Novel Calcium Release Mechanism Regulates Dendritic Cell Function

描述(申请人提供)：树突状细胞(DC)是免疫系统的多能调节器。许多重要的DC功能都涉及到钙信号转导。Ca~(2+)是真核生物中用途最广泛的细胞内信使，参与调节许多对细胞生命至关重要的过程。树突状细胞和许多其他免疫细胞中的钙离子水平在静息状态和激活过程中通过多种机制受到严格调控。趋化因子受体刺激可增加树突状细胞内钙离子浓度([Ca2+]i)和趋化作用。尽管我们对淋巴细胞内钙信号的了解取得了重要进展，但参与树突状细胞内钙离子形成的分子仍未确定。目前的范式认为，DC胞浆内钙水平的增加是由内质网(ER)钙库释放和/或通过开放质膜上由库操作的钙通道而导致的细胞外钙内流引起的。我们提出了一种新的树突状细胞的钙释放机制，这种机制是通过表达在溶酶体室内的钙通道来实现的，我们推测这一途径对于依赖钙的树突状细胞的功能是必不可少的。支持我们建议的初步数据显示：1)功能性钙离子相关的黑素相关瞬时受体电位通道(TRPM2)在树突状细胞的内溶酶体隔室独占定位。2)腺苷二磷酸核糖(ADPR)和趋化因子均可诱导树突状细胞内TRPM2介导的钙信号；3)TRPM2通道是树突状细胞内钙释放的通道。4)TRPM2缺失的DC对趋化因子的趋化反应受损。V)TRPM2基因缺陷的小鼠在感染时引起的炎症募集减少。基于这些数据，我们假设：TRPM2作为细胞内溶酶体相关的钙释放通道，调节树突状细胞的钙依赖过程。为了严格验证我们的假设，我们提出了以下具体目标：目的1.确定TRPM2通道激活钙释放的机制，并阐明该途径如何影响树突状细胞钙依赖功能。目的2.通过建立小鼠尿路感染模型，明确TRPM2介导的细胞外钙信号在炎症、免疫和细胞炎症向粘膜渗透过程中的具体作用。这项研究的发现将为深入了解TRPM2通道的细胞内功能如何影响钙离子介导的信号转导通路提供见解，这些信号转导通路在DC反应中发挥核心调节作用。这些研究的完成将揭示新的药理靶点(ADPR和/或TRPM2)在操纵DC的钙反应和功能方面的作用，并将进一步促进针对DC可能发挥重要致病作用的慢性炎症或传染病的新治疗策略的设计。 公共卫生相关性：树突状细胞在启动和调节免疫方面发挥关键作用。这项资助建议阐明一种新的钙释放机制，该机制调节钙稳态，从而调节树突状细胞中的钙依赖功能。本文提出的实验的成功完成将为树突状细胞中的信号通路提供洞察，树突状细胞具有发现新的治疗靶点的强大潜力，以及治疗慢性炎症性疾病的未被重视的方法。

DESCRIPTION (provided by applicant): Dendritic cells (DCs) are multi-potent regulators of the immune system. Many critical DC functions involve calcium (Ca2+) signaling. Ca2+ is the most versatile eukaryotic intracellular messenger that mediates regulation of many processes important to cell life. Ca2+ levels in DCs and many other immune cell types are tightly regulated through multiple mechanisms during the resting state as well as during activation. Chemokine receptor stimulation increases intracellular Ca2+ concentration ([Ca2+]i) and chemotaxis in DCs. Despite important advances in our understanding of Ca2+ signaling in lymphocytes, the molecular players involved in shaping intracellular Ca2+ in DCs remain to be characterized. The current paradigm states that increases in DC cytosolic Ca2+ levels are caused by release from endoplasmic reticulum (ER) Ca2+ stores and/or via influx of extracellular Ca2+ by opening of store-operated Ca2+ channels at the plasma membrane. We propose a novel Ca2+ release mechanism in DCs, which operates via a Ca2+ channel expressed on lysosomal compartments and, we postulate this pathway is essential for Ca2+-dependent DC functions. Preliminary data that support our proposal show: i) Exclusive localization of functional Ca2+-permeant melastatin-related transient receptor potential channel (TRPM2) in endolysosomal compartments of DCs. ii) Both adenosine diphosphoribose (ADPR) and chemokines induce TRPM2 mediated Ca2+ signals in DCs, iii) TRPM2 channel acts as a Ca2+ release channel in DCs. iv) TRPM2-deficient DCs exhibit impaired chemotactic responses to chemokines. v) TRPM2-deficient mice elicit reduced inflammatory recruitment upon infection. Based upon these data, we hypothesize that: TRPM2 functions as an intracellular lysosome- associated calcium release channel that regulates Ca2+-dependent processes in DCs. To critically test our hypothesis, we propose the following specific aims: Aim 1. Identify mechanisms of activation of Ca2+ release via TRPM2 channel, and elucidate how this pathway impacts on DC Ca2+-dependent functions. Aim 2. Define the specific roles for TRPM2-mediated Ca2+ signaling in DCs during inflammation, immunization and cellular inflammatory infiltration to the mucosa using a mouse model of urinary tract infection. The findings from this proposal will provide insights into how the intracellular function of TRPM2 channel affects Ca2+-mediated signal transduction pathways that play a central regulatory role during DC responses. The completion of these studies will unveil the utility of new pharmacological targets (ADPR and/or TRPM2) for manipulation of DCs' Ca2+responses and functions, and will further facilitate the design of new therapeutic strategies for chronic inflammatory or infectious diseases, where DCs may play important pathogenic roles. PUBLIC HEALTH RELEVANCE: Dendritic cells play a critical role in initiating and regulating immunity. This grant proposes to elucidate a novel calcium release mechanism that regulates calcium homeostasis and therefore, calcium-dependent functions in dendritic cells. The successful completion of the experiments proposed here will provide insights into a signaling pathway in dendritic cells, which has strong potential to uncover novel therapeutic targets, and unappreciated approaches for chronic inflammatory diseases.