Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells.
Expression of the inducible nitric oxide synthase (iNOS) gene in rat mesangial cells is differentially triggered by IL-1beta 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-kappaB/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-1beta inducibility. Moreover, a minimal promoter ranging from the transcriptional start site up to -111 containing a kappaB site is sufficient to confer IL-1beta-mediated iNOS promoter activation. Consistent with these findings, an electrophoretic mobility shift assay shows the appearance of an IL-1beta-inducible nuclear factor-kappaB 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-1beta. Abs against cyclic AMP-responsive element-binding protein, C/EBPbeta, and C/EBPdelta 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-1beta. Finally, we show that both cAMP and IL-1beta strongly induce steady-state levels of C/EBPbeta and C/EBPdelta mRNA levels. These data demonstrate that IL-1beta and cAMP use distinct as well as partially overlapping sets of transcriptional activators to modulate iNOS gene expression in rat mesangial cells.
Eberhardt
W
W
Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt, Germany.
Plüss
C
C
Hummel
R
R
Pfeilschifter
J
J
D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005920:Glomerular Mesangium / Q000201:enzymology*; D007375:Interleukin-1 / Q000494:pharmacology*; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D015533:Transcriptional Activation
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D009687:Nuclear Proteins; D012333:RNA, Messenger; D004247:DNA; D000242:Cyclic AMP; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat