Mechanism Of Action Of Opsin3 In Ultraviolet A-induced Photoaging

Objective:Whether opsins are expressed in human skin fibroblasts?NHDFs?is detected.To explore whether Opsin3?OPN3?is involved in the regulation of ultraviolet A?UVA?induced photoaging,and to clarify its molecular regulatory mechanisms.Methods:Human primary dermal fibroblasts were isolated and cultured by adherent method,and passed to the third generation.Cells were identified using morphological and specific vimentin and subsequent experiments were performed.Immunofluorescence was used to detect the expression and localization of OPNs on fibroblasts.Western-blot and real-time quantitative PCR?RT-PCR?were used to detect mRNA and protein levels of OPNs,respectively.After different doses of UVA were applied to the skin fibroblasts,the cell state was observed by morphology,and the cell survival rate was observed by CCK8.The changes of OPNs after irradiation were detected by Western-blot.According to the results of CCK8 and OPNs expression level,skin fibroblasts were repeatedly irradiated at a dose of 10 J/cm² to construct a photoaging model of fibroblasts.The photoaging model was established by morphology,cell cycle and?-galactosidase staining.The localization and co-expressionofOPN3,Matrixmetalloproteinase-1?MMP1?andMatrix metalloproteinase-3?MMP3?inphotoagingmodelweredetectedby immunofluorescence and Western-blot.The siRNA was targeted to silence the fibroblast OPN3 and then irradiated with UVA.The expression of MMP1 and MMP3was detected by Western-blot.The cells were irradiated with UVA and the siRNA was targeted to silence the cells OPN3 and then irradiated with UVA.The cells were treated with the PLC inhibitor u73122 and then irradiated with UVA.After treatment with the Fluo-3-AM probe,intracellular Ca²⁺concentration was measured by flow cytometry.And Western-blot was used to detect the expression of MAPK/AP-1signaling pathway-related proteins.Results:Under the phase contrast microscope,fibroblasts showed long spindle-shaped growth,and immunofluorescence staining vimentin was positive.Immunofluorescence showed that the opsin subunits?OPN1-SW,OPN2,OPN3,OPN4 and OPN5?were expressed on the fibroblast membrane in a semicircular or round form,and the immunofluorescence intensity of OPN3 was significantly higher than that of other opsins.Protein expression levels and mRNA levels of opsin were analyzed by Western-blot and RT-PCR,respectively,and the results were consistent with the results of immunofluorescence?p<0.05?.The results of Western blot analysis were consistent with opsin mRNA levels.Our results showed that UVA increases the expression of OPN3 in a dose-dependent manner.After 5J/cm² to 25J/cm² UVA single-dose irradiation of fibroblasts,the protein expression level of OPN3 was detected,increased at a dose of 5 J/cm²,peaked at 10J/cm²,and then gradually decreased.At the same time,the cell proliferation of different doses of UVA irradiated fibroblasts was tested by CCK-8 assay.More than75%of the cells remained viable after 10 J/cm² UVA irradiation.Combined with previous literature reports,we selected 10J/cm² UVA for follow-up studies.The expression of OPN3 after single dose of 10J/cm² UVA irradiation was significantly higher than that of other OPNs.The expression level of OPN3 protein in the photoaging model was also significantly higher than that in the control group?p<0.01?.In addition,the expression levels of MMP1 and MMP3 proteins in the photoaging model were also significantly higher than those in the control group?p<0.01?.We further explored the correlation between OPN3 and MMP1,MMP3;immunofluorescence staining confirmed that OPN3 was co-expressed with MMP1and MMP3 in fibroblasts,respectively.After knocked-down OPN3 in the mRNA and protein level using siRNA targeting OPN3 gene,NHDFs were irradiated with 10J/cm² UVA.48 hours later no significant changes were observed in the expression of MMP1 and MMP3 protein.Previous research has demonstrated that activation of G protein-coupled receptors?GPCRs?leads to an increase of calcium flux.After UVA irradiation of fibroblasts,the intracellular calcium levels of fibroblasts were measured using the Fluo-3AM probe,and as a result,intracellular calcium levels and related p-CAMKII,p-CREB protein levels were significantly increased.After targeting OPN3 and UVA irradiation,there was no significant change in intracellular calcium levels and related p-CAMKII and p-CREB protein levels.To understand the signaling pathways by which OPN3 regulates MMP1 and MMP3 expression,we treated fibroblasts with a pathway inhibitor?PLC inhibitor u73122?and then detected MMP1and MMP3 expression at the protein level.These results show that inhibition of the PLC pathway can reduce MMP1 and MMP3 expression.Previous studies have shown that downstream p38MAPK,ERK,and JNK signaling pathways are involved in the regulation of MMPs.We used siRNA to silence OPN3 expression in fibroblasts and then irradiated with UVA.The results showed that there was no significant change in the expression of p38MAPK,ERK and JNK signal transduction pathways;while UVA activated OPN3,p38MAPK,ERK and JNK signal transduction pathways The expression of related proteins is up-regulated,which triggers the high expression of MMP1 and MMP3 to degrade collagen fibers leading to photoaging.Conclusion:Our study demonstrates that UVA up-regulated MMP1 and MMP3 expression via OPN3-calcium-dependent signal transduction pathway and downstream MAPK/AP-1signaling-associated protein phosphorylation.It also reveals OPN3 as a key sensor of UVA and may be used as a molecular target for skin photoaging prevention or clinical treatment.