Rho-Dependent COX-2 Expression in Intestinal Cells

肠细胞中 Rho 依赖性 COX-2 表达

• 批准号: 6793977
• 负责人: Lee Warren Slice
• 金额: $ 30.72万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-21 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6793977
• 关键词: G protein; biological signal transduction; gastrin releasing peptide; gastrointestinal epithelium; gene expression; genetic transcription; guanine nucleotide binding protein; guanine nucleotide exchange factors; intestine neoplasm; northern blottings; prostaglandin endoperoxide synthase; receptor coupling; tissue /cell culture; transfection; western blottings

DESCRIPTION (provided by applicant): Prostaglandins (PGs) play a fundamental role in the physiology and pathology of the small intestine. PGs modulate various physiological processes including secretion, cytoprotection, epithelial and endothelial barrier function, and motility. Alterations in PG production is also implicated in pathological processes including intestinal carcinogenesis. PGs are produced by intestinal epithelial cells by cyclooxygenases (COX). Acute changes in PG levels in these cells are controlled by COX-2, which is rapidly induced as an immediate-early gene in response to multiple stimuli including inflammatory cytokines and growth factors. Despite the importance of COX-2 in intestinal physiology, little is known about the regulation of COX-2 gene expression in intestinal epithelial cells. Understanding the signaling pathways that regulate the expression of COX-2 in intestinal cells will be important for defining the loss of its control in pathology including intestinal cancer. The broad, long-term objective of this proposal is to elucidate the signal transduction pathways that mediate COX-2 expression in intestinal epithelial cells in response to agonists that act through G-protein coupled receptors (GPCRs). We have identified a new Rho-dependent signaling pathway, activated by agonists of GPCRs, that induces COX-2 expression in fibroblast cell lines. Therefore, we have proposed a model that envisages a novel Rho-dependent signaling cascade that is activated by GPCR agonists in their target intestinal epithelial cells. This proposal will examine the following hypotheses: 1) Agonist occupancy of GPCRs induces COX-2 expression via a Rho-dependent pathway, 2) Signaling by Rho and EGF synergistically induce COX-2 expression in intestinal cells, 3) GPCR activation of Rho is mediated by Galpha12/13 via Rho-specific guanine nucleotide exchange factors (GEFs) or Galphaq via PYK-2 and 4) COX-2 expression is mediated through unique, Rho-specific cis-acting elements on the COX-2 promoter. In order to test these hypotheses, we have formulated four specific aims, which are: 1) Demonstrate and characterize GPCR-mediated COX-2 expression in normal rat intestinal epithelial cells. 2a) Define the role of Ga2/13 signaling in COX-2 expression in intestinal epithelial cells. 2b) Define the role of Gaq and Gai signaling in COX-2 expression in intestinal epithelial cells. 3) Define the role of Rho and of Protein Kinases acting upstream and downstream of Rho in COX-2 expression in intestinal epithelial cells. 4) Characterize COX-2 promoter elements that are responsive to Rho.

描述（由申请人提供）：前列腺素（PGS）在小肠的生理学和病理学中起着基本作用。 PGS调节各种生理过程，包括分泌，细胞保护，上皮和内皮屏障功能以及运动性。 PG产生的改变还涉及包括肠道癌发生在内的病理过程。 PG是由肠上皮细胞由环氧酶（COX）产生的。这些细胞中PG水平的急性变化受COX-2的控制，COX-2迅速将其作为一种直接诱导的基因，以响应包括炎症细胞因子和生长因子（包括炎症性细胞因子和生长因子）的多种刺激。尽管COX-2在肠道生理学中的重要性，但对肠上皮细胞中Cox-2基因表达的调节知之甚少。了解调节COX-2在肠细胞中表达的信号传导途径对于定义其在包括肠癌在内的病理中的控制丧失至关重要。该提议的广泛长期目标是阐明响应通过G蛋白偶联受体（GPCR）起作用的激动剂，介导肠上皮细胞中COX-2表达的信号转导途径。我们已经确定了由GPCR的激动剂激活的新的RHO依赖性信号通路，该途径诱导了成纤维细胞细胞系中的Cox-2表达。因此，我们提出了一个模型，该模型设想了一种新型的Rho依赖性信号级联，该级联在其靶肠上皮细胞中被GPCR激动剂激活。该建议将检查以下假设：1）GPCR的激动剂占用率通过RHO依赖性途径诱导COX-2表达，2）Rho和EGF通过Galpha12/13的GEFEF介导Rho的Rho和EGF Synergistry of Rho诱导Rho的Cox-negeration（3）GPCR激活（3）GPCR激活（3）通过Rho的GEFEF介导的Cox-guef eff effa in offpha12/13） PYK-2和4）COX-2的表达是通过COX-2启动子上独特的Rho特异性顺式作用元件介导的。为了检验这些假设，我们提出了四个特定目的，即：1）在正常大鼠肠上皮细胞中演示和表征GPCR介导的COX-C-2表达。 2A）定义了Ga2/13信号在COX-2表达中的作用在肠上皮细胞中。 2b）定义了Gaq和GAI信号传导在COX-2表达中的作用。 3）定义RHO和蛋白激酶在肠上皮细胞中COX-2表达中作用于Rho上游和下游的蛋白激酶的作用。 4）表征对Rho响应的COX-2启动子元素。