Recent studies have revealed that autophagy is involved in differentiation of erythrocytes, lymphocytes, and adipocytes. Keratinocyte differentiation is also going along with activation of lysosomal enzymes and organelle clearance, expecting the contribution of autophagy in this process.
Previously, we show multiple roles of Notch signaling in the regulation of transit amplifying cells in suprabasal layers. Notch signaling induces differentiation of suprabasal cells via Hes1 independent manner, whereas Hes1 is required for maintenance of the immature status of suprabasal cells by preventing premature differentiation. In this study, we found that Hes1 directly suppressed the expression of Bnip3, whose expression is sufficient to induce terminal differentiation of keratinocytes by induction of autophagy.
Chromatin immunoprecipitation assay revealed that HES1 could directly bind to BNIP3 promoter to suppress the expression. We also found that BNIP3 was expressed in the granular layers. Consistent with the BNIP3 expression, autophagosome formation was observed in the granular layer of human epidermis. Forced expression of BNIP3 in human primary epidermal keratinocytes (HPEK) resulted in keratinocyte differentiation, whereas knockdown of BNIP3 had an opposite effect. Intriguingly, autophagy inhibitor significantly suppressed the BNIP3-stimulated differentiation of keratinocytes, suggesting that autophagy is involved in the process. Moreover, we also found that overexpression of BNIP3 induced autophagy and mitophagy in HPEK. These data clearly suggest that BNIP3 plays a crucial role in keratinocytes differentiation by inducing autophagy. Furthermore, dead cells were increased in human epidermal equivalent from BNIP3 knockdown keratinocytes, which gave us the idea that BNIP3 is also indispensable for maintenance of skin epidermis. To test the hypothesis, HPEK were irradiated with UVB. UVB irradiation stimulated BNIP3 expression and caspase-3 activation. Surprisingly, suppression of BNIP3 expression induced by UVB irradiation caused a further increase of the cleaved caspase-3 protein level, suggesting that BNIP3 has a protective effect against UVB-induced apoptosis in keratinocytes. Overall, our data shed light on functions of BNIP3, in both differentiation and maintenance of epidermal keratinocytes.