Nitric oxide (NO), enzymatically synthesized by nitric oxide synthases (NOSI, II and III), has important functions in both physiology and pathophysiology. The regulation, in particular, of NOSII, and the modulation of other genes and gene products, such as cyclooxygenase-2 (COX-2), by NO may be important in carcinogenesis. We have been studying the expression of NOSII, which is normally induced by interferon gamma (IFN-g) and lipopolysaccharide (LPS), and its regulation by a variety of factors including the chemopreventive agent dehydroepiandrosterone (DHEA), tumor suppressor proteins p53 and APC (adenomatous polyposis coli ), diet-related metabolic intermediate pyrroline-5-carboxylate (P5C) and hypoxia. We found that DHEA, a compound with known chemopreventive activity, markedly decreased the levels of NOSII in response to LPS plus IFN-g in both cultured fibroblasts and macrophages as well as in macrophages freshly isolated from mice treated with DHEA in vivo. Reduced responsiveness of NOSII induction, demonstrated by Western and Northern blots as well as by the Griess reaction, has been observed in cells missing both alleles of p53. On the other hand, truncation of APC protein due to Apc min mutation in mouse colonic epithelial cells caused elevated expression of NOSII and COX-2 in response to LPS and IFN-g. Accumulation of NO by NOSII overexpression further increased both COX-2 mRNA and protein levels as well as PGE2 generation. NO-releasing compounds SNAP and NOR-1 had similar effect on COX-2 induction and PGE2 generation. Preliminary data indicates that NO induces COX-2 expression by increasing nuclear translocation of the transcription factor -- catenin. Interestingly, although P5C may interact with NO and its degradative products directly to form nitroso-proline (NPRO), as discussed bellow, it may also play a novel role in the induction of NOSII under hypoxic condition. P5C synergistically induces NOSII along with hypoxia. The newly identified hypoxia-responsive element on NOSII gene presents this interaction the possibility of a potential novel pathway for NOSII induction. Although the formation of NOSII has been elucidated, the inactivation of NO is not well understood but inactivation is important because NO may be the source for endogenous carcinogens. We found that P5C, the precursor and degradative product of proline reacts with NO and its congeners to produce NPRO at physiological pH. Identification of NPRO from this reaction has been made by GC-MS. Although NPRO is known to be formed from degradative products of NO and excreted in the urine, its formation has not been considered a major route for NO detoxification because NPRO is formed from proline only at extremely acid conditions (pH < 4). Since P5C not only is an intracellular intermediate but also a diet-dependent constituent of human plasma, it may be a molecule for scavenging and inactivating excess NO produced under certain pathophysiological conditions, such as chronic infection and inflammation.
