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Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells.
J Immunol 1998; 160(10):4961-9JI

Abstract

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.

Authors+Show Affiliations

Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt, Germany.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9590244

Citation

Eberhardt, W, et al. "Molecular Mechanisms of Inducible Nitric Oxide Synthase Gene Expression By IL-1beta and cAMP in Rat Mesangial Cells." Journal of Immunology (Baltimore, Md. : 1950), vol. 160, no. 10, 1998, pp. 4961-9.
Eberhardt W, Plüss C, Hummel R, et al. Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells. J Immunol. 1998;160(10):4961-9.
Eberhardt, W., Plüss, C., Hummel, R., & Pfeilschifter, J. (1998). Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells. Journal of Immunology (Baltimore, Md. : 1950), 160(10), pp. 4961-9.
Eberhardt W, et al. Molecular Mechanisms of Inducible Nitric Oxide Synthase Gene Expression By IL-1beta and cAMP in Rat Mesangial Cells. J Immunol. 1998 May 15;160(10):4961-9. PubMed PMID: 9590244.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Molecular mechanisms of inducible nitric oxide synthase gene expression by IL-1beta and cAMP in rat mesangial cells. AU - Eberhardt,W, AU - Plüss,C, AU - Hummel,R, AU - Pfeilschifter,J, PY - 1998/5/20/pubmed PY - 1998/5/20/medline PY - 1998/5/20/entrez SP - 4961 EP - 9 JF - Journal of immunology (Baltimore, Md. : 1950) JO - J. Immunol. VL - 160 IS - 10 N2 - 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. SN - 0022-1767 UR - https://www.unboundmedicine.com/medline/citation/9590244/Molecular_mechanisms_of_inducible_nitric_oxide_synthase_gene_expression_by_IL_1beta_and_cAMP_in_rat_mesangial_cells_ L2 - http://www.jimmunol.org/cgi/pmidlookup?view=long&pmid=9590244 DB - PRIME DP - Unbound Medicine ER -