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

描述（由申请人提供）：本文提出的研究目的是了解黑素细胞特异性瞬时受体电位（TRP）离子通道蛋白--美司他丁的功能和调节。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Ca 2+和Mg 2+起重要作用。这些过程包括光感知、温度感知、味觉感知、机械感知、疼痛感知、肾脏中的Mg 2+稳态、平滑肌张力和血压调节。Melastatin（TRPM 1）是TRP通道蛋白TRPMs家族的创始成员，最初被鉴定为小鼠黑色素瘤转移的抑制因子。独立地，我们确定TRPM 1作为在生长停滞的人黑色素瘤细胞中诱导的基因。组织分布和启动子调节小眼转录因子（MITF）证实TRPM 1作为黑素细胞限制性TRP。在异源HEK 293细胞中使用部分长度的人TRPM 1蛋白的功能研究表明，它是组成型活性Ca 2+通道。虽然TRPM 1表达的丧失（如通过原位RNA杂交评估的）被认为与皮肤黑素瘤的侵袭性相关，但TRPM 1的确切功能以及其表达在黑素细胞中如何调节尚不清楚。为了解决这些问题，我们产生了TRPM 1表达（组成型和tet诱导型）shRNA质粒和抗体试剂。初步数据显示，在原代新生儿包皮黑素细胞中，a）通过shRNA敲低TRPM 1抑制黑色素的积累，B）TRPM 1表达通过黑素细胞分化的诱导剂上调，并通过p53下调，以及c）恶性黑素细胞不耐受全长TRPM 1的表达。这些数据表明TRPM 1参与调节表皮黑素细胞的生长和分化功能。本研究的主要目的是：（a）研究TRPM 1的生物合成、翻译后修饰、亚细胞定位和膜定位，并建立TRPM 1的结构-功能关系;（B）利用稳定的shRNA敲除策略研究TRPM 1在正常和恶性黑素细胞生长和色素沉着中的作用;（c）研究TRPM 1的调控机制。了解TRPM 1功能的这些研究在TRP通道功能、TRP基因突变与遗传性疾病的关联以及靶向TRP用于治疗由其故障引起的疾病的潜力方面的兴趣日益增长的背景下是重要的。 黑色素细胞是皮肤和毛发色素沉着的来源。改变黑素细胞功能的遗传缺陷和环境因素导致色素性疾病，包括皮肤癌黑色素瘤。该提案研究了一种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新的治疗色素性疾病的方法。

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.