BMAL1/CLOCK In Regulating UVB-induced Apoptosis,DNA Damage,and Inflammatory Responses In Human Keratinocytes

A diverse array of biological processes is under circadian control.In mouse skin,ultraviolet rays(UVR)-induced apoptosis and DNA damage responses are time-of-day dependent,which is regulated by core clock proteins.BMAL1 and CLOCK are the core circadian clock genes.Recent findings suggested that BMAL1 and CLOCK genes expression could be altered in human keratinocytes under UVB irradiation.In addition,the activity of BMAL1 can be regulated by ROR?.However,the functions of BMAL1 and CLOCK in human keratinocytes is still unknown under UVB irradiation.Objective:This study investigates the role of BMAL1,CLOCK and ROR?in regulating human keratinocytes responses under UVB irradiation;the molecular mechanism of BMAL1 and ROR?in human keratinocytes under UVB exposed.And this study will provide new program to preventing and treating skin related diseases including cancers.Methods:1.In vitro cultured immortal human keratinocytes(HaCat)and primary human keratinocytes(HKCs).2.The expression of circadian clock genes,ROR?and other genes which are related to DNA damage responses in HaCat and HKCs were detected by real time RT-PCR and western blot.3.To detect the functions of circadian clock genes in human keratinocytes under UVB radiation,siRNA targeting BMAL1 or CLOCK was used for silencing its expression.4.Lentiviral shRNA targeting ROR?was used in ROR?silencing.5.Apoptotic response was detected by western blot and Annexin V staining FACs analysis.6.Real time RT-PCR,western blot and immunofluorescence methods were used to analyze human keratinocytes DNA damage responses under UVB radiation.Result:1.The expression of BMAL1,CLOCK and ROR?genes was altered by low dose(5 mJ/cm~2)of UVB in human keratinocytes.2.The expression of pro-inflammatory cytokines were altered by low dose(5 mJ/cm~2)of UVB irradiation in HaCat cells.3.Knockdown of BMAL1,CLOCK and ROR?genes causing reduced apoptotic and immune responses observed in human keratinocytes following UVB irradiation.4.In HaCat cells,although depletion of BMAL1 or CLOCK inhibited UVB induced expression of DNA damage marker?-H2AX and cell cyle inhibitor p21.5.In HKCs,UVB induced p53 protein accumulation was blocked by depletion of CLOCK.6.Depletion of either BMAL1 or CLOCK gene triggered early keratinocyte differentiation of HKCs at their steady state.7.Overexpression or knockdown of ROR?gene abrogated the expression of BMAL1 under UVB irradiation condition in HaCat cells.Conclusion:Above all,these results suggested that UVB induced apoptosis,DNA damage responses and inflammation are controlled by circadian clock genes,but the mechanisms are different between HaCat keratinocytes and primary human keratinocytes.ROR?has the similar function with BMAL1 or CLOCK in HaCat cells following UVB irradiation,but the mechanisms need further more investigation.Given that UV is the leading cause of skin diseases,elucidation of clock gene functions in regulating UVB response in the human skin should be important and beneficial for prevention of skin aging and photo-carcinogenesis.