Histone deacetylases augment cytokine induction of the iNOS gene.
The inducible nitric oxide synthase (iNOS) gene plays an important role in renal diseases. Transcription is the principal mode of regulation. This study explores the role of acetylation in cytokine-mediated iNOS induction in cultured murine mesangial cells and RAW 264.7 cells. Nitric oxide production was measured by the Griess reaction. The activity of the iNOS promoter and a nuclear factor-kappa B (NF-kappa B) element promoter were assessed in transient transfection assays. Gel shift and supershift assays were used to identify NF-kappa B in nuclear extracts. Protein-protein interactions were assayed by co-immunoprecipitation and GST pull-down assays. Treatment with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and overexpression of HDAC isoforms were used to assess the impact of acetylation status on iNOS and NF-kappa B element promoter activity. TSA inhibited induction of endogenous NO production and iNOS as well as NF-kappa B element promoter activity in response to interleukin-1 beta (IL-1 beta) or lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) in both cell types without altering NF-kappa B DNA binding activity. Overexpression of specific HDAC isoforms enhanced cytokine induction of both the iNOS and the NF-kappa B element promoter. HDAC2 and NF-kappa B p65 co-immunoprecipitated from mesangial cell nuclear extracts, and in vitro translated HDAC2 specifically interacted with an NF-kappa B p65 GST fusion protein. Hyperacetylation diminishes cytokine induction of iNOS transcription activity, at least partially, by limiting the functional efficacy of NF-kappa B. The specific recruitment of HDAC2 to NF-kappa B at target promoters and the consequent effects on acetylation status may play an important role in regulating iNOS as well as other NF-kappa B-dependent genes involved in inflammation.
Departments of Internal Medicine and Department of Integrative Biology, Pharmacology and Physiology, The University of Texas Medical School at Houston, Houston Texas 77030, USA.
Zhiyuan
Z
Yu
Wenzheng
W
Zhang
Bruce C
BC
Kone
D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000502:physiology*; D004338:Drug Combinations; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000502:physiology*; D005920:Glomerular Mesangium / Q000201:enzymology; D006655:Histone Deacetylases / Q000502:physiology*; D006877:Hydroxamic Acids / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D016328:NF-kappa B / Q000502:physiology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D051996:Transcription Factor RelA
D016207:Cytokines; D004338:Drug Combinations; D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D007375:Interleukin-1; D007527:Isoenzymes; D008070:Lipopolysaccharides; D016328:NF-kappa B; D051996:Transcription Factor RelA; D009569:Nitric Oxide; C012589:trichostatin A; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D006655:Histone Deacetylases