Prostaglandin production is a hallmark of inflammation in many tissues, including the airway (i.e. asthma) and gastrointestinal tract (inflammatory bowel disease). Prostaglandin secretion is modulated by cytokines, endotoxin and other inflammatory response mediators in macrophage, epithelial cells and fibroblasts. Until recently the mechanism of induced prostaglandin synthesis in the inflammatory response was unknown; although cytokine or endotoxin challenge increased synthesis of immunoprecipitable prostaglandin synthase (PGS), the mRNA for the known PGS (PGS-1; EC.1.14.99.1), was not coordinately induced. We have cloned a cDNA and gene, TIS10/PGS-2, for a second prostaglandin synthase. TIS10/PGS-2 message and protein are rapidly and transiently induced in macrophage, epithelial and fibroblast cells, in response to growth factors, cytokines, and endotoxin. Moreover, induction of both prostaglandin synthesis and TIS10/PGS-2 expression in response to inflammatory mediators and growth factors is blocked by glucocorticoids. Nitric oxide has recently been shown to also play a major role in mediating inflammatory responses. We have now demonstrated that the calcium-independent form of nitric oxide synthase (iNOS) is induced in many of the same cell as TIS10/PGS-2. We believe that interplay between prostaglandin and nitric oxide production, regulated by the induced expression of the TIS10/PGS-2 and iNOS genes, plays a critical role in immune inflammatory reactions in the respiratory and gastrointestinal tracts. In this project we will (i) characterize the iNOS gene, (ii) prepare antisera to the iNOS protein, (iii) determine the molecular mechanism(s) of INOS induction and its regulation by glucocorticoids and regulatory cytokines in macrophage, epithelial cells and fibroblasts, in comparison with similar studies with TIS10/PGS-2, and (iv) characterize the effect of polycyclic aromatic hydrocarbons on the expression of TIS10/PGS-2 and iNOS, in both the absence and presence of other inflammatory mediators, such as cytokines. We will also (v) create mice with homologous deletions of the iNOS gene, and characterize the inflammatory response phenotype of mice deleted for iNOS, for TIS10/PGS- 2, and for both genes.
