Molecular Mechanisms of Inducible Nitric Oxide Synthase Gene Expression by IL-1β and cAMP in Rat Mesangial Cells

Abstract

Expression of the inducible nitric oxide synthase (iNOS) gene in rat mesangial cells is differentially triggered by IL-1β and cAMP predominantly at the transcriptional level. The 5′-flanking region of the rat iNOS gene contains several binding sites for transcription factors potentially involved in cytokine and cAMP signaling such as nuclear factor-κB/Rel, CCAAT/enhancer-binding protein (C/EBP), and cyclic AMP-responsive element-binding protein/ATF. We tested promoter activities of serial and site-directed deletion mutants of iNOS-chloramphenicol acetyltransferase reporter genes after transient transfection and stimulation of mesangial cells. A region between bp −277 and −111 bearing a CCAAT/enhancer-binding protein-response element was found to be critical for cAMP-mediated gene induction but dispensable for IL-1β inducibility. Moreover, a minimal promoter ranging from the transcriptional start site up to −111 containing a κB site is sufficient to confer IL-1β-mediated iNOS promoter activation. Consistent with these findings, an electrophoretic mobility shift assay shows the appearance of an IL-1β-inducible nuclear factor-κB p50/p65 heterodimeric complex. Using probes containing C/EBP-binding sites from the iNOS gene revealed further binding of different complexes, all of which were strongly inducible by cAMP and to a lower extent also by IL-1β. Abs against cyclic AMP-responsive element-binding protein, C/EBPβ, and C/EBPδ were able to partially supershift single complexes, suggesting the participation of these transcription factors in the regulation of iNOS gene expression by cAMP and IL-1β. Finally, we show that both cAMP and IL-1β strongly induce steady-state levels of C/EBPβ and C/EBPδ mRNA levels. These data demonstrate that IL-1β and cAMP use distinct as well as partially overlapping sets of transcriptional activators to modulate iNOS gene expression in rat mesangial cells.