Store-operated cAMP signaling in colon pathophysiology

描述(申请人提供)：分泌性腹泻通常是肠道和结肠上皮细胞内不适当的第二信使信号(cAMP和/或钙)的结果。这些信号被认为激活了顶端依赖于钙和cAMP的阴离子传导通路(例如，cftr等氯离子通道)，促使液体分泌进入肠腔，导致潜在威胁生命的水分和营养物质的损失。许多引起腹泻的不同因素(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)也导致内质网(ER)释放钙。我们最近描述了一种新的信号通路，它将内质网腔内游离钙浓度与产生cAMP的酶-腺苷环化酶(AC)的激活联系起来。这一过程完全不依赖于细胞内的钙离子，需要内质网钙离子受体蛋白STIM1的转位。我们的中心假设是，持续性的钙库枯竭导致与腹泻疾病相关的异常阴离子分泌，而不是通过胞浆钙的升高，而是通过“库操作的”cAMP信号。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独的蛋白质组分及其相互之间的空间关系来进一步表征这一新的途径。该项目的具体目标是：(I)确定参与这一过程的特定AC异构体(S)，并定位它们在ER和STIM蛋白中的定位及其空间关系；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产生的推测微域；(Ii)评估STIM1的其他结合伙伴(如钙通道Orai家族)在这一过程中的参与；以及(Iii)使用我们实验室产生的新工具来控制cAMP水平，以评估cAMP在储备耗尽后产生的驱动结肠上皮细胞氯-分泌的程度。公共卫生相关性：腹泻是全球婴儿死亡的第二大原因，也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，因为商店操作的环核苷酸途径的概念与传统上被接受的用于解释分泌性腹泻的起源的理论有很大的不同。这些范式转变的研究最终可能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，也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，因为商店操作的环核苷酸途径的概念与传统上被接受的用于解释分泌性腹泻的起源的理论有很大的不同。这些范式转变的研究最终可能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. )