The Critical Role And Regulation Mechanisms Of P2RY6 In Skin Pigmentation

Background:Skin color is one of the most important human traits,and the proportion of melanin mainly determines our skin color.In addition to physical chemical and other external factors,genetic factors are one of the important internal factors affecting human skin color.Although numerous genes related to skin color have been recently reported,the regulation of G protein-coupled receptors（GPCR）in skin pigmentation has become the focus of rising concern.The P2RY6 receptor（encoded by the P2RY6 gene）is an extracellular nucleotide G-protein-coupled receptor,which is expressed in a variety of human tissues and cells types.P2RY6 is related to the occurrence and development of diseases such as immunity,inflammation and cancer.However,the role of P2RY6 in skin pigmentation has not yet been elucidated.Objective:In this study,we researched the effect of P2RY6 on skin pigmentation and the related molecular mechanism.Method and results:In this study,by using a combination of NaOH lysis,Western Blot and Masson Fontana melanin staining,we found that the the level of melanin content in the ears and tail skin of P2ry6^-/-mice was significantly higher than that of P2ry6^+/+mice.Moreover,5μM P2RY6 inhibitor MRS-2578 could promote the pigmentation of zebrafish.The data showed that deletion of P2YR6 could result in pigmentation of mouse skin and increased melanin content in zebrafish.In vitro,based on the QPCR experimental results that P2RY6 is highly expressed in HaCaT cells and poorly expressed in melanocytes,we speculate that P2RY6 gene deletion promotes melanin deposition in skin by affecting the ability of keratinocytes to absorb melanin.We constructed P2RY6 knock-down cell line in HaCaT cells.Then we use conditioned medium,cell co-culture and other systems,the results showed that the ability of uptake of melanin by HaCaT-sh P2RY6 was obviously enhanced,and—0.25,0.5 and 1μM MRS-2578,promoted the uptake of melanin by HaCaT cells in different degrees.PAR-2 is one of the major genes that is involved in melanin uptake.We then examined the expression of m RNA and protein of PAR-2 and its related downstream PI3K/AKT signaling pathway by using real-time fluorescence quantitative PCR,Western Blot,immunofluorescence and other techniques.The data showed that the transcriptional expression and protein level of PAR-2 was significantly increased in HaCaT-sh P2RY6 cells and MRS-2578-treated HaCaT cells,increasing the expression of two molecules in the PI3K/AKT signaling pathway,PI3K（110α）and p-AKT proteins.However,the effect of low expression of P2RY6 on promoting melanin uptake by HaCaT cells was reversed with activity inhibition of PAR-2 or AKT molecules by AZ3451 or MK2206.We also found that the expressions of PAR-2 and PI3K（110α）and p-AKT proteins in the ears and tail skin of P2ry6^-/-mice were significantly higher than those of P2ry6^+/+mice.Because pigment deposition is also closely related to abnormal autophagy of keratinocytes,we used Rapamycin to induce autophagy in HaCaT cells,and explored the relationship between P2RY6 and autophagy-induced pigment loss by Western Blot,cellular immunofluorescence,MDC staining and lysosomal probe labeling.The results showed that the autophagy level and lysosomal activity in sh P2RY6 cells were significantly reduced under the induction of rapamycin at the same concentration,suggesting that P2RY6 could effectively inhibit cytochrome deficiency caused by autophagy.P2ry6^-/-mice also showed low autophagy levels in the skin of ears and tails.In terms of molecular mechanism,we found that knocking down P2RY6 in HaCaT cells upregulated the activity of PI3K（110α）,p-AKT and p-m TOR,which were related to PI3K/AKT/m TOR signaling pathway,a key pathway to regulate autophagy.The results showed that P2RY6 gene deletion affected the autophagy of skin keratinocytes by activating PI3K/AKT/m TOR signaling pathway.Conclusion:In this study,we found a new gene that is related to skin pigmentation—P2RY6.The results showed that the deletion of P2ry6 promoted pigmentation in the skin of the ears and tail of mice and in the body of zebrafish.We also found two ways in which P2RY6 affects pigmentation:（1）P2RY6 deletion promotes the uptake of melanin by keratinocytes by up-regulating the expression of PAR-2 and the proteins expression of PI3K（110α）and p-AKT;（2）P2RY6 deletion up-regulate the expression of PI3K（110α）,p-AKT and p-m TOR proteins,thereby inhibiting the autophagy induced pigmentation loss in keratinocytes.Our results highlight the important role of P2RY6 in regulating skin pigmentation and,provide novel evidence for further exploring the treatment and clinical application of P2RY6 in skin pigmentation.