An ErbB receptor-mediated AP-1 regulatory network is modulated by STAT3 and c-MYC during calcium-dependent keratinocyte differentiation

A calcium gradient in skin epidermis is known to regulate epidermal differentiation. In cultures of human epidermal keratinocytes (NHEK), induction of calcium-dependent differentiation is associated with phosphorylation of ErbB receptors, including the epidermal growth factor receptor (EGFR). The activation of EGFR triggers the induction of activator protein 1 (AP-1) proteins necessary for keratinocyte terminal differentiation. Interestingly, an in vitro long-term calcium treatment revealed the activation of different ErbB receptors with different timings, which is consistent with the differential localization of each receptor in the skin layers in vivo. In the current study, the regulatory relationship between ErbB receptor activation and induction of AP-1 proteins in calcium-dependent keratinocyte differentiation was analysed. For this purpose, we used a mathematical method to predict molecular regulations from time-course proteomic data of 30 target components. The predicted network showed that the ErbB receptor might regulate AP-1 protein expression via two pathways: positive regulation by c-MYC and negative regulation by signal transducer and activator of transcription 3 (STAT3) pathways. Experimental validation analysis revealed that ErbB receptor inhibition resulted in defective induction of AP-1 proteins and suppressed terminal differentiation of keratinocytes. Inhibition of STAT3, however, affected expression of a partial set of AP-1 proteins and resulted in premature differentiation. Studies using RNAi to diminish expression of each ErbB receptor and the AP-1 proteins strongly suggested that STAT3 established a balance that maintains the appropriate set of AP-1 proteins and participates in a complex network for regulating keratinocyte differentiation.