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

描述（由申请人提供）：这里提出的研究目标是了解黑色素细胞特异性瞬时受体电位（TRP）离子通道蛋白melastatin的功能和调控。哺乳动物TRP通道蛋白家族由近30个成员组成。TRPs对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+发挥重要作用。这些过程包括光觉、热感觉、味觉、机械感觉、痛觉、肾内Mg2+稳态、平滑肌张力和血压调节。美拉他汀（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因子。独立地，我们发现TRPM1是一个基因诱导生长受阻的人类黑色素瘤细胞。小眼症转录因子（MITF）的组织分布和启动子调控证实了TRPM1是黑色素细胞限制性TRP。在异种HEK293细胞中使用部分长度的人TRPM1蛋白进行功能研究表明，它是一个组成性活性的Ca2+通道。尽管TRPM1表达的缺失（通过原位RNA杂交评估）被认为与皮肤黑色素瘤的侵袭性有关，但TRPM1的确切功能及其在黑色素细胞中的表达是如何调节的尚不清楚。为了解决这些问题，我们生成了TRPM1表达（组成型和可诱导型）shRNA质粒和抗体试剂。初步数据显示，在初生新生儿人包皮黑色素细胞中，a) shRNA敲低TRPM1可抑制黑色素色素的积累，b) TRPM1的表达可被黑色素细胞分化诱导剂上调，而p53和c)恶性黑色素细胞不耐受全长TRPM1的表达。这些数据表明，TRPM1参与调节表皮黑色素细胞的生长和分化功能。在本课题中，我们将对TRPM1的生物合成、翻译后修饰、亚细胞定位和膜取向进行表征，建立TRPM1的结构-功能关系；b)利用shRNA稳定敲低策略研究TRPM1在正常和恶性黑色素细胞生长和色素沉着中的作用；c)研究TRPM1的调控机制。这些了解TRPM1功能的研究在TRP通道功能、TRP基因突变与遗传疾病的关联以及靶向TRP治疗由其功能障碍引起的疾病的潜力日益增加的背景下具有重要意义。黑素细胞是皮肤和头发色素沉着的来源。遗传缺陷和环境因素会改变黑素细胞的功能，导致包括皮肤癌黑色素瘤在内的色素紊乱。本研究探讨了一种独特的钙转运蛋白在维持人体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新的治疗色素紊乱的方法。

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.