p63 Knockdown Induces GRHL2 Downregulation and EMT In Human Keratinocytes

Objectives: Grainyhead-like 2 (GRHL2), a transcription factor responsible for regulating epithelial proliferation, is known to play an important role in epithelial cancers and has been found to regulate epithelial-mesenchymal transition (EMT). We found that EMT is regulated by transcription factor p63 in human keratinocytes. In the current study, we investigated the interplay between GRHL2 and p63 and its role in regulating EMT, stemness, and multipotency in normal human epithelial keratinocytes (NHEK). Methods: NHEK were transiently transfected with siRNA targeting p63 (si-p63). Onset of EMT was assessed by altered cellular morphology and expression of EMT markers, including fibronectin (FN), E-Cadherin (E-Cad), and ZEB proteins. Stemness was assessed by change in expression of cell surface markers, e.g., CD44, CD73, CD90, and CD105. Expression of p63 and GRHL2 were assessed in si-p63/NHEK. Multipotent capacity of these cells was assessed by assaying for Alkaline Phosphatase (ALP) activity, Alizarin Red staining, and Oil-Red-O staining for odontogenic, osteogenic, and adipogenic differentiation, respectively. Results: si-p63/NHEK exhibited EMT-associated morphological changes, e.g. elongated, spindle-shaped morphology, as well as enhanced FN and loss of E-Cad expression. These cells also exhibited enhanced ZEB1, CD44, CD90, and CD105 expression, and suppressed CD73 levels. GRHL2 expression was inhibited in NHEK by p63 knockdown, and p63 was suppressed by GRHL2 knockdown, indicating mutual regulation. si-p63/NHEK exhibited strong ALP, Alizarin Red, and Oil-Red-O staining. Conclusion: These results indicate that knockdown of p63 in NHEK induces EMT by downregulation of GRHL2, and that knockdown of p63 induces stemness and multipotency in NHEK.