Store-operated cAMP signaling in colon pathophysiology

描述（由申请人提供）：分泌性腹泻通常是肠道和结肠上皮细胞中不适当的细胞内第二信使信号（cAMP和/或Ca2+）的结果。这些信号被认为激活顶端Ca2+和camp依赖的阴离子传导途径（如Cl-通道如CFTR），驱动液体分泌到肠腔，导致潜在的危及生命的水和营养物质的损失。许多引起腹泻的不同药物（肠道病原体、化疗药物、生物活性膳食成分、胆汁酸、炎症介质和环境毒素）也会导致Ca2+从内质网（ER）释放。我们最近描述了一种新的信号通路，将内质网腔内Ca2+的游离浓度与产生cAMP的酶（腺苷酸环化酶（AC））的激活联系起来。这一过程完全独立于细胞质Ca2+，需要ER Ca2+传感器蛋白STIM1的易位。我们的中心假设是，持续的Ca2+储存消耗导致与腹泻疾病相关的阴离子分泌异常，而不是通过细胞质Ca2+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，我们将通过识别其单个蛋白质成分及其在亚细胞水平上彼此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：(i)确定参与这一过程的特定AC异构体，并查明它们的定位及其与ER和STIM蛋白的空间关系；这将包括使用TIRF和靶向fret的cAMP传感器在亚细胞水平测量假定的cAMP生产微域；（ii）评估STIM1的其他结合伙伴（如Ca2+通道的Orai家族）在这一过程中的参与情况；（iii）使用我们实验室生成的新工具来控制cAMP水平，以评估储存耗尽后产生的cAMP驱动结肠上皮细胞Cl-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性/开发资金，因为储存操作环核苷酸途径的概念与用于解释分泌性腹泻起源的传统接受的学说有很大的不同。这些范式转换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. )