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.