Study On The Mechanism Of TRPA1 Channels Regulating UVR-Induced Melanogenesis And Melanosome Homeostasis

BackgroundMelanin pigments are responsible for the color of human skin and play a photo-protective role against the carcinogenic damage caused by exposure to ultraviolet radiation（UVR）from sunlight.Melanin is synthesized and deposited within a unique,membrane-bound organelle termed melanosome,which is produced in melanocytes located predominantly in the basal layer of skin epidermis.The homeostasis of melanosome is crucial to the melanogenesis process taken place in its cavity.The homeostasis of melanosomes can be affected by the balance of various ions,including calcium homeostasis and p H homeostasis determined by the H⁺balance.The melanosome is a lysosome-related organelle that requires a number of specific enzymatic and structural proteins to mature and become competent in order to produce melanin.Tyrosinase（TYR）is the most predominant rate-limiting enzyme among critical enzymes that affect the quantity and quality of melanin,whilst the melanosome-specific Pmel17（also known as gp100 or SILV;referred to here as gp100）is a critical structural protein.Importantly,the activity of TYR is greatly reduced at acidic p H.Melanosomal p H,rather than TYR expression level,has been shown to regulate TYR activity and the amount of melanin produced in melanocytes from different skin types.Melanosomes from melanocytes of fair-skinned individuals are significantly more acidic and display low TYR activity,whereas melanosomes in dark skin melanocytes are less acidic or neutral and present higher levels of TYR activity.Thus,revealing how the luminal p H of melanosome is controlled is crucial for understanding the biological process of skin pigmentation in humans.However,the relevant mechanism and regulatory factors involved have not been fully elucidated.Transient receptor potential（TRP）ion channels were first characterized in Drosophila phototransduction as calcium(Ca²⁺)-permeable ion channels.More recently,members of the TRP family have been implicated in a wide range of sensory functions,including photosensation,chemosensation,thermosensation,and nociception.TRPA1channel is the only member of the TRP ankyrin（TRPA）subfamily so far identified in mammals.Previous description of tissue distribution for TRPA1 channel is restricted to sensory neurons,while recent studies have confirmed that TRPA1 channel is also expressed in non-neuronal cells in skin,such as keratinocytes,fibroblasts,and melanocytes.In human epidermal melanocytes physiological doses of UVR activate a retinal-dependent current and a rapid Ca²⁺influx mediated by TRPA1 ion channels.Remarkably,TRPA1 channel is essential for a unique extraocular phototransduction pathway in human melanocytes that is activated by UVR and may result in early melanin synthesis.Nevertheless,the detailed mechanism of TRPA1-associated melanin increase remains as yet unknown.Melanogenesis is vital to protect the skin.Abnormal skin melanin homeostasis results in refractory pigmentary diseases,including hyperpigmentary disorders,such as melasma,and hypopigmentary disorders,such as vitiligo.Hence,a thorough understanding of melanogenesis is indispensable for the development of targeted drugs and the clinical treatment of such diseases.In the current study,TRPA1 channel suppressed by its specific antagonist HC-030031 mitigated UVB-induced facultative pigmentation in the dorsal skin of guinea pigs,as well as melanin production in normal human epidermal melanocytes（HEMs）and B16-F10 murine melanoma cells culture.While,promoted by its agonist JT010,TRPA1channel increased the UVB-induced pigmentation in vivo and in vitro.Moreover,we found that melanosomal p H can be regulated by TRPA1 channel,which is an important link in the process of increased skin pigmentation caused by intracellular calcium flow through activation of TRPA1 channel by UVR.The results suggest TRPA1 channel as a potential therapeutic target in the treatment of skin pigmentary disorders that are at high risk under UV radiation.In summary,we suggest that TRPA1 channel,an important calcium channel protein,can promote UVR-induced skin pigmentation by promoting intracellular calcium ion concentration,elevating the luminal p H of melanosomes,affecting the homeostasis within the melanosome,and enhancing TYR enzyme activity upon UVR activation.Further in-depth studies of the TRPA1 channel pathway in the field of skin photobiology may help to reveal the upstream pathogenesis of UVR-related pigmentary disorders,and provide a molecular basis and an important experimental basis for the development of targeted drugs.Purposes1.To study the effect of TRPA1 channels on melanin synthesis in melanocytes under UVR activation and its possible molecular mechanisms.2.To elucidate the effect of TRPA1 channels on intracellular calcium concentration in melanocytes under UVR activation and its possible molecular mechanisms.3.To investigate the effect of TRPA1 channels on the melanosome luminal p H and the homeostasis of melanosomes within melanocytes and its possible mechanisms.4.To verify the effect of TRPA1-related topical agents on UVR-induced skin pigmentation in guinea pigs,and provide an important molecular and experimental basis for the development of targeted drugs for UVR-related pigmented skin diseases.Methods1.In vitro study methods:（1）Primary culture of human epidermal melanocytes（HEMs）and mouse melanoma B16-F10 melanocyte cell line.（2）The eukaryotic expression vector of TRPA1 channel with HA tag was constructed,and the recombinant plasmids were extracted and purified by molecular cloning assay.（3）The plasmid was transfected into B16-F10 melanocytes by liposome method to up-regulate the protein expression of TRPA1 channels.（4）Infection of B16-F10 melanocytes with TRPA1-sh RNA adenovirus to down-regulate the protein expression of TRPA1 channels.（5）SDS polyacrylamide gel electrophoresis（Western blot）to detect the protein expression levels of TRPA1 and gp100.（6）Immunofluorescence specific staining to observe the localization between TRPA1/gp100 and TRPA1/TYR antigens in B16-F10 cells.（7）Protein immunoprecipitation to analyze the interaction between TRPA1/TYR proteins（8）Na OH method to detect the content of melanin in B16-F10 cells.（9）Masson-Fontana melanin staining to observe the number and distribution of melanin granule synthesis in B16-F10 cells.（10）Fluo-4 AM calcium ion fluorescence probe and confocal microscopy observation to detect the changes of intracellular calcium ion concentration.（11）Transfection of melanosomal p H fluorescent indicator MELOPS plasmid and fluorescence microscopy observation to detect changes of melanosomal p H.2.In vivo study methods:（1）Establish a guinea pig model of UVR-induced skin pigmentation.（2）The L（light）values of skin lightness and darkness in the dorsal skin of guinea pigs before and after the experiment were detected and recorded by colorimeter.（3）The skin tissue biopsy was carried out and treated with HE staining to observe the histopathological changes and Masson-Fontana staining to observe the deposition of melanin granules.（4）Immunohistochemical staining was performed to detect the protein expressions of TRPA1 and TYR in skin tissues.Results（A）Study on the mechanism of the regulatory effect of TRPA1 channels on UVR-induced melanin synthesis in epidermal melanocytes.1.Primary cultured human epidermal melanocytes were observed microscopically,which conformed to the typical morphology of melanocytes,with obvious multi-level dendritic protrusions,elongated axial filaments,and high cell purity.The expression of melanosome signature protein gp100 was detected in HEMs and mouse B16-F10melanocytes by Western blot and immunofluorescence.2.The protein expressions of TRPA1 channels were detected by Western blot and immunofluorescence in both HEMs and B16-F10 melanocytes.3.The eukaryotic expression vector of TRPA1 channel was constructed,and the protein expression of TRPA1 channels was successfully up-regulated by the plasmid transfection into melanocytes.4.TRPA1-sh RNA adenovirus infection successfully down-regulated the protein expression of TRPA1 channels in melanocytes.5.Immunofluorescence staining and immunoprecipitation results showed endogenous molecular interactions between TRPA1 and TYR in B16-F10 melanocytes.6.It was observed by both Western blot,Na OH method and Masson-Fontana staining that agonistic TRPA1 activity or up-regulation of TRPA1 expression in melanocytes significantly increased melanin synthesis;inhibition of TRPA1 activity or down-regulation of TRPA1 expression in melanocytes significantly decreased melanin synthesis.（B）Mechanistic studies on the role of TRPA1 channels in regulating the homeostasis of melanosmes in epidermal melanocytes1.The activation of TRPA1 channel in HEMs by UVB irradiation increased the intracellular calcium concentration,which can be inhibited by TRPA1 antagonist HC-030031.Moreover,TRPA1 channel agonist JT010 activated TRPA1 channels in melanocytes and significantly increased intracellular calcium concentration.2.JT010 activated TRPA1 channels in B16-F10 melanocytes,which significantly increased the p H level in melanosomes.However,inhibition of TRPA1 channels by HC-030031,or TRPA1-sh RNA adenovirus infection significantly reduced the p H level in melanosomes.（C）In vivo studies on the mechanism of TRPA1’s regulatory effect on UVB-induced skin pigmentation.1.The regulatory effect of topical treatment of TRPA1 channel agonist JT010 on UVB-induced skin pigmentation.（1）It was observed that topical application of TRPA1-specific agonist JT010 deepened UVB-induced skin pigmentation in guinea pigs.（2）The results of Masson-Fontana staining showed that the epidermal melanin granules were significantly increased in the group with topical JT010 combined with UVB compared with the vehicle group and the control group.（3）Immunohistochemical results showed that topical application of JT010 could enhance the expressions of TRPA1 and TYR in epidermis,and the promoting effect was more significant in combination with UVB irradiation.2.The regulatory effect of topical treatment of TRPA1 channel antagonist HC-030031 on UVB-induced skin pigmentation.（1）After topical application of TRPA1-specific antagonist HC-030031,it was observed that HC-030031 reduced UVB-induced skin pigmentation in guinea pigs.（2）The results of Masson-Fontana staining showed that the epidermal melanin granules were significantly reduced in the group with topical HC-030031 combined with UVB compared with the vehicle group and the control group.（3）Immunohistochemical results showed that topical application of HC-030031 could inhibit the expressions of TRPA1 and TYR in epidermis even under the irradiation of UVB.Conclusions1.The protein expression of TRPA1 ion channels was confirmed in melanocytes,and TRPA1 ion channel may play a positive regulatory role in regulating melanogenesis of melanocytes through endogenous interaction with TYR.2.TRPA1 ion channels can play an important positive regulatory role in regulating the calcium homeostasis of melanocytes and the p H homeostasis of melanosomes under the activation of UVR,thereby promoting melanogenesis of melanocytes.3.Topical application of TRPA1 ion channel regulators can promote/mitigate UVB-induced pigmentation in guinea pig epidermis,suggesting that TRPA1 channels may become an important target for topical therapy of UVR induced pigmented skin diseases.