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。与其他哺乳动物的皮肤不同，人类皮肤对紫外线的反应是表皮黑色素细胞产生黑色素并将其转移到角质形成细胞，从而增加色素沉着。黑色素通过直接吸收紫外线和中和自由基来保护黑素细胞和角质细胞的核DNA，从而防止遗传损伤和癌症的形成。人体皮肤如何检测和响应紫外线辐射？地球表面的紫外线包括95%的长波紫外线（UVA）和5%的短波紫外线（UVB）。虽然UVB引起DNA损伤并导致暴露后超过一天的黑色素合成延迟的机制已经被很好地描述，但UVA的影响却知之甚少。高剂量的UVA会引起氧化损伤，但目前还没有专门的途径来检测生理剂量的UVA。我们的初步结果描述了一种新的uva激活的黑素细胞信号通路，它是视网膜依赖和g蛋白偶联的。生理剂量的UVA激活该途径导致瞬时受体电位A1 （TRPA1）离子通道激活，细胞内Ca2+快速增加，黑色素含量在快速时间尺度上增加（UVA暴露后约一小时开始）。本提案的目标是表征UVA引起Ca2+反应（目的1）和细胞黑色素浓度增加（目的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中识别由UVA激活的G蛋白途径，表征细胞信使，并确定导致快速和短暂的Ca2+反应的机制。在Aim 2中，我们将测试uva介导的细胞去极化是持续的Ca2+反应和持续的PKC激活所必需的假设，这导致了早期黑色素的产生。在Aim 3中，我们将验证UVA和UVB信号通路同时被太阳UVR激活的假设，它们协同作用以调节黑色素的产生和色素沉着。我们将使用具有“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，并表征介导这种相互作用的分子机制。这些研究将识别和表征一种新的UVA光导途径，并确定其在皮肤中的功能，并将显著推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.