Role of TRPM1 (Melastatin1) in the Biology of Human Melanocytes

描述（由申请人提供）：此处提出的研究目标是了解黑素细胞特异性瞬时受体电位（TRP）离子通道蛋白（melastatin）的功能和调节。哺乳动物TRP通道蛋白家族由近30个成员组成。 TRP 对于广泛的生理过程的功能至关重要，其中细胞二价阳离子，特别是 Ca2 和 Mg2 发挥着重要作用。这些过程包括光觉、热觉、味觉、机械感觉、疼痛觉、肾脏中的镁离子稳态、平滑肌张力和血压调节。褪黑素 (TRPM1) 是 TRP 通道蛋白新家族 TRPM 的创始成员，最初被确定为小鼠黑色素瘤转移的抑制剂。我们独立地确定了 TRPM1 是在生长停滞的人类黑色素瘤细胞中诱导的基因。组织分布和小眼转录因子 (MITF) 的启动子调控证实 TRPM1 是黑素细胞限制性 TRP。在异源 HEK293 细胞中使用部分长度的人 TRPM1 蛋白进行的功能研究表明，它是一个组成型活性 Ca2 通道。尽管 TRPM1 表达缺失（通过原位 RNA 杂交评估）被认为与皮肤黑色素瘤的侵袭性相关，但 TRPM1 的确切功能以及其表达在黑色素细胞中的调节方式尚不清楚。为了解决这些问题，我们生成了 TRPM1 表达（组成型和 tet 诱导型）shRNA 质粒和抗体试剂。初步数据表明，在原代新生儿人包皮黑素细胞中，a）shRNA敲低TRPM1可抑制黑色素的积累，b）TRPM1表达被黑素细胞分化诱导剂上调，并被p53下调，c）恶性黑素细胞不耐受全长TRPM1的表达。这些数据表明TRPM1参与表皮黑素细胞的生长和分化功能的调节。在本提案中，我们将a）表征TRPM1的生物合成、翻译后修饰、亚细胞定位和膜取向，并建立TRPM1的结构功能关系，b）使用稳定的shRNA敲低策略研究TRPM1在正常和恶性黑素细胞生长和色素沉着中的作用，以及c）研究TRPM1的调节机制。人们对 TRP 通道功能、TRP 基因突变与遗传性疾病的关联以及靶向 TRP 治疗其功能障碍引起的疾病的潜力越来越感兴趣，这些关于了解 TRPM1 功能的研究具有重要意义。 公共卫生相关性 黑素细胞是皮肤和毛发色素沉着的来源。改变黑色素细胞功能的遗传缺陷和环境因素会导致色素性疾病，包括皮肤癌黑色素瘤。该提案研究了独特的钙转运蛋白在维持人类皮肤黑素细胞正常功能中的作用。从这些研究中获得的知识可以帮助开发治疗色素障碍的新疗法。

DESCRIPTION (provided by applicant): The goal of studies proposed here is to understand the functions and regulation of the melanocyte-specific transient receptor potential (TRP) ion channel protein, melastatin. Mammalian TRP channel protein family consists of nearly 30 members. TRPs are critical for functions of wide ranging physiological process, where cellular divalent cations, specifically Ca2+ and Mg2+ play important roles. These processes include photoperception, thermosensation, taste perception, mechanosensation, pain perception, Mg2+ homeostasis in kidney, smooth muscle tone and blood pressure regulation. Melastatin (TRPM1), the founding member of a novel family of TRP channel proteins known as TRPMs, was originally identified as suppressor of mouse melanoma metastasis. Independently, we identified TRPM1 as a gene induced in growth arrested human melanoma cells. Tissue distribution and promoter regulation by microphthalmia transcription factor (MITF) confirmed TRPM1 as a melanocyte-restricted TRP. Functional studies using a partial length human TRPM1 protein in heterologous HEK293 cells suggested that it is a constitutively active Ca2+ channel. Although loss of TRPM1 expression (as assessed by in situ RNA hybridization) is thought to correlate with aggressiveness of cutaneous melanoma, the exact function(s) of TRPM1 and how its expression is regulated in melanocytes are not known. To address these issues, we generated TRPM1 expression (constitutive and tet-inducible) shRNA plasmids and antibody reagents. Preliminary data showed that in primary neonatal human foreskin melanocytes a) knockdown of TRPM1 by shRNA inhibits accumulation of melanin pigment, b) TRPM1 expression is up-regulated by inducers of melanocyte differentiation and down regulated by p53 and c) malignant melanocytes do not tolerate expression of full-lengthTRPM1. These data suggested that TRPM1 is involved in regulation of growth and differentiated functions of epidermal melanocytes. In this proposal we will a) characterize biosynthesis, posttranslational modification, subcellular localization and membrane orientation and establish structure-function relationship of TRPM1, b) investigate the role of TRPM1 in growth of normal and malignant melanocytes and pigmentation using stable shRNA knockdown strategy, and c) investigate mechanism of regulation of TRPM1. These studies on understanding TRPM1 functions are significant in the context of growing interest in functions of TRP channels, association of mutations in TRP genes with genetic disorders and the potential of targeting TRPs for treatment of diseases arising from their malfunction. PUBLIC HEALTH RELEVANCE Melanocytes are the source of skin and hair pigmentation. Genetic defects and environmental factors that alter melanocyte function cause pigmentary disorders including skin cancer melanoma. This proposal investigates the role of a unique calcium transporting protein in maintaining proper functioning of human skin melanocytes. Knowledge gained from these studies could help develop new therapies for pigmentary disorders.