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.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor involved in the regulation of many cellular processes. We and others have previously shown that PPARγ activators display anti-inflammatory and chondroprotective properties in vitro and improve the clinical course and histopathological features in an experimental animal model of osteoarthritis (OA). However, the expression and regulation of PPARγ expression in cartilage are poorly defined. This study was undertaken to investigate the quantitative expression and distribution of PPARγ in normal and OA cartilage and to evaluate the effect of IL-1β, a prominent cytokine in OA, on PPARγ expression in cultured chondrocytes. Immunohistochemical analysis revealed that the levels of PPARγ protein expression were significantly lower in OA cartilage than in normal cartilage. Using real-time RT-PCR, we demonstrated that PPARγ1 mRNA levels were about 10-fold higher than PPARγ2 mRNA levels, and that only PPARγ1 was differentially expressed: its levels in OA cartilage was 2.4-fold lower than in normal cartilage (p < 0.001). IL-1 treatment of OA chondrocytes downregulated PPARγ1 expression in a dose- and time-dependent manner. This effect probably occurred at the transcriptional level, because IL-1 decreases both PPARγ1 mRNA expression and PPARγ1 promoter activity. TNF-α, IL-17, and prostaglandin E2 (PGE2), which are involved in the pathogenesis of OA, also downregulated PPARγ1 expression. Specific inhibitors of the mitogen-activated protein kinases (MAPKs) p38 (SB203580) and c-Jun N-terminal kinase (SP600125), but not of extracellular signal-regulated kinase (PD98059), prevented IL-1-induced downregulation of PPARγ1 expression. Similarly, inhibitors of NF-κB signaling (pyrrolidine dithiocarbamate, MG-132, and SN-50) abolished the suppressive effect of IL-1. Thus, our study demonstrated that PPARγ1 is downregulated in OA cartilage. The pro-inflammatory cytokine IL-1 may be responsible for this downregulation via a mechanism involving activation of the MAPKs (p38 and JNK) and NF-κB signaling pathways. The IL-1-induced downregulation of PPARγ expression might be a new and additional important process by which IL-1 promotes articular inflammation and cartilage degradation.

Stem cells have been the focus of intense research opening up new possibilities for the treatment of various diseases. Mesenchymal stromal cells (MSCs) are multipotent cells with relevant immunomodulatory properties and are thus considered as a promising new strategy for immune disease management. To enhance their efficiency, several issues related to both MSC biology and functions are needed to be identified and, most importantly, well clarified. The sources from which MSCs are isolated are diverse and might affect their properties. Both clinicians and scientists need to handle a phenotypic-characterized population of MSCs, particularly regarding their immunological profile. Moreover, it is now recognized that the tissue-reparative effects of MSCs are based on their immunomodulatory functions that are activated following a priming/licensing step. Thus, finding the best ways to pre-conditionate MSCs before their injection will strengthen their activity potential. Finally, soluble elements derived from MSC-secretome, including extracellular vesicles (EVs), have been proposed as a cell-free alternative tool for therapeutic medicine. Collectively, these features have to be considered and developed to ensure the efficiency and safety of MSC-based therapy. By participating to this Special Issue “Mesenchymal Stem/Stromal Cells in Immunity and Disease”, your valuable contribution will certainly enrich the content and discussion related to the thematic of MSCs.

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.

Conclusion. PGD 2 inhibits IL-1-induced production of MMP-1 and MMP-13 by chondrocytes through the DP1/cAMP/PKA signaling pathway. These data also suggest that modulation of PGD 2 levels in the joint may have therapeutic potential in the prevention of cartilage degradation.