Store-operated cAMP signaling in colon pathophysiology

描述（由申请人提供）：分泌性平滑肌瘤通常是肠和结肠上皮细胞中不适当的细胞内第二信使信号传导（cAMP和/或Ca 2+）的结果。这些信号被认为激活顶端Ca 2+和cAMP依赖性阴离子传导途径（例如Cl-通道，如CFTR），驱动液体分泌到肠腔中，导致潜在的危及生命的水分和营养物质损失。许多引起腹泻的不同物质（肠道病原体、化疗药物、生物活性饮食成分、胆汁酸、炎症介质和环境毒素）也会导致内质网（ER）释放Ca 2+。我们最近描述了一种新的信号通路，其将ER内腔中的游离Ca 2+浓度与产生cAMP的酶（腺苷酸环化酶（AC））的激活相连接。这一过程是完全独立的胞质Ca 2+，并需要一个ER Ca 2+传感器蛋白，STIM 1的易位。我们的中心假设是，持续的Ca 2+储存耗竭导致与牙周病相关的异常阴离子分泌，不是通过胞质Ca 2+的升高，而是通过“储存操作的”cAMP信号传导。为了评估这种可能性，我们将通过在亚细胞水平上识别其单个蛋白质组分及其相互之间的空间关系来进一步表征这种新途径。该项目的具体目标是：（一）确定参与这一过程的特定AC同种型，并查明它们相对于ER和STIM蛋白的定位和空间关系;这将包括使用TIRF和基于靶向FRET的cAMP传感器在亚细胞水平上测量cAMP产生的推定微域;（二）评估科技创新机制其他具有约束力的伙伴的参与情况1（如奥赖家族的Ca 2+通道）;和（iii）使用我们实验室产生的用于控制cAMP水平的新工具，以评估在何种程度上产生的cAMP后存储耗尽驱动Cl-分泌在结肠上皮细胞。公共卫生相关性：腹泻是全球婴儿死亡的第二大原因，并且是美国发病率和死亡率的重要来源，特别是在脆弱的患者群体中。我们正在为这个项目寻求探索/开发资金，因为存储操作的环核苷酸途径的概念是一个戏剧性的偏离传统上接受的学说，用于解释分泌型大肠杆菌的起源。这些范式转变的研究可能最终导致治疗和预防腹泻和其他疾病的新途径。 公共卫生相关性：腹泻是全球婴儿死亡的第二大原因，并且是美国发病率和死亡率的重要来源，特别是在脆弱的患者群体中。我们正在为该项目寻求探索/开发资金，因为储存操作的环核苷酸途径的概念与用于解释分泌型核糖核酸的起源的传统理论有很大的不同。这些范式转变的研究可能最终导致治疗和预防腹泻和其他疾病的新途径。)

DESCRIPTION (provided by applicant): Secretory diarrheas are frequently the consequence of inappropriate intracellular second messenger signaling (cAMP and/or Ca2+) in intestinal and colonic epithelial cells. These signals are thought to activate apical Ca2+- and cAMP-dependent anion-conducting pathways (e.g. Cl- channels such as CFTR) that drive fluid secretion into the gut lumen, resulting in potentially life-threatening loss of water and nutrients. Many of the diverse agents (intestinal pathogens, chemotherapy drugs, bioactive dietary constituents, bile acids, inflammatory mediators and environmental toxins) that cause diarrhea also cause the release of Ca2+ from the endoplasmic reticulum (ER). We recently described a novel signaling pathway that connects the free concentration of Ca2+ in the lumen of the ER to the activation of the enzyme that generates cAMP, adenylyl cyclase (AC). This process is completely independent of cytosolic Ca2+, and requires the translocation of an ER Ca2+ sensor protein, STIM1. Our central hypothesis is that persistent Ca2+ store depletion contributes to the abnormal anion secretion associated with diarrheal diseases not through elevation in cytosolic Ca2+, but rather through "store- operated" cAMP signaling. In order to evaluate this possibility, we will further characterize this novel pathway by identifying its individual protein components and their spatial relationships with one another at the subcellular level. The specific aims of this project are: (i) To identify the specific AC isoform(s) involved in this process, and to pinpoint their localization and their spatial relationships with respect to the ER and the STIM proteins; this will include measurements of putative micro-domains of cAMP production at the subcellular level using TIRF and targeted FRET-based cAMP sensors; (ii) To evaluate the participation of other binding partners of STIM1 (such as the Orai family of Ca2+ channels) in this process; and (iii) Using novel tools generated in our lab for controlling cAMP levels, to evaluate the extent to which the cAMP produced following store depletion drives Cl- secretion in colonic epithelial cells. Public Health Relevance: Diarrhea is the second leading cause of death in infants worldwide and is a significant source of morbidity and mortality in the U.S., particularly in vulnerable patient populations. We are seeking exploratory/development funding for this project because the concept of a store-operated cyclic nucleotide pathway is a dramatic departure from the conventionally accepted doctrine used to explain the genesis of secretory diarrheas. These paradigm-shifting studies may ultimately lead to new avenues for treatment and prevention of diarrhea and other diseases. PUBLIC HEALTH RELEVANCE: Diarrhea is the second leading cause of death in infants worldwide and is a significant source of morbidity and mortality in the U.S., particularly in vulnerable patient populations. We are seeking exploratory/development funding for this project because the concept of a store- operated cyclic nucleotide pathway is a dramatic departure from the conventionally accepted doctrine used to explain the genesis of secretory diarrheas. These paradigm-shifting studies may ultimately lead to new avenues for treatment and prevention of diarrhea and other diseases. )