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.     

Regulation of ΔNp63α by NFκΒ

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be down regulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NF-κΒ in presence of cisplatin. We find that NF-κΒ promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NF-κΒ leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NF-κΒ with siRNA-mediated silencing NF-κΒ expression attenuates chemotherapy induced degradation of ΔNp63α . These data demonstrate that NF-κΒ plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NF-κΒ may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.     

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.     

Cell density-dependent acetylation of ΔNp63α is associated with p53-dependent cell cycle arrest

ΔNp63α is a p63 isoform that is predominantly expressed in the epidermal stem cells and in cancer. To find the regulatory pathways of ΔNp63α, we assessed whether ΔNp63α is acetylated and determined the functional implications of acetylation. First, the hinge region of p63 was shown to be acetylated by PCAF, similarly to other p53 family members. Second, acetylation synergistically induced cytoplasmic localization of ΔNp63α. Finally, acetyl-ΔNp63α was induced during high-density culture, suggesting that acetylation of ΔNp63α may reinforce cell cycle arrest upon cell contact. Altogether, these findings suggest that acetylation of ΔNp63α contributes to the epidermal homeostasis.     

Dysregulated ΔNp63α inhibits expression of Ink4a/arf, blocks senescence, and promotes malignant conversion of keratinocytes

p63 is critical for squamous epithelial development, and elevated levels of the ΔNp63α isoform are seen in squamous cell cancers of various organ sites. However, significant controversy exists regarding the role of p63 isoforms as oncoproteins or tumor suppressors. Here, lentiviruses were developed to drive long-term overexpression of ΔNp63α in primary keratinocytes. Elevated levels of ΔNp63α in vitro promote long-term survival and block both replicative and oncogene-induced senescence in primary keratinocytes, as evidenced by the expression of SA-β-gal and the presence of nuclear foci of heterochromatin protein 1γ. The contribution of ΔNp63α to cancer development was assessed using an in vivo grafting model of experimental skin tumorigenesis that allows distinction between benign and malignant tumors. Grafted lenti-ΔNp63α keratinocytes do not form tumors, whereas lenti-GFP/v-ras(Ha) keratinocytes develop well-differentiated papillomas. Lenti-ΔNp63α/v-ras(Ha) keratinocytes form undifferentiated carcinomas. The average volume of lenti-ΔNp63α/v-ras(Ha) tumors was significantly higher than those in the lenti-GFP/v-ras(Ha) group, consistent with increased BrdU incorporation detected by immunohistochemistry. The block in oncogene-induced senescence corresponds to sustained levels of E2F1 and phosphorylated AKT, and is associated with loss of induction of p16(ink4a)/p19(arf). The relevance of p16(ink4a)/p19(arf) loss was demonstrated in grafting studies of p19(arf)-null keratinocytes, which develop malignant carcinomas in the presence of v-ras(Ha) similar to those arising in wildtype keratinocytes that express lenti-ΔNp63α and v-ras(Ha). Our findings establish that ΔNp63α has oncogenic activity and its overexpression in human squamous cell carcinomas contributes to the malignant phenotype, and implicate its ability to regulate p16(ink4a)/p19(arf) in the process.     

Culture of limbal stem cells on human amniotic membrane

Limbal stem cells (LSC) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell (HLECs) transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. A major challenge is the ability to identify LSC in vitro and in situ, and one of the major controversies in the field relates to reliable LSC markers. This study was carried out to evaluate the culture of a limbal biopsy on human amniotic membrane (HAM): directly on the chorionic side and on intact epithelium, and the expression of the stem cell associated markers: ABCG2, p63. HAM has been extensively used for ocular surface reconstruction and has properties which facilitate the growth of epithelial cells controlling inflammation and scarring.     

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.     

Ionizing radiation-induced TAp63α phosphorylation at C-terminal S/TQ motifs requires the N-terminal transactivation (TA) domain

TAp63α, a homolog of p53 and one of six alternatively spliced p63 isoforms, is a critical mediator of the ionizing radiation (IR)-induced DNA damage response in female germ cells and also tumor suppression in somatic cells. The ΔNp63α isoform, lacking the N-terminal transactivation (TA) domain, is associated with oncogenic potential. The mechanism of p63 functional regulation is not well understood. TAp63α is phosphorylated by ionizing radiation (IR)-induced DNA damage and gene transactivation is likely to be involved. Based on information gleaned from studies on p53, we explored the possibility that TAp63α S/TQ sites may be phosphorylated by IR-induced DNA damage. Our findings show a wortmanin-sensitive kinase phosphorylates TAp63α at C-terminal Ser-Gln and Thr-Gln (S/TQ) sites but not N-terminal S/TQ sites. ΔNp63α, lacking the TA domain, and TAp63γ, lacking C-terminal domains, including S/TQ sites, fail to undergo IR-induced phosphorylation. We propose a model for TA domain-dependent C-terminal phosphorylation drawing from previously described self-inactivating intramolecular interaction between N-terminal TA domain and C-terminal Transactivation Inhibitory Domain (TID) of TAp63α. A specific topology adopted only by TAp63α, but not possible for ΔNp63α or TAp63γ, may lead to TAp63α-specific kinase recruitment, phosphorylation and self-inactivation release. TID-lacking TAp63γ, like p53, is constitutively active and thus may forgo phosphorylation-dependent activation. Thus, p53 is regulated by protein stabilization and TAp63α by protein activation but both appear to involve S/TQ phosphorylation. The difference in phosphorylation potential of TAp63α and ΔNp63α may in part help explain why the two similar isoforms have diametrically opposite tumor suppression and oncogene functions, respectively.