Interferon Regulatory Factor 6 is required for keratinocyte differentiation

Mutations in the transcription factor IRF6 cause Van der Woude and popliteal ptergyrium syndromes, two orofacial clefting diseases. Among other epithelial tissues, Irf6 is expressed in the spinous layer of the epidermis. Mice deficient for Irf6 exhibited oral and limb abnormalities, and showed epidermal dysfunction, including continuous proliferation and failure to undergo keratinocyte differentiation. These data suggested that Irf6 functions to regulate keratinocyte proliferation and differentiation. To test our hypothesis, we cultured murine keratinocyte from Irf6−/− and Irf6+/+ epidermis. Under low calcium culture conditions, Irf6−/− keratinocytes showed increased proliferation and colony forming efficiency compared to wildtype cells. In vitro differentiation was achieved by increasing the calcium concentration to 0.15mM. Morphological analysis shows in vitro stratification of Irf6+/+ keratinocytes, whereas Irf6−/− cells exhibited a single monolayer, suggesting their inability to differentiate in vitro. These results were confirmed by Western blot analysis. Irf6−/− keratinocytes infected with adenovirus carrying Irf6 stratify in vitro and express keratinocyte differentiation markers. Overexpression of Irf6, however, did not induce differentiation in low calcium medium. These results identify Irf6 as a potential switch for epidermal differentiation.