The Role And Mechanism Of Opsin 3 In Melanin Cap Formation On The Nucleus Of Human Epidermal Keratinocytes Induced Via Ultraviolet A

Objective:Skin cancer is one of the cancers with the highest incidence rate and mortality in the world,and has become a major public health problem.Solar ultraviolet（UV）radiation-induced DNA damage is a major risk factor for skin cancer development.UV-induced redistribution of melanin near keratinocyte nuclei leads to the formation of a supranuclear cap,which acts as a natural sunscreen and protects DNA by absorbing and scattering UV radiation.Therefore,our in-depth understanding of the molecular mechanism of UV induced melanin cap formation in human skin is crucial for protecting DNA.Opsins（OPN）are a large group of light-sensitive G protein–coupled receptors（GPCRs）that use retinene molecule as a ligand and trigger signaling cascades upon distinct wavelength of light.OPN,primarily found in light-detecting cells such as the retinal photoreceptors,are widely known for their key role in visual transduction.However,it is unclear whether OPN acts as a UV photoreceptor to mediate the formation of melanin caps in keratinocytes.Methods:1.We used Dispase enzyme digestion to extract human primary keratinocytes（HEK）and human primary melanocytes（MC）from the children foreskin tissue.Simultaneously,we use HEK co-cultured with MC to observe the formation of melanin caps in keratinocytes after UVA irradiation.In addition,melanin produced by melanocytes was fed into HaCaT（without melanin）,and the presence of feeding melanin in HaCaT was confirmed by Masson-Fontana（MF）staining,indicating successful feeding.Next,we irradiated the co-culture model of MC and HaCaT irradiated with UVA to observe the formation of melanin caps in HaCaT.In order to further determine whether UVA can induce the formation of melanin cap,we used new skin explants as a model in vitro to study.After UVA irradiation the skin explants model,we observed the distribution of melanin in Keratinocyte under fluorescence microscope.Simultaneously,the melanin distribution in keratinocytes were also detected by transmission electron microscopy（TEM）.2.In order to explore the role of cytoplasmic dynein and dynactin in UVA induced melanin cap formation,after UVA irradiation of HEK and HaCaT,respectively.we used Western blot（WB）and Quantitative Real-time PCR（qRT-PCR）to analyze the changes of protein expression level and mRNA expression level of cytoplasmic dynamin intermediate chain 1（Dync1i1）and dynactin（DCTN1）,respectively.Simultaneously,after UVA irradiation of skin explant,we also used immunofluorescence method to analyze the changes of Dync1i1 and DCTN1 protein expression level in the skin explant keratinocytes.In addition,we designed four pairs of small interfering RNA（siRNA）that inhibit the expression of DCTN1,and used the transfection reagent lipo2000 to transfect HEK,qRT-PCR and WB to analyze the transfection efficiency at the mRNA expression level and protein expression level respectively.And screened out a pair of RNAi-DCTN1 with the highest inhibition efficiency for follow-up research.We then constructed the co-culture model of RNAi-DCTN1 HEK and MC and the co-culture model of RNAi-DCTN1 HaCaT and MC.After UVA irradiation of these two models,we observed the distribution of melanin in HEK and HaCaT under the microscope.3.We further explore the UVA photoreceptor in human epidermal keratinocytes.First,we analyzed the expression of OPN1,OPN2,OPN3,OPN4 and OPN5 in skin explant keratinocytes by immunofluorescence double staining.Meanwhile,we analyzed the protein expression level and mRNA expression level of OPN1,OPN2,OPN3,OPN4 and OPN5 in HEK and HaCaT by WB and qRT-PCR,respectively.The localization of OPN1,OPN2,OPN3,OPN4 and OPN5 in HEK and HaCaT was analyzed by immunofluorescence double staining.In addition,after irradiation of HEK and HaCaT with different doses of UVA,the expression level of OPN protein was analyzed by WB and immunofluorescence,and the expression level of OPN mRNA was analyzed by qRT-PCR.4.Based on the RNAi-OPN3 previously designed by our team,after transfecting HEK with the transfection reagent lipo2000,we used qRT-PCR and WB to analyze the transfection efficiency at the mRNA expression levels and protein expression levels respectively.And screened out the siRNA dose with the highest inhibition efficiency for subsequent experimental research.In addition,we constructed lentivirus silencing OPN3 expression（sh OPN3#1,sh OPN3#2）,and empty lentivirus（sh NC）as control group,we transfected HaCaT with lentivirus according to the operation instructions provided by the supplier,and screened the stable strain with purinomycin.And qRT-PCR and WB analyzed the inhibition efficiency of OPN3 at the mRNA expression levels and protein expression levels respectively,and screened the sh OPN3with the highest inhibition efficiency for subsequent test and research.We constructed a lentivirus that overexpressed OPN3（LV-OPN3）,and the empty lentivirus（LV-control）as control group.According to the operation instructions,HaCaT was transfected with LV-OPN3 and stable strains were screened with purinomycin.qRT-PCR and WB also analyzed the transfection efficiency at the mRNA expression levels and protein expression levels respectively.5.We further used sh OPN3-HaCaT for transcriptome sequencing,and carried out the enrichment analysis of differential genes,functions and signal pathways by thermography,GO and KEGG.In addition,we analyzed the effect of the inherent function of OPN3 on the survival of keratinocytes using stable cell lines with knockdown or overexpression of OPN3.For example,we used Ed U proliferation test to analyze cell proliferation changes,wound healing test to analyze cell migration changes and Annexin V-PE to detect cell apoptosis changes.6.To analyze whether OPN3 mediates UVA-induced melanin cap formation,we constructed the co-culture model of RNAi-OPN3 HEK and MC and the co-culture model of sh OPN3 HaCaT and MC.After UVA irradiation of these two models,we observed the distribution of melanin in keratinocytes under the microscope.Meanwhile,WB and qRT-PCR were used to analyze the effect of silencing OPN3expression on the expression levels of Dync1i1 and DCTN1 in UVA induced keratinocytes.In addition,we further analyzed the effect of overexpression OPN3 on the expression level of Dync1i1 and DCTN1 in UVA-induced keratinocytes by WB and qRT-PCR.Finally,fluorescence microscopy and flow cytometry were used to analyze the intracellular ROS production induced by UVA in human keratinocytes.7.We explore the specific signal pathway molecular mechanism of UVA-induced melanin cap formation.After UVA irradiation of HEK and HaCaT,Fluo 3-AM calcium fluorescence probe was incubated with cells,the changes of intracellular Ca²⁺were observed by fluorescence microscope,and the intracellular Ca²⁺influx was further quantitatively analyzed by flow cytometry.And we used WB to detect the expression level changes of p-Ca MKII and p-CREB.In addition,After UVA irradiation of RNAi-OPN3 HEK,the change of intracellular Ca²⁺influx was also analyzed by fluorescence microscope and flow cytometry,and the expression level of p-Ca MKII and p-CREB was analyzed by WB.We further treated the cells with PTX and U73122 respectively,and analyzed the effect of UVA-induced Ca²⁺in keratinocytes by fluorescence microscope and flow cytometry,and analyzed the expression level changes of p-Ca MKII,p-CREB,Dync1i1,DCTN1 by WB.Finally,after we treated cells with MK-2206,WB analyzed the changes of Dync1i1 and DCTN1 expression levels.We use Quantity one software to analyze the gray value of each protein band,and Prism software to carry out statistical analysis.Results:1.We observed that the melanin particles in human keratinocytes irradiated by UVA increased and tended to gather around the nucleus.Transmission electron microscopy（TEM）was used to observe the phenomenon that melanin particles in human keratinocytes aggregated to the nucleus to form melanin caps.WB and qRT-PCR analysis showed that UVA upregulated the expression of Dync1i1 and DCTN1in human keratinocytes.And down-regulating the expression of DCTN1 will inhibit UVA-induced melanin accumulation around the nucleus in keratinocytes.2.The tissue immunofluorescence double staining analysis found that OPN1,OPN2,OPN3,OPN4,and OPN5 were stained positive in pan Cytokeratin（pan-CK）labeled human skin tissue keratinocytes.Simultaneously,RT-PCR and WB analysis showed the expression of OPN1,OPN2,OPN3,OPN4 and OPN5 in HEK and HaCaT,and OPN3 mRNA expression level was significantly higher than other opsins mRNA expression level.OPN1-5 localization in human keratinocyte membrane and cytoplasm by cell immunofluorescence double staining analysis.WB and RT-PCR analysis showed that 3 J/cm²UVA can up-regulate the expression of OPN3.3.RT-PCR and WB analysis showed that 60n M RNAi-OPN3 significantly inhibited the expression of OPN3 in keratinocytes.Moreover,we also successfully constructed the co-culture model of RNAi-OPN3 HEK and MC,and further analysis showed that down-regulation of OPN3 expression would inhibit the formation of UVA-induced HEK melanin cap.Moreover,the co-culture model of sh OPN3 HaCaT and MC was constructed.And further analysis also found that down-regulating the expression of OPN3 inhibited the formation of UVA-induced HaCaT melanin cap.In addition,according to the transcriptome sequencing results,the expression of DCTN1is related to OPN3.After UVA irradiation on RNAi-OPN3 HEK and sh OPN3 HaCaT respectively,WB and RT-PCR detected the changes of the expression level of Dync1i1 and DCTN1,and the results showed that inhibiting the expression of OPN3would down-regulate the increase of the expression of Dync1i1 and DCTN1 induced by UVA.We further investigated and found that overexpression of OPN3 promoted UVA induced elevation of Dync1i1 and DCTN1 expression.4.We analyzed the impact of regulating the expression of OPN3 on the physiological function of keratinocytes.The Ed U cell proliferation experiment analysis showed that compared with the sh NC group,the cell proliferation ability in the sh OPN3 group seemed to be slightly weakened,but the difference in cell proliferation ability was not statistically significant（P>0.05）;There was no statistically significant difference in the cell proliferation ability between the LV-OPN3 group and the LV-control group（P>0.05）.Moreover,there was no significant difference in cell migration ability between the sh OPN3 group or LV-OPN3 group and the control group（P>0.05）.There was no statistically significant difference in the apoptosis rate between the sh OPN3 group or LV-OPN3 group and the control group（P>0.05）.5.Our results showed that UVA increased the intracellular Ca²⁺level and upregulated the phosphorylation of Ca MKII and CREB.When OPN3 expression was silent,UVA-induced influx of Ca²⁺decreased through the immunofluorescence and flow cytometry detecting technology in HEK,and we found that after UVA exposure,upregulation expression of the above calcium-related proteins was restrained after decreased expression of OPN3.UVA-induced intracellular Ca²⁺reaction was blocked by PTX,and UVA exposure led to increased p-Ca MKII and p-CREB levels.This effect was reduced when Gαi was inhibited.Since G proteins can cause Ca2+release via phospholipase Cβ（PLCβ）activation,we tested whether UVA induced PLC expression.UVA upregulates the expression of PLC,and this effect was reduced when Gαi was inhibited.In addition,UVA-induced PLC expression was decreased when OPN3 was inhibited.Treatment of HEK with U73122 significantly inhibited UVA-induced Ca²⁺transients.Simultaneously,although UVA exposure increased p CAMKII,p-CREB,Dync1i1,and DCTN1 expression levels,this effect was blocked when Ca²⁺release was inhibited.6.We conducted enrichment analysis of KEGG signal pathway through transcriptome sequencing results,and showed that OPN3 was related to Akt pathway.WB analysis showed that UVA induced Akt phosphorylation,and this effect was inhibited when OPN3 expression was silenced.Simultaneously,Furthermore,UVAinduced Akt phosphorylation was eliminated when Ca²⁺release was blocked by U73122.We further investigated whether Akt is involved in the regulation of Dync1i1and DCTN1 expression after UVA irradiation.UVA-regulated Dync1i1 and DCTN1expression levels were eliminated when Akt phosphorylation was inhibited by MK-2206（2HCl）.Conclusions:1.UVA up-regulates the expression of Dync1i1 and DCTN1 and mediates melanin cap formation in human skin epidermal keratinocytes.2.OPN3,as a key UVA photoreceptor,relies on Gαi protein-coupled receptor up-regulates the expression of Dync1i1 and DCTN1 through Ca²⁺/Akt signaling pathway,thus mediating the formation of melanin cap in skin epidermal keratinocytes.3.The findings presented here expand our understanding of OPN3 function and its role as an extra-ocular opsin in human skin.Since OPN3 was shown to be a key sensor for UVA-induced melanin cap formation in human skin keratinocytes,it can be used as a target to protect against DNA damage.