126 EphA2 is a novel regulator of autolysosome recycling at end-stage autophagy and a key regulator in epidermal proliferation

Abstract

Abnormal autophagy is observed in several skin diseases associated with hyperproliferation. We have shown that Beclin1 is increased in psoriatic lesions and when Beclin1 is conditionally knocked out in the epidermis, imiquimod(IMQ)-induced hyperproliferation is markedly attenuated. This indicates that autophagy plays a positive role in psoriasis and contributes to epidermal hyperproliferation in psoriatic lesions. However, how autophagy is regulated in the epidermis of psoriatic lesions is a knowledge gap that needs to be filled. We show that loss of EphA2, a receptor tyrosine kinase, reduces proliferation in mouse epidermis and human keratinocytes. Importantly, expression of EphA2 was shown to be increased in psoriatic lesions. We demonstrate that knockdown of EphA2 in HEKs results in an increase in: (i) large vacuoles; and (ii) LC3II protein, a marker for autophagosomes. To further explore this defect in autophagy, we measured autophagy flux by two well-established approaches: LC3II turnover and p62 levels, and found that loss of EphA2 reduced LC3II turnover in HEKs; and increased p62 in HEKs as well as in mouse epidermis, indicating an inhibition of autophagy flux. To determine the novel interactors of EphA2 in autophagy, we conducted BioID and co-immunoprecipitation assays and confirmed that among the novel binding partners of EphA2 was phospholipase D1 (PLD1). We have shown that PLD1 is a critical regulator of autolysosome recycling at end-stage autophagy. We have also demonstrated that the activity of dynamin1 (one of key players in autolysosome recycling at end-stage autophagy) and its association with autolysosomes is inhibited by PDL1/PKC pathway in HEKs. EphA2 affects autolysosome recycling via inhibiting PLD1/PKC signaling and thus ensuring dynamin1 activity and its association with autolysosome. Thus, we conclude that upregulation of EphA2 in the “activated” epidermis contributes to enhanced autophagy and consequently a hyperproliferative state.