No abstract

SummaryThe ccd operon of the F plasmid encodes CcdB, a toxin targeting the essential gyrase of Escherichia coli, and CcdA, the unstable antidote that interacts with CcdB to neutralize its toxicity. Although work from our group and others has established that CcdA and CcdB are required for transcriptional repression of the operon, the underlying mechanism remains unclear. The results presented here indicate that, although CcdA is the DNA-binding element of the CcdA -CcdB complex, the stoichiometry of the two proteins determines whether or not the complex binds to the ccd operator -promoter region. Using electrophoretic mobility shift assays, we show that a (CcdA)2 -(CcdB)2 complex binds DNA. The addition of extra CcdB to that protein -DNA complex completely abolishes DNA retardation. Based on these results, we propose a model in which the ratio between CcdA and CcdB regulates the repression state of the ccd operon. When the level of CcdA is superior or equal to that of CcdB, repression results. In contrast, derepression occurs when CcdB is in excess of CcdA. By ensuring an antidote -toxin ratio greater than one, this mechanism could prevent the harmful effect of CcdB in plasmid-containing bacteria.

These data indicate that HDAC inhibitors suppressed IL-1-induced NO and PGE(2) synthesis, iNOS and COX-2 expression, as well as proteoglycan degradation. The suppressive effect of HDAC inhibitors is not due to impaired DNA-binding activity of NF-kappaB. These findings also suggest that HDAC inhibitors may be of potential therapeutic value in the treatment of OA.

Membrane-associated prostaglandin (PG) E 2 synthase-1 (mPGES-1) catalyzes the conversion of PGH 2 to PGE 2 , which contributes to many biological processes. Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a ligand-activated transcription factor and plays an important role in growth, differentiation, and inflammation in different tissues. Here, we examined the effect of PPAR␥ ligands on interleukin-1␤ (IL-1␤)-induced mPGES-1 expression in human synovial fibroblasts. PPAR␥ ligands 15-deoxy-⌬ 12,14 prostaglandin J 2 (15d-PGJ 2 ) and the thiazolidinedione troglitazone (TRO), but not PPAR␣ ligand Wy14643, dose-dependently suppressed IL-1␤-induced PGE 2 production, as well as mPGES-1 protein and mRNA expression. 15d-PGJ 2 and TRO suppressed IL-1␤-induced activation of the mPGES-1 promoter. Overexpression of wild-type PPAR␥ further enhanced, whereas overexpression of a dominant negative PPAR␥ alleviated, the suppressive effect of both PPAR␥ ligands. Furthermore, pretreatment with an antagonist of PPAR␥, GW9662, relieves the suppressive effect of PPAR␥ ligands on mPGES-1 protein expression, suggesting that the inhibition of mPGES-1 expression is mediated by PPAR␥. We demonstrated that PPAR␥ ligands suppressed Egr-1-mediated induction of the activities of the mPGES-1 promoter and of a synthetic reporter construct containing three tandem repeats of an Egr-1 binding site. The suppressive effect of PPAR␥ ligands was enhanced in the presence of a PPAR␥ expression plasmid. Electrophoretic mobility shift and supershift assays for Egr-1 binding sites in the mPGES-1 promoter showed that both 15d-PGJ 2 and TRO suppressed IL-1␤-induced DNA-binding activity of Egr-1. These data define mPGES-1 and Egr-1 as novel targets of PPAR␥ and suggest that inhibition of mPGES-1 gene transcription may be one of the mechanisms by which PPAR␥ regulates inflammatory responses.