The Retinoic Acid Receptor-related Orphan Receptor RORα Regulates Differentiation And Survival Of Keratinocytes During Hypoxia

Low O2 pressures present in the microenvironment of epidermis control keratinocyte differentiation and epidermal barrier function through hypoxia inducible factors（HIFs）dependent gene expression.The retinoic acid receptor-related orphan receptor alpha（RORα）belongs to the steroid nuclear hormone receptor superfamily and is widely expressed in a variety of tissues.Current research has found that the transcription of RORA gene could be up-regulated by hypoxia in a panel of cell lines derived from different tissues,albeit none from the skin and the role of RORαin keratinocyte differentiation and epidermal barrier formation is unclear under hyouxia.Objective:This study focuses on investigating relations of the retinoic acid receptor-related orphan receptor alpha（RORα）to HIF-1αin keratinocytes under hypoxic conditions;this study will provide new targets for the treatment of cutaneous diseases related to defective epidermal barrier functions.Methods:1.We detect the role of hypoxia in the expression of RORαand other genes related to differentiation in immortal human keratinocyte cell line HaCat and murine keratinocyte PAM212 using the Real-time PCR and Western blotting.2.To detect the function of RORαin keratinocyte differentiation under hypoxia,we adopted the loss-of-function approach.Two siRNA or two lenti-viral shRNAs targeting all RORαisoforms silence the expression of RORαor HIF-1α.3.Using caspase 3/7 activity analysis and Annexin V staining-FACS analysis to detect the protective action of RORαin hypoxic human keratinocytes.4.Western blotting and immunofluorescence technique were used to detecte the role of hypoxia-induced RORαin the expression of HIF-1αprotein and the accumulation of the nucleus.Then luciferase assay is used to detect the effect of RORαon transcriptional activity of HIF-1α.Result:1.Hypoxia induces RORαexpression and late differentiation in keratinocytes.2.Knock down of RORαor HIF-1αcan significantly inhibit late differentiation of keratinocytes and epidermal barrier formation.3.We also found that HIF1A gene silencing also abrogated the hypoxic expression of RORαat both mRNA and protein levels,these outcomes suggest that HIF-1αlikely functions upstream of RORαin the pathway that leads to transactivation of genes associated with late differentiation and epidermal barrier function.4.RORαcan inhibit hypoxia-induced keratinocytes apoptosis.This suggests that the pro-survival function of RORαin keratinocytes under hypoxic conditions.5.Hypoxia-induced RORαincreases the stability of HIF-1αprotein and promote its accumulation in the cell nucleus and then enhance the transcription activity HIF-1α.Conclusion:In summary,we have revealed that hypoxia–induced RORαcan promote the late terminal differentiation,epidermal barrier function and survival of keratinocytes under low O₂ tension through enhancing HIF transcriptional activity by stabilizing the expression of HIF-1αprotein and promoting accumulation of HIF-1αprotein in the nucleus.