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DeltaNp63? protein triggers epithelial-mesenchymal transition and confers stem cell properties in normal human keratinocytes.

ABSTRACT: p63 is a p53 family protein required for morphogenesis and postnatal regeneration of epithelial tissues. Here we demonstrate that ?Np63?, a p63 isoform lacking the N-terminal transactivation domain, induces epithelial-mesenchymal transition (EMT) in primary human keratinocytes in a TGF-?-dependent manner. Rapidly proliferating normal human epidermal keratinocytes (NHEK) were infected with retroviral vector expressing ?Np63? or empty vector and serially subcultured until replicative senescence. No phenotypic changes were observed until the culture reached senescence. Then the ?Np63?-transduced cells underwent morphological changes resembling mesenchymal cells and acquired the EMT phenotype. Treatment with exogenous TGF-? accelerated EMT in presenescent ?Np63?-transduced cells, whereas the inhibition of TGF-? signaling reversed the EMT phenotype. TGF-? treatment alone led to growth arrest in control NHEK with no evidence of EMT, indicating that ?Np63? altered the cellular response to TGF-? treatment. ?Np63?-transduced cells acquiring EMT gained the ability to be differentiated to osteo-/odontogenic and adipogenic pathways, resembling mesenchymal stem cells. Furthermore, these cells expressed enhanced levels of Nanog and Lin28, which are transcription factors associated with pluripotency. These data indicate that EMT required ?Np63? transduction and intact TGF-? signaling in NHEK.

SUBMITTER: Oh JE

PROVIDER: S-EPMC3207403 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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DeltaNp63α protein triggers epithelial-mesenchymal transition and confers stem cell properties in normal human keratinocytes.

Oh Ju-Eun JE Kim Reuben H RH Shin Ki-Hyuk KH Park No-Hee NH Kang Mo K MK

The Journal of biological chemistry 20110831 44

p63 is a p53 family protein required for morphogenesis and postnatal regeneration of epithelial tissues. Here we demonstrate that ΔNp63α, a p63 isoform lacking the N-terminal transactivation domain, induces epithelial-mesenchymal transition (EMT) in primary human keratinocytes in a TGF-β-dependent manner. Rapidly proliferating normal human epidermal keratinocytes (NHEK) were infected with retroviral vector expressing ΔNp63α or empty vector and serially subcultured until replicative senescence. N ...[more]

PMID: 21880709

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Project description:Background:Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients. Several treatment modalities have been put forth, but as yet no satisfactory approach to the prevention or treatment of keloids has been identified. The process of epithelial-to-mesenchymal transition (EMT) has been implicated in keloid scarring, as keloid keratinocytes display an EMT-like phenotype. This study investigated the potential of pirfenidone, an antifibrotic agent, to counteract EMT-like alterations in keloid keratinocytes, including gene expression, cell migratory and proliferative functions. Methods:Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues, respectively. Cells were quiesced for 24 h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1 (TGF-?1; 10 ng/mL), with or without pirfenidone (400 ?g/mL), for an additional 24 h. The effects of pirfenidone on cytotoxicity, cell migration, cell proliferation, and on expression of genes and proteins involved in EMT were assayed. Statistical significance was determined by two-way ANOVA using Sigma Plot. Results:We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000 ?g/mL. A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000 ?g/mL. Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes, but a significant decrease in TGF-?1-induced cell migration was seen only in keloid keratinocytes. Significant inhibition of the expression of TGF-?1-induced core EMT genes, namely hyaluronan synthase 2, vimentin, cadherin-11, and wingless-type MMTV integration site family, member 5A along with fibronectin-1, was observed in both normal and keloid keratinocytes treated with pirfenidone. In addition, the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone (400 ?g/mL) in both normal and keloid keratinocytes. Conclusions:For the first time, this study shows the efficacy of pirfenidone in inhibiting the EMT-like phenotype in keratinocytes derived from keloids, suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence.

Dataset Information

DeltaNp63? protein triggers epithelial-mesenchymal transition and confers stem cell properties in normal human keratinocytes.

Publications

DeltaNp63α protein triggers epithelial-mesenchymal transition and confers stem cell properties in normal human keratinocytes.

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