Histone Deacetylases Regulate The Transcriptional Activity Of CIITA In Macrophages And Smooth Muscle Cells

Objectives: Atherogenesis is a pathophysiological event involving complex interplay between different cells and humoral factors. Two major events during this process are macrophage-triggered chronic inflammation and smooth muscle cell-initiated vascular remodeling. At the molecular level, major histocompatibility class II (MHC II) transactivator (CIITA) is a key mediator of these processes through transcriptional regulation of interferon gamma (IFN-γ) induced MHC II activation and type I collagen repression. The ability of CIITA to modulate transcription is fine-tuned by a complex layer of regulatory pathways, ranging from promoter selectivity to mRNA stability to multiple post-translational modifications (PTMs). PTMs refer to the reversible reaction that covalently transfers to or removes from proteins certain chemical groups (e.g., phosphate) and small polypeptides (e.g., ubiquitin). PTMs may regulate CIITA transcriptional activity by altering CIITA subcellular compartmentation, target selection, and/or protein stability. The importance of PTM in modulating CIITA activity is increasingly being recognized. Therefore, we further studied how histone deacetylases (HDAC2, SIRT1)–mediated lysine deacetylation regulates the transcriptional activity of CIITA and the pathophysiological significance in atherosclerosis.Methods: We examined the interaction between CIITA and histone deacetylases (HDAC2, SIRT1) in smooth muscle cells and macrophages by co-immunoprecipitations. Using HDAC inhibitor trichostatin A (TSA) or SIRT1 activator resveratrol treatment and over-expression of siHDAC2 in cultured cells, we evaluated the effect of histone deacetylases on CIITA acetylation by immunoprecipitations and Western blotting analysis. Next, to assess the effect of histone deacetylases on the activity of CIITA target promoters (MHC II, COL1A2), the luciferase reporter assays were performed in cultured cells. Furthermore, we evaluated the effect of histone deacetylases on the mRNA levels of CIITA target genes by quantitative PCR. To further assess the effect of HDAC2 on the transcriptional activity of CIITA, we examined whether HDAC2 modulated the recruitment of CIITA to MHC II as well as type I collagen promoters by chromatin immunoprecipitation assays. Finally, we probed the effect of HDAC2 on protein stability of CIITA. Using TSA and proteasomal inbibitor MG132 treatment or over-expression of siHDAC2 in cultured cells, we examined the degradation of CIITA protein by Western blotting analysis and the recruitment of CIITA to the target promoters by chromatin immunoprecipitation assays.Results: HDAC2 interacted with and deacetylated CIITA in macrophages and smooth muscle cells, targeting it to proteasomal degradation. HDAC2 suppressed the transcriptional activity of CIITA by reducing the recruitment of CIITA to its target promoters in cultured cells. On the other hand, SIRT1 interacted with and deacetylated CIITA. SIRT1 up-regulated the CIITA target promoter activity and increased the CIITA target gene mRNA levels in cultured cells.Conclusion: Modulating CIITA transcriptional activity by targeting histone deacetylases (HDAC2, SIRT1) may yield novel anti-atherogenic strategies. Therefore, our findings provide potential target for the treatment of atherosclerosis.