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.
