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

结肠上皮功能中新型 STIM1 依赖性环 AMP 信号通路

• 批准号: 8398937
• 负责人: ALDEBARAN M HOFER
• 金额: --
• 依托单位: VA BOSTON HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8398937
• 关键词: Address; Adenylate Cyclase; Affect; Aging; Anions; Apoptosis; Bile Acids; Biological; Calcium; Candidate Disease Gene; Cell Line; Cell division; Cell membrane; Cell model; Cell physiology; Cell surface; Cells; Chromosomes; Chronic; Colon; Colon Carcinoma; Cultured Cells; Cyclic AMP; Diarrhea; Digestive Physiology; Disease; Elements; Endoplasmic Reticulum; Epithelial; Epithelial Cells; Epithelium; Fluid Balance; Fluids and Secretions; Fluorescence Resonance Energy Transfer; Flushing; Functional disorder; Funding; G-Protein-Coupled Receptors; Gastrointestinal tract structure; Gene Expression; Generations; Health; Healthcare; Human; Image; Imaging Techniques; Individual; Interphase Cell; Ion Transport; Ions; Irritants; Laboratories; Large Intestine; Libraries; Life; Link; Liquid substance; Malignant Neoplasms; Measurement; Mitosis; Modality; Molecular; Mucous Membrane; Mutate; Names; Paint; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Physiological; Population; Premalignant; Process; Production; Property; Proteins; RNA Interference; Rattus; Receptor Activation; Reporter; Route; STIM1 gene; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Tissues; Toxin; Veterans; Villus; Viral; adenoma; base; cohort; commensal microbes; crypt cell; genome-wide; interdisciplinary approach; knock-down; monolayer; novel; pathogen; public health relevance; response; sensor; tumor

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.

描述（由申请人提供）： 环腺苷酸（cAMP）是结肠上皮细胞用来控制基本细胞功能（例如增殖、凋亡、分化、液体分泌和极化）的基本信号分子之一，其最终有助于粘膜屏障的功能和完整性。通常，cAMP信号通过细胞表面G蛋白偶联受体（GPCR）的激活而产生，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鉴定了一种以前未知的信号传导途径，该途径将内质网（ER）钙传感器STIM 1与结肠隐窝衍生细胞中cAMP的产生连接起来。类似于钙库操作或“容量性”Ca 2+进入，这种GPCR非依赖性cAMP信号通路可通过导致ER腔内游离[Ca 2 +]减少的任何操作直接激活，并且绝对独立于胞质[Ca 2 +]。它需要STIM 1从大体积ER到与质膜紧密贴壁的位点的易位和聚集，导致通过未知过程激活常规跨膜腺苷酸环化酶。我们的总体假设是，这种新的cAMP产生的“储存操作”途径代表了一种保护机制，其在ER Ca 2+储存的非生理性、灾难性损失后参与。随后的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预计会导致基因表达和细胞分裂的改变，最终可能破坏上皮的功能，甚至引起腺瘤形成。在这里，我们建议确定STIM 1内的结构域，负责激活腺苷酸环化酶后，ER Ca 2+商店耗尽（具体目标#1）。为此，我们将使用分子方法来改变STIM 1蛋白和敏感的FRET为基础的报告成像cAMP遵循突变的STIM 1在单个NCM 460结肠上皮细胞的影响。然后，我们将研究STIM 1的磷酸化如何调节这一途径（具体目标#2）。在具体目标#3中，我们将使用全基因组RNAi文库来敲除单个候选基因，以鉴定商店操作的cAMP信号级联的其他元件。为此目的，我们的实验室已经开发了一种高通量的cAMP动态功能筛选，可以在活细胞中使用基于FRET的cAMP报告。最后，我们建议调查在何种程度上存储消耗依赖性cAMP生成调节大鼠大肠的天然上皮细胞的离子转运特性（具体目标#4）。对退伍军人医疗保健的潜在影响：结肠疾病，包括结肠癌，是老年退伍军人普遍存在的健康问题。所述的钙库操作途径可能受到结肠腔中成分的激活，例如胆汁酸、肠道细菌的代谢物、摄入的药物和病原体（病毒和细菌），其可以引起ER Ca 2+钙库的非生理释放。由于cAMP影响隐窝细胞的增殖、凋亡、分化、液体分泌和极化，因此预期随后的cAMP信号将影响消化生理学和屏障功能的广泛方面，包括控制上皮细胞沿着隐窝-绒毛轴的成熟和引起腹泻的液体分泌活性。