Ion Channel and Calcium Signaling in Ultraviolet Light Transduction in Human Skin

描述(申请人提供)：皮肤是人体最大的器官，经常暴露在太阳紫外线辐射(UVR)下，这是一种强大的环境致癌物质。与其他哺乳动物的皮肤不同，人类皮肤对UVR的反应是由于表皮黑素细胞产生黑色素并将其转移到角质形成细胞而导致色素沉着增加。黑色素通过直接吸收UVR和中和自由基来保护黑素细胞和角质形成细胞的核DNA，从而防止遗传损伤和癌症形成。人体皮肤是如何检测和响应紫外线的？地球表面的紫外线由95%的长波紫外线(UVA)和5%的短波紫外线(UVB)组成。尽管UVB导致DNA损伤并导致暴露一天多后黑色素合成延迟的机制已经得到了很好的描述，但UVA的影响却知之甚少。高剂量的UVA会引起氧化损伤，但目前还没有确定检测UVA生理剂量的具体途径。我们的初步结果描述了黑素细胞中一种新的UVA激活的信号通路，它是视网膜依赖的和G蛋白偶联的。生理剂量的UVA激活这一途径导致瞬时受体电位A1(TRPA1)离子通道的激活，细胞内钙离子的快速增加，以及黑色素含量的快速增加(从UVA暴露后约一小时开始)。这项建议的目标是描述UVA引起钙离子反应(目标1)和细胞黑色素浓度增加(目标2)的分子步骤。利用活细胞荧光成像、电生理学和生物化学，我们将在Aim1中鉴定由UVA激活的G蛋白途径，表征细胞信使，并确定导致快速和瞬时钙反应的机制。在目标2中，我们将测试假设，即UVA介导的细胞去极化是持续的钙反应和持久的PKC激活所必需的，这导致早期黑色素的产生。在目标3中，我们将测试UVA和UVB信号通路的假设，这两个信号通路同时被太阳UVR激活，协同作用调节黑色素的产生和着色。我们将使用“人源化”皮肤的小鼠来研究UVA和UVB激活途径之间的相互作用，并表征这种相互作用的分子机制。这些研究将确定和表征一种新的UVA光传导途径，并确定其在皮肤中的功能，并将极大地促进我们对黑素细胞功能和皮肤对太阳UVR反应的理解。

DESCRIPTION (provided by applicant): Skin, the largest organ of the human body, is constantly exposed to solar ultraviolet radiation (UVR), a powerful environmental carcinogen. Unlike the skin of other mammals, human skin responds to UVR by increased pigmentation resulting from melanin production in epidermal melanocytes and its transfer to keratinocytes. Melanin protects the nuclear DNA of melanocytes and keratinocytes by directly absorbing UVR and neutralizing free radicals, thus preventing genetic damage and cancer formation. How does human skin detect and respond to UVR? UVR at the earth's surface consists of 95% long wavelength UVR (UVA) and 5% short wavelength UVR (UVB). Although the mechanism by which UVB causes DNA damage and results in delayed melanin synthesis more than a day after exposure is well characterized, the effects of UVA are poorly understood. High UVA doses cause oxidative damage, but no specific pathway for detecting physiological doses of UVA has been characterized. Our preliminary results describe a novel UVA-activated signaling pathway in melanocytes that is retinal-dependent and G-protein coupled. Activation of this pathway by physiological doses of UVA leads to the activation of transient receptor potential A1 (TRPA1) ion channels, a rapid increase in intracellular Ca2+, and increased melanin content on a rapid time scale (beginning about an hour after UVA exposure). The goal of this proposal is to characterize the molecular steps by which UVA causes a Ca2+ response (Aim 1) and an increase in cellular melanin concentration (Aim 2). Using live-cell fluorescence imaging, electrophysiology, and biochemistry, we will identify in Aim1 the G protein pathway activated by UVA, characterize the cellular messengers and determine the mechanisms that lead to a rapid and transient Ca2+ response. In Aim 2 we will test the hypothesis that UVA-mediated cellular depolarization is required for a sustained Ca2+ response and persistent PKC activation, which leads to early melanin production. In Aim 3 we will test the hypothesis that UVA and UVB signaling pathways, which are simultaneously activated by solar UVR, interact synergistically to regulate melanin production and pigmentation. We will use mice with "humanized" skin to investigate the interaction between the UVA and UVB activated pathways and characterize the molecular mechanisms mediating this interaction. The proposed studies will identify and characterize a novel UVA phototransduction pathway and determine its function in skin, and will significantly advance our understanding of melanocyte function and of the skin's response to solar UVR.