CHARACTERIZATION AND FUNCTIONAL-ANALYSIS OF THE HUMAN INDUCIBLE NITRIC-OXIDE SYNTHASE GENE PROMOTER

Background: Nitric oxide has a wide variety of homeostatic and patholo gical effects. Control of the production of nitric oxide by the induci ble form of the enzyme resides in the 5' promoter region of the gene. Although control of the murine isoform has been investigated, little i s known about the functional aspects of the human analog. Materials an d Methods: A 3.9-kb 5' nontranslated region of the human gene was clon ed, sequenced, and several reporter constructs prepared. The promoter- reporter constructs were transfected into human or murine monocytoid c ells and reporter expression quantified following cytokine activation of the cells. The production of nitric oxide was also monitored. Resul ts: Although a murine promoter-reporter functioned efficiently in both human and mouse cells, the human constructs functioned only in human cells. The activity of the mouse construct increased progressively wit h the addition of activating cytokines, but the human promoter-reporte r did nor. Although interleukin 1 beta drove expression of the human i nducible nitric oxide synthase reporter, actual expression of nitric o xide required both interleukin 1 beta and interferon-gamma. Conclusion s: The data indicate that despite the significant homology between the human and mouse inducible nitric oxide synthase promoter sequence, co ntrol of the two genes is quite different. In addition to being more e fficient in promoter activity, the murine promoter responds increasing ly to cytokines that are not effective for the human analog. It is als o apparent that human inducible nitric oxide synthase is controlled at both the level of transcription and post-translationally.