Histone Deacetylases Augment Cytokine Induction of the iNOS Gene

Abstract

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

Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

“…The lack of reappearance of total-IκBa after HDAC inhibitor exposure in our study was associated with a decrease in IκBα phosphorylation and no change in NFκB DNA binding activity after 1 h of stimulation, suggesting that HDAC influences NFκB-dependent gene expression down-stream of DNA binding in beta cells, probably by modulating the chromatin structure of NFκB-dependent genes, leading to increased NFκB transactivation, promoter activity and iNOS protein production, as has been demonstrated in cultured mesangial cells [4,50].…”

“…In addition, the anti-inflammatory potential of these compounds has also been demonstrated in animal models of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus [4,[7][8][9][10].…”

“…However, the underlying mechanisms seem to depend on the cell type investigated and include on the one hand an HDAC-inhibitor-mediated continual degradation of de novo synthesised IκBα protein (possibly by prolonged activation of the upstream IκB-kinase) causing prolonged presence and DNA-binding of NFκB in the nucleus and enhancing NFκB-dependent transactivation, or on the other hand a reduction in proteasomal degradation of IκBα, preventing NFκB nuclear translocation and DNA binding [4,8,[46][47][48][49].…”

“…Inhibition of HDACs results in hyper-acetylation and unravelling of DNA permitting gene activation. There are, however, exceptions to this general rule [4][5][6].…”

Epigenetic Mechanisms in Cerebral Ischemia

“…Effects can differ extensively, or even be opposed e.g., in microglial cells versus macrophages. 98,99 In general, the administration of either HDACi, VPA, or SB, leads to a significant suppression of microglia activation 47,53,100 and additionally to the inhibition of further inflammatory markers like monocytes and macrophages in stroke. 47 On the gene level, HDAC inhibition impacts the expression of heat shock protein (HSP)70, a heat shock/stress protein that assists proper protein folding, and is involved in inflammatory processes.…”