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）： 环状AMP（CAMP）是结肠上皮细胞使用的基本信号分子之一，以控制基本的细胞功能（例如增殖，凋亡，分化，流体分泌和极化），最终有助于粘膜障碍的功能和完整性。通常，CAMP信号是通过激活细胞表面G蛋白偶联受体（GPCR）而产生的，该过程受到严格调节。但是，在上一家资金期间提出的发现导致鉴定了先前未知的信号通路，该信号通路将内质网（ER）钙传感器STIM1连接到结肠隐窝衍生细胞中的CAMP的产生。类似于商店经营或“电容性” Ca2+进入，这种与GPCR无关的cAMP信号通路直接被任何导致ER腔内的自由[Ca2+]的机动直接激活，并且绝对独立于胞质[CA2+]。它需要将STIM1从块状ER转移到与质膜相关的位点的转运和聚类，从而导致通过未知过程激活常规的跨膜腺苷酸环化酶。我们的总体假设是，这种新的“商店经营”的营地生产途径代表了一种保护机制，在非生理，灾难性损失的ER CA2+商店损失后，它会参与其中。随之而来的营地高程会触发自适应反应，包括cAMP刺激的离子和流体分泌。然而，从长远来看，cAMP的不适当或持续升高有望导致基因表达和细胞分裂的改变，这可能最终破坏上皮的功能，甚至会导致腺瘤形成。在这里，我们建议确定STIM1中负责激活ER Ca2+存储后腺苷酸环化酶激活的域（特定目标＃1）。为此，我们将使用分子方法来改变STIM1蛋白和基于敏感的FRET的记者成像营地，以遵循突变的STIM1在单个NCM460结肠上皮细胞中的影响。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）。在特定的目标＃3中，我们将使用全基因组RNAi库来确定商店经营的CAMP信号级联的其他元素，以击倒单个基因候选物。为此，我们的实验室开发了一个高通量功能的CAMP动力学功能屏幕，可以使用基于FRET的CAMP记者在活细胞中执行。最后，我们建议研究储存耗尽依赖性的cAMP产生的程度，调节大鼠大肠的天然上皮的离子转运特性（特定目标＃4）。对老兵的潜在影响：包括结肠癌在内的结肠疾病是老年退伍军人人口普遍存在的健康问题。所描述的商店操作的途径可能会受到结肠内腔中成分的激活，例如胆汁酸，共生细菌的代谢产物，摄取的药物和病原体（病毒和细菌），这些（病毒和细菌）可能导致ER CA2+商店的非生理学释放。由于营地会影响加密细胞的增殖，凋亡，分化，流体分泌和极化，因此随后的cAMP信号有望影响消化生理学和屏障功能的广泛范围，包括控制上皮细胞沿着密码 - villus轴和流体分泌活性的成熟，这是基于杜比二世的流体分泌活动。