228 Loss of EPHA2 represses GATA-3 function and causes a terminal differentiation defect

Abstract

Epidermal morphogenesis and differentiation require the coordination of complex signal transduction networks. These signal relays are often initiated at the plasma membrane and transmitted to the nucleus to control gene expression. Receptor tyrosine kinases (RTKs) are integral in orchestrating communication cascades to induce differentiation. We report loss of EPHA2 RTK causes a terminal differentiation defect in 3D human skin equivalents (3D HSE) resulting in loss of stratification and ablation of the granular and cornified layers. In EPHA2-deficient (shEPHA2) 3D HSE, we show significant loss (P<0.05) of loricrin, filaggrin, and involucrin protein and mRNA, indicating EPHA2 signaling can impact keratinocyte differentiation at the transcriptional level. The transcription factor GATA-3 is a key driver of differentiation-associated gene expression in epidermis, although the upstream signals regulating its activity are unknown. GATA-3 is expressed in the nuclei of suprabasal keratinocytes in mature 3D HSE, mimicking the pattern in normal human skin (R2=0.99). In 2D cultures, GATA-3 accumulates in the nucleus following 24 h exposure to 1.2 mM calcium. However, GATA-3 protein and transcript expression are lost in shEPHA2 3D HSE and 2D cultures (P<0.001). Further, there is a decrease of GATA-driven transcription indicated by a significant loss in GATA binding activity in a promoter luciferase reporter assay (P<0.001). Re-expression of nuclear GATA-3 using a retroviral construct in EPHA2-deficient 3D HSE restored differentiation. Taken together, these results indicate that EPHA2 promotes GATA-3 nuclear accumulation to positively regulate the transcription of terminal differentiation genes in epidermal keratinocytes.