Novel STIM1-dependent cyclic AMP signaling pathway in colonic epithelial function

描述(由申请人提供)： 环磷酸腺苷(CAMP)是结肠上皮细胞用来控制基本细胞功能(如增殖、凋亡、分化、液体分泌和极化)的基本信号分子之一，最终有助于粘膜屏障的功能和完整性。正常情况下，cAMP信号通过激活细胞表面G蛋白偶联受体(GPCRs)产生，这是一个受到严格调控的过程。然而，在之前的资助期间所做的发现导致发现了一条先前未知的信号通路，该通路将内质网(ER)钙传感器STIM1与结肠隐窝来源细胞中cAMP的产生联系起来。类似于储存操作或“容量性”钙离子内流，这种不依赖于gpr的cAMP信号通路可以被任何动作直接激活，导致内质网内的游离[Ca~(2+)]减少，并且完全不依赖于胞浆[Ca~(2+)]。它需要STIM1从块状内质网转移和聚集到与质膜紧密相对的位置，导致传统的跨膜腺苷酸环化酶以一种未知的过程激活。我们的总体假设是，这种新的“商店操作的”cAMP产生途径代表了一种保护机制，该机制在内质网钙离子储存的非生理性、灾难性丧失后开始参与。随之而来的cAMP升高触发适应性反应，包括cAMP刺激的离子和液体分泌。然而，从长远来看，cAMP的不适当或持续升高可能会导致基因表达和细胞分裂的改变，最终可能扰乱上皮功能，甚至引发腺瘤的形成。在这里，我们建议确定STIM1中负责激活内质网钙库耗尽后的腺酰环化酶的结构域(特定目标#1)。为此，我们将使用分子方法改变STIM1蛋白，并使用敏感的基于FRET的报告进行cAMP成像，以跟踪单个NCM460结肠上皮细胞中突变的STIM1的影响。然后，我们将研究STIM1的磷酸化如何调节这一途径(特定目标2)。在具体目标#3中，我们将使用全基因组RNAi文库识别存储操作的cAMP信号级联的其他元件，以敲除单个候选基因。为此，我们的实验室开发了一种高通量的cAMP动态功能筛选，可以使用基于FRET的cAMP记者在活细胞中进行操作。最后，我们建议调查依赖存储耗竭的cAMP生成在多大程度上调节大鼠大肠天然上皮的离子运输特性(特定目标#4)。对退伍军人和S的潜在影响医疗保健：包括结肠癌在内的结肠疾病是老年退伍军人中普遍存在的健康问题。所描述的存储操作途径可能受到结肠腔中的成分的激活，例如胆汁酸、共生细菌的代谢物、摄入的药物和病原体(病毒和细菌)，它们可以导致ER钙存储的非生理性释放。由于cAMP影响隐窝细胞的增殖、凋亡、分化、液体分泌和极化，因此，随后的cAMP信号有望影响消化生理和屏障功能的广泛方面，包括控制隐窝绒毛轴上皮细胞的成熟和导致腹泻的液体分泌活动。

DESCRIPTION (provided by applicant): Cyclic AMP (cAMP) is one of the fundamental signaling molecules used by colonic epithelial cells to control basic cellular functions (e.g. proliferation, apoptosis, differentiation, fluid secretion and polarization) that ultimately contribute to the function and integrity of the mucosal barrier. Normally, cAMP signals arise through activation of cell surface G-protein coupled receptors (GPCRs), a process that is tightly regulated. However, findings made during the prior funding period led to the identification of a previously unknown signaling pathway that connects the endoplasmic reticulum (ER) calcium sensor STIM1 to the production of cAMP in colon crypt- derived cells. Similar to store-operated or "capacitative" Ca2+ entry, this GPCR-independent cAMP signaling pathway is directly activated by any maneuver that causes free [Ca2+] within the ER lumen to become reduced, and is absolutely independent of cytosolic [Ca2+]. It requires translocation and clustering of STIM1 from the bulk ER to sites closely apposed to the plasma membrane, resulting in activation of conventional transmembrane adenylyl cyclases by an unknown process. Our overall hypothesis is that this new "store-operated" avenue to cAMP production represents a protective mechanism that becomes engaged following non- physiological, catastrophic loss of the ER Ca2+ store. The ensuing cAMP elevation triggers adaptive responses, including cAMP-stimulated ion and fluid secretion. Over the long term, however, inappropriate or persistent elevation of cAMP is expected to cause alterations in gene expression and cell division that may ultimately disrupt the function of the epithelium, or even elicit adenoma formation. Here we propose to determine the domains within STIM1 that are responsible for activation of adenylyl cyclase following depletion of ER Ca2+ stores (Specific Aim #1). For this purpose we will use molecular approaches for altering the STIM1 protein and sensitive FRET-based reporters for imaging cAMP to follow the effects of mutated STIM1 in single NCM460 colonic epithelial cells. We will then investigate how phosphorylation of STIM1 regulates this pathway (Specific Aim #2). In Specific Aim #3 we will identify other elements of the store-operated cAMP signaling cascade using a genome-wide RNAi library to knock down individual gene candidates. For this purpose our lab has developed a high-throughput functional screen of cAMP dynamics that can be performed in live cells using FRET-based cAMP reporters. Finally, we propose to investigate the extent to which store depletion-dependent cAMP generation regulates the ion transport properties of the native epithelium of the rat large intestine (Specific Aim #4). Potential Impact on Veteran<s Health Care: Diseases of the colon, including colon cancer, are pervasive health issues in the aging veteran population. The store- operated pathway described may be subject to activation by constituents in the colonic lumen, such as bile acids, metabolites of commensal bacteria, ingested drugs and pathogens (viral and bacterial) that can cause non-physiological release of the ER Ca2+ store. Because cAMP influences proliferation, apoptosis, differentiation, fluid secretion and polarization of the crypt cell, the ensuing cAMP signals are expected to impact wide-ranging aspects of digestive physiology and barrier function, including control of the maturation of epithelial cells along the crypt-villus axis and fluid secretory activity that underlies diarrhea.