Women exposed to diethylstilbestrol (DES) in utero develop abnormalities, including cervicovaginal adenosis that can lead to cancer. We report that transient disruption of developmental signals by DES permanently changes expression of p63, thereby altering the developmental fate of Müllerian duct epithelium. The cell fate of Müllerian epithelium to be columnar (uterine) or squamous (cervicovaginal) is determined by mesenchymal induction during the perinatal period. Cervicovaginal mesenchyme induced p63 in Müllerian duct epithelium and subsequent squamous differentiation. In p63(-/-) mice, cervicovaginal epithelium differentiated into uterine epithelium. Thus, p63 is an identity switch for Müllerian duct epithelium to be cervicovaginal versus uterine. P63 was also essential for uterine squamous metaplasia induced by DES-exposure. DES-exposure from postnatal day 1 to 5 inhibited induction of p63 in cervicovaginal epithelium via epithelial ERalpha. The inhibitory effect of DES was transient, and most cervicovaginal epithelial cells recovered expression of p63 by 2 days after discontinuation of DES-treatment. However, some cervicovaginal epithelial cells failed to express p63, remained columnar and persisted into adulthood as adenosis.     

Loss of ΔNp63α promotes mitotic exit in epithelial cells

Protein p63 is a key regulator in cell proliferation and cell differentiation in stratified squamous epithelium. ΔNp63α is the most commonly expressed p63 isoform, which is often overexpressed in human tumor. In the present work we report the potential involvement of ΔNp63α in cell cycle regulation. ΔNp63α accumulated in mitotic cells but its expression decreased during mitotic exit. Moreover, ΔNp63α knockdown promoted mitotic exit. ΔNp63α shares a conserved destruction box (D-box) motif with other potential targets of the Anaphase-Promoting Complex/Cyclosome (APC/C). Overexpression of APC/C coactivator Cdh1 destabilized ΔNp63α. Our results suggest that ΔNp63α level is cell cycle-regulated and may play a role in the regulation of mitotic exit.     

Over-expression of ΔNp63α facilitates rat corneal wound healing in vivo

To investigate the roles of ΔNp63α during corneal wound healing and the genes regulated by ΔNp63α in limbal epithelial cells. Adenovirus or shRNA targeting ΔNp63α were pre-injected into the anterior chamber of rat eyeballs and the central corneal epithelium was then wounded with NaOH. The effects of ΔNp63α expression during wound healing were observed by propidium iodide staining. In addition, limbal epithelial cells were cultured and ectopically expressed ΔNp63α by transfecting Ad-ΔNp63α. Total RNA was extracted from transfected epithelial cells and subjected to a gene expression microarray assay. The results showed that over-expression of ΔNp63α accelerated the process of corneal wound healing while knockdown of ΔNp63α impaired the process. ΔNp63α positively up-regulated several cell growth promoter genes and could be referred as a positive regulator of limbal epithelial cell proliferation. It might also inhibit cell differentiation and cell death by differential target gene regulation.     

Overexpression of ΔNp63 in a human nasopharyngeal carcinoma cell line downregulates CKIs and enhances cell proliferation

p63 belongs to a member of the tumor suppressor protein p53 family. Due to alternative promoter usage, two types of p63 proteins are produced. The ΔNp63 isoform lacks the N‐terminal transactivation domain and is thought to antagonize TAp63 and p53 in target gene regulation. ΔNp63 has been found to be overexpressed in numerous human squamous cell carcinomas, including nasopharyngeal carcinoma (NPC). However, the role of ΔNp63 overexpression in NPC pathogenesis has not been clear. In this study, we use a ΔNp63 overexpressing human NPC cell line (NPC‐076) to explore the possible roles of ΔNp63 in cell proliferation and cell‐cycle regulation. We found that the proliferation of NPC‐076 cell is greatly suppressed when the overexpressed ΔNp63 is silenced by specific ΔNp63 siRNA. Further studies show that ΔNp63 silencing results in the upregulation of CKIs, including p27^kip1 and p57^kip2 in both mRNA and protein levels. Cell‐cycle analysis shows that ΔNp63 silencing also results in an increased G1 phase cell and apoptotic cell population. Our findings indicate that ΔNp63 plays important roles in the regulation of NPC‐076 cell‐cycle progression, and may play a role in the maintenance of NPC‐076 tumor cell phenotype. J. Cell. Physiol. 219: 117–122, 2009. © 2008 Wiley‐Liss, Inc.     

ROCK‐dependent phosphorylation of NUP62 regulates p63 nuclear transport and squamous cell carcinoma proliferation

p63, more specifically its ΔNp63α isoform, plays essential roles in squamous cell carcinomas (SCCs), yet the mechanisms controlling its nuclear transport remain unknown. Nucleoporins (NUPs) are a family of proteins building nuclear pore complexes (NPC) and mediating nuclear transport across the nuclear envelope. Recent evidence suggests a cell type‐specific function for certain NUPs; however, the significance of NUPs in SCC biology remains unknown. In this study, we show that nucleoporin 62 (NUP62) is highly expressed in stratified squamous epithelia and is further elevated in SCCs. Depletion of NUP62 inhibits proliferation and augments differentiation of SCC cells. The impaired ability to maintain the undifferentiated status is associated with defects in ΔNp63α nuclear transport. We further find that differentiation‐inducible Rho kinase reduces the interaction between NUP62 and ΔNp63α by phosphorylation of phenylalanine–glycine regions of NUP62, attenuating ΔNp63α nuclear import. Our results characterize NUP62 as a gatekeeper for ΔNp63α and uncover its role in the control of cell fate through regulation of ΔNp63α nuclear transport in SCC.     

ΔNp63α promotes cellular quiescence via induction and activation of Notch3

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of self-renewing capacity in the stem cell compartments of diverse epithelial structures; however, the underlying cellular and molecular mechanisms remain incompletely defined. Cellular quiescence is a common feature of adult stem cells that may account for their ability to retain long-term replicative capacity while simultaneously limiting cellular division. Similarly, quiescence within tumor stem cell populations may represent a mechanism by which these populations evade cytotoxic therapy and initiate tumor recurrence. Here, we present evidence that ΔNp63α, the predominant TP63 isoform in the regenerative compartment of diverse epithelial structuresm, promotes cellular quiescence via activation of Notch signaling. In HC11 cells, ectopic ΔNp63α mediates a proliferative arrest in the 2N state coincident with reduced RNA synthesis characteristic of cellular quiescence. Additionally, ΔNp63α and other quiescence-inducing stimuli enhanced expression of Notch3 in HC11s and breast cancer cell lines, and ectopic expression of the Notch3 intracellular domain (N3^ICD) was sufficient to cause accumulation in G₀/G₁ and increased expression of two genes associated with quiescence, Hes1 and Mxi1. Pharmacologic inhibition of Notch signaling or shRNA-mediated suppression of Notch3 were sufficient to bypass quiescence induced by ΔNp63α and other quiescence-inducing stimuli. These studies identify a novel mechanism by which ΔNp63α preserves long-term replicative capacity by promoting cellular quiescence and identify the Notch signaling pathway as a mediator of multiple quiescence-inducing stimuli, including ΔNp63α expression.     

MSX2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol

In utero exposure to diethylstilbestrol (DES) leads to patterning defects in the female reproductive tract (FRT) and a propensity to the development of vaginal adenocarcinomas in humans. In the mouse, DES treatment similarly induces a plethora of FRT developmental defects, including stratification of uterine epithelium and presence of glandular tissue in cervix and vagina. Uterine abnormalities are associated with repression of the homeobox gene Msx2, and DES leads to an altered uterine response in Msx2 mutants including a dilated uterine lumen. Here we investigate the role of Msx2 in normal vaginal development and in FRT response to DES. During vaginal development, Msx2 is required for Tgfbeta2 and Tgfbeta3 expression and for proper vaginal epithelial differentiation. Moreover, Msx2 is involved in caudal Wolffian duct regression by promoting apoptosis. Consistently, neonatal DES exposure represses Msx2 expression in the Wolffian duct epithelium and inhibits its apoptosis and subsequent regression. Intriguingly, although DES treatment also represses Msx2 expression in the vaginal epithelium, a much more severe DES-induced vaginal phenotype was observed in Msx2 mutant mice, including a complete failure of Müllerian vaginal epithelial stratification and a severely dilated vaginal lumen, accompanied by loss of p63 and water channel protein expression. These results demonstrate a critical role for Msx2 in counteracting the effect of DES on FRT patterning and suggest that the response to DES may be highly variable depending on the genotype of an individual.     

NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs

Self-renewal of human embryonic stem cells (ESCs) is promoted by FGF and TGFbeta/Activin signaling, and differentiation is promoted by BMP signaling, but how these signals regulate genes critical to the maintenance of pluripotency has been unclear. Using a defined medium, we show here that both TGFbeta and FGF signals synergize to inhibit BMP signaling; sustain expression of pluripotency-associated genes such as NANOG, OCT4, and SOX2; and promote long-term undifferentiated proliferation of human ESCs. We also show that both TGFbeta- and BMP-responsive SMADs can bind with the NANOG proximal promoter. NANOG promoter activity is enhanced by TGFbeta/Activin and FGF signaling and is decreased by BMP signaling. Mutation of putative SMAD binding elements reduces NANOG promoter activity to basal levels and makes NANOG unresponsive to BMP and TGFbeta signaling. These results suggest that direct binding of TGFbeta/Activin-responsive SMADs to the NANOG promoter plays an essential role in sustaining human ESC self-renewal.