The Effects Of Angiotensin Ⅱ On Melanogenesis In Human Melanocytes And Its Mechanism

In humans, abnormal pigmentation is commonly seen following skin damage and during the wound healing response, such as post-inflammatory hyperpigmentation after burns, wounds, or laser surgery, and is a major concern for most people because it induces significant cosmetic problems that affect their quality of life. However, treating abnormal pigmentation remains challenging and the results are discouraging. Recently, many investigations have focused on both pigment cells and wound healing, but knowledge of skin repigmentation following cutaneous injury remains sparse.Abnormal pigmentation after wound healing is complex and controlled by many extrinsic and intrinsic factors,such as post-traumatic changes in skin blood circulation, local inflammation, physical and chemical factors that lead to local metabolic disorders, melanocyte dysfunction or loss and other factors. Skin melanogenesis is controlled by complex regulatory mechanisms, such as by multiple genes, multiple transcription factors participate in the regulation, the physical and chemical properties of melanocytes ambient change, cytokines in vivo, a variety of hormones and so on. However, tyrosinase gene family is a key regulatory factor in the melanin synthesis, tyrosinase is the key regulatory enzyme in melanin biosynthesis. Studies have confirmed that the tyrosinase gene family proteins are melanocyte-specific proteins, mainly by small eyes associated transcription factor(MITF) regulation. MITF combined with the tyrosinase family by the the special parts of the promoter, which can contribute to TRP 1 and TRY expression in melanocytes and stimulate the transcriptional activity of the promoter, subsequence regulate the melanogenesis. Thus, MITF plays a fundamental role in the transcriptional regulation of melanogenesis. Mean while, the research on signal pathway of melanin synthesis has also been focused on melanocyte cell research. Many studies have shown that c AMP/PKA and diacylglycerol(DAG)/PKC pathways are important signal transduction pathways that participate in melanogenesis regulation. Park et al. demonstrated that the c AMP pathway also upregulates PKC-β expression via MITF.Previous reports showed that angiotensin II(Ang II) may be involved in all stages of wound healing, including inflammatory cell invasion, cell proliferation, cell migration, neovascularization, and fibrosis. Ang II has been shown to increase vascular permeability and recruit inflammatory cells as well as promote keratinocyte proliferation and dermal wound closure. Previous studies described the expression and putative role of Ang II in human skin; regulation of keratinocyte, dermal myofibroblast, and endothelial cell proliferation was reported in the early period. Previous studies reported that human skin expresses Ang II type 1(AT1) and type 2(AT2) receptors, which were suggested to also play an important role in skin wound healing. Some study recently reported that AT1 and AT2 receptor expression was strongly enhanced within the epidermal and dermal areas of scars. In addition, it is reported that inhibition of the AT1 receptor limited murine melanoma growth by reducing tumor volume and microvessel density, demonstrating the importance of AT1 receptors in melanoma growth. A previous study reported the expression of AT1 m RNA, but not AT2 m RNA, in cultured primary melanocytes.Although several functional roles of Ang II have been suggested, whether Ang II induces abnormal pigmentation by regulating the melanocyte system during wound healing remains to be elucidated. In the present study, the functional role of Ang II in human melanocytes was investigated, and alterations in human melanocytes were characterized.In the present study, we aimed to investigate the function of Ang II on melanogenesis in human melanocytes and to explore the relationship with its role and the AT1 receptor(AT1 R), tyrosinase, MITF, PKC, respectively. The study is divided into three parts contents:Part One: Effects of Ang II on the melanogenesis in human melanocyts and the migration of melanocytesMethods: Reference to preliminary experiments concentration of Ang II, we selected different 100 n M, 10 n M, 1 n M, 0.1 n M, 0.01 n M concentrations of Ang II on melanocytes 24 h, then tetrazolium blue(MTT) colorimetric assay and Na OH dissolving method were used to evaluate cell viability and melanin contents in human melanocytes respectively, spectmphotometer to determine dopa oxidase activity of tyrosinase. Transwell assays were performed to observe the effect of Ang II on the migration of melanocyte.Results: There was a small but non-significant increase in cells proliferation(P>0.05). Tyrosinase activity and melanin content increased significantly in a dose-dependent manner following Ang II treatment. There was a significant increase in tyrosinase activity and melanin content when cells were incubated with increasing concentrations of Ang II(0.1~100 n M) and Ang II(10~100 n M) for 24 h, respectively. The maximal increase was achieved with 100 n M Ang II(P < 0.01). Under 0.01 n M Ang II, there was no difference in tyrosinase activity compared with the control. At 0.01~1 n M Ang II, there was no difference in melanin content compared with the control. Transwell assay revealed a promoting effect of Ang II on melanocytes migration.Conclusion: There was a small but non-significant increase in Ang II on melanocyte proliferation; but Ang II can promote the melanogenesis and the migration of melanocytes.Part Two: Ang II regulation of AT1 R and TYR activity and protein expression in human melanocytesMethod: With 100 n M, 10 n M, 1 n M, 0.1 n M, 0.01 n M different concentration of Ang II effected on melanocytes respectively, the semi-quantitative RT-PCR and Western blot methods detected Ang II on m RNA transcription of AT1 R, TRY and the corresponding protein under different concentrations. Meanwhile, reference to preliminary experiments concentration of AT1 receptor antagonists(LOS), the melanocytes were exposed to 1000 n M LOS in addition to Ang II, and/or a combination of LOS for 30 min before Ang II stimulation, subsequently detected the m RNA transcription of AT1 R, TRY and the corresponding protein expression and the migration of the melanocytes.Result: At 0.1~100 n M, Ang II increased AT1 and tyrosinase m RNA levels and protein expression in human melanocytes. Pretreatment with LOS prevented Ang II-induced tyrosinase and AT1 m RNA and protein expression. But the LOS can not inhibit the effect of Ang II promote the migration of the melanocyte.Conclusion: The AT1 receptor mediates the Ang II induced increase of tyrosinase in human melanocytes and that it is impossible that AT1 R related to tyrosinase signaling. Ang II stimulates melanogenesis via AT1 R, but promotes the migration of melanocytes via a non-AT1 R.Part Three: MITF and PKC is involved in Ang II regulating tyrosinase and melanogenesisMethod: After the intervention of different concentrations Ang II, the RT-PCR and Western blot methods were used to detect m RNA transcription and protein levels of MITF. Then we used specific PKA inhibitor H89 and specific PKC inhibitor Ro-32-0432 interverntion melanocytes for 1h, then 100 n M Ang II effected on melanocytes cells for 24 h, measured the changes of melanin content and tyrosinase activity. Follow the results, we also detected TYR m RNA and protein expression following the combined treatment of 100 n M Ang II and Ro-32-0432. ELISA detected the PKC activity in melanocytes.Result: Ang II significant increase the m RNA and the protein levels of MITF. The effect of Ang II in promoting melanogenesis, increasing tyrosinase activity and m RNA expression level were partly reversed by the PKC inhibitor(Ro-32-0432), not by the PKA inhibitor(H89). Ang II can enhance the intracellular PKC activity.Conclusion: Ang II on melanogenesis is probable regulated by two ways, one hand by regulating the transcription of MITF and expression; on the other hand by activating PKC pathway.In summary, Ang II has a certain promotion to melanogenesis and play a role by regulating melanin contents, tyrosinase acivity, tyrosinase m RNA and protein expression via AT1 R. Meanwile, the MITF and PKC also play important role on Ang II stimulates melanogenesis in human melanocyts. Notably, the results of the present study suggested a potential role for Ang II in tyrosinase regulation through the AT1 receptors, and supported an association between Ang II, tyrosinase and AT1 receptor activation, supported MITF and PKC also was involved in Ang II regulating tyrosinase and melanogenesis, which may be involved in abnormal pigmentation by upregulating tyrosinase in melanocytes. The identification of functional AT1 receptors in human melanocytes has provided novel insights into melanocyte physiology. Their additional functional activities and their regulatory mechanism require further investigation to assess the role of Ang II in cutaneous pathophysiology.