The gastrointestinal tract provides an excellent model system in which to study the regulation of growth and differentiation. The continuous process of intestinal epithelial proliferation and differentiation is tightly regulated and organized into specific compartments. The molecular mechanisms which underlie intestinal epithelial growth regulation and differentiation are poorly understood. Our broad long-term goal is to obtain a better understanding of the molecular basis of intestinal epithelial growth regulation and transformation. Recently our laboratory cloned an intestinal mitogen inducible cyclooxygenase gene which is upregulated by transforming growth factor alpha (TGFalpha) and tumor promoters. Treatment of rat intestinal epithelial cells with cyclooxygenase inhibitors blocks TGFalpha stimulated mitogenesis. The recombinant RS-2 protein has cyclooxygenase activity which is completely inhibited by sulindac sulfide, a potent non steroidal anti-inflammatory drug (NSAID). Treatment of the recombinant enzyme with another commonly used NSAID (aspirin) converts it from a cyclooxygenase to a lipoxygenase which produces almost exclusively 15(R)-HETE, a novel eicosanoid product. Several animal and epidemiologic studies demonstrate that NSAID use is linked to a 50-60% reduction in colon cancer. The mitogen inducible cyclooxygenase mRNA is increased in some human colon cancers. What is the role of the inducible cyclooxygenase gene and the eicosanoid products it produces in intestinal epithelial growth regulation and transformation? What are the molecular mechanisms regulating its expression? We propose to sequence the full length mitogen inducible cyclooxygenase-related gene (RS-2) from growth factor stimulated intestinal epithelial cells and prepare large quantities of the RS-2 recombinant protein for further characterization. We are particularly interested in determining if selected NSAIDs preferentially inhibit RS-2. We will isolate and characterize the 5' flanking sequences of the RS-2 gene and determine the TGFalpha and eicosanoid cis regulatory sequences in the Zif268 and RS-2 promoters. This will give us a better understanding for the molecular basis for the regulation of the induction of these genes in intestinal epithelial cells. We will determine the role of RS-2 in intestinal epithelial growth regulation and transformation utilizing antisense and transgenic approaches. We will design antisense oligonucleotides to specifically inhibit the expression of RS-2 and/or Zif268 and then determine what effect this has on TGFalpha-induced eicosanoid production and mitogenesis. This will help to precisely determine the importance of these genes in intestinal epithelial growth regulation. We will link the RS-2 coding region downstream of the villin and metallothionine promoters and prepare transgenic mice which will overexpress this gene in various regions of the intestine and then determine what effect this has on growth regulation. We will add an initiator to transgenic mice and determine if tumor formation is accelerated in mice overexpressing the RS-2 gene relative to control mice. These studies should provide insights about the role of the mitogen inducible cyclooxygenase gene and the eicosanoids it produces in intestinal epithelial growth regulation, differentiation and transformation.
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