Calcium Signal Triggers Melanosome Transfer: Mechanism In Responses To Ultraviolet A Or Ultraviolet B Irradiation

Backgrounds and ObjetivesRepeated exposure to UVR is the main reason that induces skin damage,aging and skin cancer.The melanocytes in the base layer of epidermis synthesize melanin after UVR radiation,and then transfer it to the nearby keratinocytes.This physiological activitity controls the skin pigmentation and makes photoprotection for the skin.This complicated mechanism is completed by melanocytes and keratinocytes,in which the melanocytes make contacts to neighboring keratinocytes via its dendritic tips,and it is the precondition for melanosome's transfer between two kinds of cells.UVR exposure stimulates epidermical keratinocytes directly.How can the melanocytes in the base layer receive light signals and complete melanin synthesis in the base layer?Whether do keratinocytes emit signals to melanocytes in advance?All of these mechanisms are still unclear.We conjecture that there is a photosensitive protein which initiates this rapid photosensitive reaction in the epidermis.Many biological responses involve Ca²⁺ as an intracellular messenger.It requires the participation of calcium channel for the malanosome's transfer to the neighboring keratinocytes via the dendritic tips.As a transient receptor potential calcium channel protein,TRPM1 mediates calcium influx and it promotes the melanin synthesis in melanocytes in early studies.However,it is less reported about the research on the mechanism of TRPM1 inmelanosome's transfer.This study includes three parts:1.Observe the phenomenon of calcium oscillation in melanocytes dynamically using calcium probe after UVR stimulation.Detect the change of the OPN3 and OPN5 protein expression in melanocytes.2.Explore the melanosome's transfer rate in co-culture system of melanocytes and HaCaT cells after UVA/UVB irradiation.3.Detect the TRPM1 protein expression after UVA/UVB irradiation in melanocytes.Explore the impacts of melanosome's transfer in melanocytes and HaCaT cells co-culture system after knockdown TRPM1 expression using small interfering RNA,or treating with TRPM1 inhibitor,G protein inhibitor and PLC inhibitor.Methods and Results1.We cultured the human primary melanocytes in vitro,marked the Ca²⁺ by the calcium probe,and then observed the calcium influx before and after UVA/UVB irradiation using confocal microscope dynamically.The result suggests that both UVA?3 J/cm²?and UVB?20mJ/cm²?could induce the calcium influx,but a single dose of 3 J/cm² UVA resulted in an early Ca²⁺ influx in MCs?with a peak intensity at 7.5 min?while exposure to 20 mJ/cm² UVB induced a delayed Ca²⁺ influx in MCs?with a peak intensity at 12.5 min?.The western blotting,RT-PCR and immunofluorescence staining showed that no significant change of OPN3 was found in melanocytes exposed to UVA?3,10,30 J/cm²?or UVB?5,10,20 mJ/cm²?irradiation,only the expression level of OPN5 was increased significantly in a dose-dependent manner in UVB-exposed cells.2.We established the melanocytes and HaCaT cells co-culture system in vitro,and explored the initial events of melanosome's transfer,cells which were seed on coverslips were immediately harvested following 3 J/cm² UVA irradiation and were stained using a Fontana-Masson stain kit.The result suggests that UVA also could stimulate melanosome's transfer and predominantly released them from the tips of the dendrites of MCs.The flow cytometry detection showed low efficiency of melanosome transfer?19%+9?in absence of UV irradiation.Then we evaluated the efficacies of melanosome's transfer in MC-KC co-cultures at two times?10 and 30 min?following UVA?3 J/cm²?or UVB?20mJ/cm²?irradiation using the flow cytometry.The result shows that the efficacy of melanosome's transfer in UVA-exposed co-cultures was increased significantly compared with UVB-exposed co-cultures at 10 min post-irradiation.In addition,UVB was superior to UVA in stimulating melanosome's transfer at 30 min post-irradiation?P<0.05?.It was also evidence that the calcium chelating agent of Bapta and EGTA dramatically inhibited melanosome stransfer in co-cultures exposed to both types of UV radiation.3.We cultured the human primary melanocytes in vitro,and RT-PCR detected the expression of TRPM1 mRNA expression in different time after UVA?3J/cm²?and UVB?20 mJ/cm²?irradiation,we found that the TRPM1 mRNA expression gradually increased,especially UVB irradiation,and Western blotting detected TRPM1 protein expression after 24h for different doses of UVA?3,10,30 J/cm²?and UVB?5,10 and 20 mJ/cm²?irradiation.The result of western blotting shows that both UVA and UVB can up-regulate TRPM1 protein expression in a dose-dependent manner.We further tested the effects of UVA?3J/cm²?or UVB?20mJ/cm²?on MCs using immunofluorescent staining.Increased expression of TRPM1 was condensed in the tips of MC dendrites,especially after UVB exposure.We knockdown TRPM1 used siRNA and treated the co-cultures with voriconazole?a potent TRPM1 channel blocker?,a G protein inhibitor?suramin?and a phospholipase C?PLC?antagonist?U73122?before UVA and UVB irradiation.The flow cytometry showed that the melanosome's transfer rate is significantly decreased after these interferences?P<0.05 or P<0.01?.Conclusions1.The different peaks of Ca²⁺ influx in response to UVA and UVB irradiation may provide a persistent impetus that drives melanosome's transfer in the solar tanning process.2.The different melanosome's transfer rates after UVA and UVB iradiation may be related to the different mechanism of melanin sythesis.3.The TRPM1 as a calcium channel protein to mediate Ca²⁺ influx plays an important role in adjusting melanosome's transfer.