Histone Deacetylase-1 Is Enriched at the Platelet-derived Growth Factor-D Promoter in Response to Interleukin-1β and Forms a Cytokine-inducible Gene-silencing Complex with NF-κB p65 and Interferon Regulatory Factor-1

Mary Y Liu,Levon M Khachigian

doi:10.1074/jbc.m109.061903

Abstract

Understanding the mechanisms governing cytokine control of growth factor expression in smooth muscle cells would provide invaluable insight into the molecular regulation of vascular phenotypes and create future opportunities for therapeutic intervention. Here, we report that the proinflammatory cytokine interleukin (IL)-1beta suppresses platelet-derived growth factor (PDGF)-D promoter activity and mRNA and protein expression in smooth muscle cells. NF-kappaB p65, induced by IL-1beta, interacts with a novel element in the PDGF-D promoter and inhibits PDGF-D transcription. Interferon regulatory factor-1 (IRF-1) is also induced by IL-1beta and binds to a different element upstream in the promoter. Immunoprecipitation and chromatin immunoprecipitation experiments showed that IL-1beta stimulates p65 interaction with IRF-1 and the accumulation of both factors at the PDGF-D promoter. Mutation of the IRF-1 and p65 DNA-binding elements relieved the promoter from IL-1beta-mediated repression. PDGF-D repression by IL-1beta involves histone deacetylation and interaction of HDAC-1 with IRF-1 and p65. HDAC-1 small interfering RNA ablates complex formation with IRF-1 and p65 and abrogates IRF-1 and p65 occupancy of the PDGF-D promoter. Thus, HDAC-1 is enriched at the PDGF-D promoter in cells exposed to IL-1beta and forms a cytokine-inducible gene-silencing complex with p65 and IRF-1.

Highlights

Phenotypes and create future opportunities for therapeutic and is secreted as a disulfide-linked homodimer and is a specific intervention
histone deacetylases (HDACs)-1 siRNA blocks the IL-1␤-inducible interaction of Interferon regulatory factor-1 (IRF-1) with p65, as demonstrated by either pulling down with IRF-1 antibodies and immunoblotting with p65 antibodies or pulling down with p65 antibodies and immunoblotting for IRF-1 (Fig. 4A, right). These results indicate that HDAC-1 is enriched at the platelet-derived growth factor (PDGF)-D promoter in cells exposed to IL-1␤ and forms a cytokine-inducible gene silencing complex with p65 and IRF-1 (Fig. 6E)
Wof VCAM-1, which, as expected, are A induced by the cytokine. p65, R induced by IL-1␤, interacts with a ITHD1, 2010 novel element in the PDGF-D

Summary

EXPERIMENTAL PROCEDURES

Of 2ϫ SYBR Green Master Mix (Applied Biosystems), 0.2 ␮l of 10 ␮M of forward and reverse primer (Sigma) and 3.6 ␮l of. 35102 JOURNAL OF BIOLOGICAL CHEMISTRY nuclear extracts were incubated with 2 ␮l (4 ␮g) of the rabbit aortic SMCs were incubated with IL-1␤ (10 ng/ml) for various polyclonal antibody in binding buffer for 20 min at room tem- times (0 –24 h) and PDGF-D mRNA levels were measured by perature before adding probe and incubated with probe reverse-transcription PCR. For EMSA using recombinant human proteins, reactions itively regulated by IL-1␤ in an NF-␬B-dependent manner in were conducted in binding buffer CGA AAA GTG AAA GCT ACG C-3Ј (putative IRF-1 binding bition of PDGF-D mRNA levels was dose-dependent (Fig. 1C) site underlined); and mOligo DϪ847/Ϫ823, 5Ј-AGT TCG CGA with no biphasic effect. EMSA performed with 32P-labeled Oligo reverse, 5Ј-GGA GGA AGT GGG AGA GGG TA-3Ј

RESULTS

DISCUSSION

Cytokine or growth factor suppression of growth factor

ADDITIONS AND CORRECTIONS