Receptor Tyrosine Kinase EPHA2 Drives Epidermal Differentiation through Regulation of EGFR Signaling.

Intricate signaling systems are required to maintain homeostasis and promote differentiation in the epidermis. Receptor tyrosine kinases are central in orchestrating these systems in epidermal keratinocytes. In particular, EPHA2 and EGFR transduce distinct signals to dictate keratinocyte fate, yet how these cell communication networks are integrated has not been investigated. Our work shows that loss of EPHA2 impairs keratinocyte stratification, differentiation, and barrier function. To determine the mechanism of this dysfunction, we drew from our proteomics data of potential EPHA2 interacting proteins. We identified EGFR as a high-ranking EPHA2 interactor and subsequently validated this interaction. We found that when EPHA2 is reduced, EGFR activation and downstream signaling are intensified and sustained. Evidence indicates that prolonged SRC association contributes to the increase in EGFR signaling. We show that hyperactive EGFR signaling underlies the differentiation defect caused by EPHA2 knockdown because EGFR inhibition restores differentiation in EPHA2-deficient 3-dimensional skin organoids. Our data implicate a mechanism whereby EPHA2 restrains EGFR signaling, allowing for fine tuning in the processes of terminal differentiation and barrier formation. Taken together, we purport that crosstalk between receptor tyrosine kinases EPHA2 and EGFR is critical for epidermal differentiation.

MicroRNAs-103/107 Regulate Autophagy in the Epidermis.

We have shown that microRNAs-103 and -107 (miRs-103/107) positively regulate end-stage autophagy by ensuring dynamin activity in cultured keratinocytes. Most work in end-stage autophagy has been conducted using in vitro model systems. In vivo regulation of end-stage autophagy in epidermis remains unknown. Here, we used antagomirs to subcutaneously knock down miR-107 in the skin; conversely, we delivered miR-107 mimic subcutaneously via in vivo transfection to increase this miR. We found that antagomir-107 treatment in epidermis: (i) depleted endogenous miR-107; (ii) increased GFP-LC3 puncta in epidermal basal layers of GFP-LC3 transgenic mice, indicative of an accumulation of autophagosomes; (iii) inhibited LC3 turnover and increased p62, suggesting an inhibition of autophagy flux; and (iv) increased phosphorylated dynamin (p-dynamin, an inactive form), a key enzyme in end-stage autophagy. Conversely, miR-107 mimic treatment in mouse epidermis: decreased GFP-LC3 puncta in basal layer, as well as p62 protein levels; and diminished p-dynamin, indicative of activation of this enzyme. In human epidermal keratinocytes, antagos-103/107 cause the formation of large vacuoles and an increase in p-dynamin, which can be rescued by inhibition of protein kinase C pathway. Collectively, these results suggest that the miR-103/107 family has a critical role in regulating end-stage autophagy in mouse epidermis via PLD1/2-protein kinase C-dynamin pathway.