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-kappaB (NF-kappaB) element promoter were assessed in transient transfection assays. Gel shift and supershift assays were used to identify NF-kappaB 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-kappaB element promoter activity. TSA inhibited induction of endogenous NO production and iNOS as well as NF-kappaB element promoter activity in response to interleukin-1beta (IL-1beta) or lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) in both cell types without altering NF-kappaB DNA binding activity. Overexpression of specific HDAC isoforms enhanced cytokine induction of both the iNOS and the NF-kappaB element promoter. HDAC2 and NF-kappaB p65 co-immunoprecipitated from mesangial cell nuclear extracts, and in vitro translated HDAC2 specifically interacted with an NF-kappaB p65 GST fusion protein. Hyperacetylation diminishes cytokine induction of iNOS transcription activity, at least partially, by limiting the functional efficacy of NF-kappaB. The specific recruitment of HDAC2 to NF-kappaB at target promoters and the consequent effects on acetylation status may play an important role in regulating iNOS as well as other NF-kappaB-dependent genes involved in inflammation.