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. 2010 Sep;53(3):519-27.
doi: 10.1016/j.jhep.2010.03.025. Epub 2010 Jun 2.

The NF-kappaB p50:p50:HDAC-1 repressor complex orchestrates transcriptional inhibition of multiple pro-inflammatory genes

Ahmed M Elsharkawy  1 , Fiona Oakley, Feng Lin, Graham Packham, Derek A Mann, Jelena Mann
Affiliations

The NF-kappaB p50:p50:HDAC-1 repressor complex orchestrates transcriptional inhibition of multiple pro-inflammatory genes

Ahmed M Elsharkawy et al. J Hepatol. 2010 Sep.

Abstract

Background & aims: The pro-inflammatory functions of NF-kappaB must be tightly regulated to prevent inappropriate tissue damage and remodelling caused by activated inflammatory and wound-healing cells. The p50 subunit of NF-kappaB is emerging as an important repressor of immune and inflammatory responses, but by mechanisms that are poorly defined. This study aims to delineate p50 target genes in activated hepatic stellate cells and to outline mechanisms utilised in their repression.

Methods: Hepatic stellate cells were isolated from nfkb1(p50)-deficient or Wt mice and gene expression compared using microarray. Target genes were verified by qRT-PCR and p50-mediated HDAC-1 recruitment to the target genes demonstrated using chromatin immunoprecipitation.

Results: We identify p50 as transcriptional repressor of multiple pro-inflammatory genes including Ccl2, Cxcl10, Gm-csf, and Mmp-13. These genes are over-expressed in nfkb1(p50)-deficient mice suffering from chronic hepatitis and in fibrogenic/inflammatory hepatic stellate cells isolated from nfkb1(-/-) liver. We identify Mmp-13 as a bona-fide target gene for p50 and demonstrate that p50 is required for recruitment of the transcriptional repressor histone deacetylase (HDAC)-1 to kappaB sites in the Mmp-13 promoter. Chromatin immunoprecipitations identified binding of HDAC-1 to specific regulatory regions of the Ccl2, Cxcl10, Gm-csf genes that contain predicted kappaB binding motifs. Recruitment of HDAC-1 to these genes was not observed in nfkb1(-/-) cells suggesting a requirement for p50 in a manner similar to that described for Mmp-13.

Conclusions: Recruitment of HDAC-1 to inflammatory genes provides a widespread mechanism to explain the immunosuppressive properties of p50.

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Figures

Fig. 1
Fig. 1
Genespring analysis of the ontologies of genes that are up or down-regulated innfkb1−/− activated HSCs as measured by Amersham 10K murine microarray.
Fig. 2
Fig. 2
Verification of differentially expressed genes in nfkb1−/−HSCs. (A) CXCL10, CCL2, GM-CSF, and MMP-13 mRNA levels were quantified by qRT-PCR in wild type and nfkb1−/− activated HSCs. Results are expressed as relative transcriptional difference as compared to the control ±SEM; n = 5. (B) Fibromodulin mRNA levels were quantified by qRT-PCR in wild type and nfkb1−/− activated HSCs. Results are expressed as already stated ±SEM; n = 5. (C) MMP3 mRNA levels were quantified by qRT-PCR in wild type and nfkb1−/− activated HSCs. Results are expressed as already stated ±SEM; n = 5.
Fig. 3
Fig. 3
Quantification of up-regulated gene targets in chronically CCl4-injured whole liver samples. (A) CXCL10, CCL2, fibromodulin, and MMP-13 mRNA levels were quantified by qRT-PCR in wild type and nfkb1−/− chronic CCl4-injured livers. Results are expressed as relative transcriptional difference as compared to the control ±SEM; n = 5.
Fig. 4
Fig. 4
Quantification of endogenous levels of MMP-13 in 3T3 cells transfected with p50. Ten centimetres of dishes of 3T3 cells was transfected with 3 μg of p50-Flag expression vector or control. Cells were harvested 48 h later, RNA prepared and used in qRT-PCRs with primers specific for MMP-13; n = 4.
Fig. 5
Fig. 5
p50 suppresses MMP-13 promoter activity. (A) Human HSC cell line LX2 was transfected with 1 μg of −721 bp long (NF-κB binding site containing) or −227 bp short (no NF-κB site) MMP-13 promoter luciferase construct and co-transfected with 2 μg p50-Flag expression or control vector. Transfections were harvested 48 h later and luciferase assay carried out on cell lysates. Results are normalized to protein concentration; n = 3. (B) Schematic representation of MMP-13 promoter-luciferase reporters used in A and C. (C) Human HSC cell line LX2 was transfected with 1 μg of −721 bp long (NF-κB binding site containing) MMP-13 promoter luciferase construct, and co-transfected with 2 μg p50-Flag expression or control vector in triplicate. Each set of transfections was either treated with 10 ng/ml LPS (8 h prior to harvest) or TNF-α (4 h prior to harvest) or left untreated. Transfections were harvested 48 h after transfection and luciferase assay carried out on cell lysates. Results are normalized to protein concentration; n = 3.
Fig. 6
Fig. 6
p50 and HDAC-1 bind to MMP-13 promoterin vivo. (A) Twenty micrograms of crosslinked chromatin obtained from rat myofibroblasts was incubated with 10 μg of anti-p50 or p65 antibody or non-specific control; ChIP assay was carried out and MMP-13 promoter amplified. (B) Human HSC cell line LX2 was transfected with 1 μg of p50-Flag expression vector, transfection harvested at 48 h, and crosslinked chromatin prepared. One hundred micrograms of obtained chromatin was incubated with 10 μg of anti-p50 antibody or non-specific control; ChIP assay was carried out and MMP-13 promoter amplified. (C) Human HSC cell line LX2 was transfected with 1 μg of p50-Flag, EM1 or EM2 expression vector, transfection harvested at 48 h and crosslinked chromatin prepared. One hundred micrograms of obtained chromatin was incubated with 10 μg of anti-p50 antibody or non-specific control; ChIP assay was carried out and MMP-13 promoter amplified. (D) Human HSC cell line LX2 was transfected with 1 μg of p50-Flag, EM1 or EM2 expression vector, transfection harvested at 48 h and nuclear extracts prepared. Five micrograms of nuclear extract was used in EMSA with NF-κB double-stranded γ-32P-labeled oligonucleotide probe. The gel shown is representative of two independent experiments. (E) Chromatin was prepared from human HSC cell line LX2. One hundred micrograms of obtained chromatin was incubated with 10 μg of anti-HDAC-1 antibody or non-specific control; ChIP assay was carried out and MMP-13 promoter amplified. (F) Human HSC cell line LX2 was transfected with 1 μg of HDAC-1 expression vector, transfection harvested at 48 h and crosslinked chromatin prepared. One hundred micrograms of obtained chromatin was incubated with 10 μg of anti-HDAC-1 antibody or non-specific control; ChIP assay was carried out and MMP-13 promoter amplified. (G) Crosslinked chromatin was prepared from Wt or nfkb−/− HSCs. Twenty micrograms of chromatin was incubated with 10 μg of anti-HDAC-1 antibody or non-specific control; ChIP assay was carried out and MMP-13 promoter amplified.
Fig. 7
Fig. 7
Supressive effects of p50 on MMP-13 promoter are reversible using HDAC-1 inhibitor trichostatin A (TSA). (A) Human HSC cell line LX2 was transfected with 0.5 μg of −721 bp long (NF-κB binding site containing) MMP-13 promoter luciferase construct and co-transfected with 1 μg p50-Flag expression/control vector along with 1 μg HDAC-1 expression/control vector. Transfections were harvested 48 h later and luciferase assay carried out on cell lysates. Results are normalized to protein concentration; n = 3. (B) Human HSC cell line LX2 was transfected with 0.5 μg of −721 bp long (NF-κB binding site containing) MMP-13 promoter luciferase construct and co-transfected with 1 μg p50-Flag expression/control vector. At 24 h, one-half of transfections containing p50 expression/control vector were treated with 300 nM TSA for 24 h. Cells were harvested at 48 h, luciferase assay carried out on cell lysates. Results are normalized to protein concentration; n = 3. (C) Wt mouse HSCs were treated with 300 nM TSA for 4 and 8 h, cells harvested and RNA prepared. MMP-13 mRNA levels were quantified by qRT-PCR, results are expressed as relative transcriptional difference as compared to the control ±SEM; n = 3.
Fig. 8
Fig. 8
HDAC-1 binds to GM-CSF, CCL2, and CXCL10 NF-κB sitesin vivo. (A) Schematic representation of putative NF-κB site within regulatory regions of GM-CSF, CCL2, and CXCL10 genes. (B) Crosslinked chromatin was prepared from Wt or nfkb−/− HSCs. Twenty micrograms of chromatin was incubated with 10 μg of anti-HDAC-1 antibody or non-specific control; ChIP assay was carried out and regions surrounding putative NF-κB sites within GM-CSF promoter (B), CCL2 promoter (C), and CXCL10 promoter (D) were amplified. Several sites were found to bind HDAC-1 which are shown in the figure. Error bars represent ±SEM; n = 3.
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