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

描述(申请人提供)：这里提出的研究目的是了解黑素细胞特异性瞬时受体潜力(Trp)离子通道蛋白Melastatin的功能和调节。哺乳动物色氨酸通道蛋白家族由近30个成员组成。TRPs在广泛的生理过程中起着至关重要的作用，其中细胞内的二价阳离子，特别是钙和镁离子起着重要的作用。这些过程包括光感受、温度感觉、味觉感觉、机械感觉、疼痛感觉、肾脏中的镁稳态、平滑肌张力和血压调节。Melastatin(TRPM1)是一个新的Trp通道蛋白家族的创始成员，最初被发现是小鼠黑色素瘤转移的抑制因子。独立地，我们确定TRPM1是一个诱导生长受阻的人黑色素瘤细胞的基因。组织分布和小眼球转录因子(MITF)对启动子的调控证实TRPM1是一种黑素细胞限制性色氨酸蛋白。使用部分长度的人TRPM1蛋白在异种HEK293细胞中进行的功能研究表明，它是一个结构性的活性钙通道。尽管TRPM1的表达缺失(通过原位RNA杂交评估)被认为与皮肤黑色素瘤的侵袭性有关，但TRPM1的确切功能(S)及其在黑素细胞中的表达如何调节尚不清楚。为了解决这些问题，我们产生了TRPM1表达(结构性和tet诱导的)shRNA质粒和抗体试剂。初步数据显示，在原代新生人类包皮黑素细胞中，a)shRNA下调TRPM1抑制黑色素的积累，b)黑素细胞分化诱导剂上调TRPM1的表达，而p53下调TRPM1的表达，c)恶性黑素细胞不耐受全长TRPM1的表达。这些数据表明，TRPM1参与了表皮黑素细胞的生长和分化功能的调节。在这个方案中，我们将a)鉴定TRPM1的生物合成、翻译后修饰、亚细胞定位和膜定位，并建立结构-功能关系；b)利用稳定的shRNA敲除策略研究TRPM1在正常和恶性黑素细胞生长和色素形成中的作用；以及c)研究TRPM1的调控机制。这些了解TRPM1功能的研究在人们对Trp通道的功能、Trp基因突变与遗传疾病的关联以及靶向TRPs治疗因其功能障碍引起的疾病的可能性的背景下具有重要意义。与公共健康相关的黑素细胞是皮肤和头发色素沉着的来源。基因缺陷和环境因素会改变黑素细胞的功能，导致包括皮肤癌和黑色素瘤在内的色素紊乱。这项建议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方面的作用。从这些研究中获得的知识可能有助于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.