Peroxisome Proliferator-activated Receptor-{gamma} Down-regulates Chondrocyte Matrix Metalloproteinase-1 via a Novel Composite Element

Interleukin-1{beta} (IL-1{beta}) induces degradation via hyperexpression of an array of genes, including metalloproteinases (MMP), in cartilage cells during articular degenerative diseases. In contrast, natural ligands for peroxisome proliferator-activated receptors (PPARs) display protective anti-cytokine effects in these cells. We used the PPAR agonist rosiglitazone (Rtz) to investigate PPAR-{gamma} isotype on IL-1{beta}-target genes. Immunocytochemistry, electrophoretic mobility shift, and transient transfection assays revealed a functional PPAR-{gamma} in chondrocytes in vitro. Rtz displayed significant inhibition of IL-1{beta} effects in chondrocytes. Low Rtz concentrations (close to Kd values for PPAR-{gamma}, 0.1 to 1 µM) inhibited the effects of IL-1{beta} on 35S-sulfated proteoglycan production and gelatinolytic activities and downregulated MMP1 expression at mRNA and protein levels. We have investigated the mechanism of action of Rtz against IL-1{beta}-mediated MMP1 gene hyperexpression. Rtz effect occurs at the transcriptional level of the MMP1 promoter, as observed in transiently transfected cells with pMMP1-luciferase vector. Transient expression of wild type PPAR-{gamma} enhanced Rtz inhibitory effect in chondrocytes, whereas a mutated dominant negative PPAR-{gamma} abolished it, supporting the role of PPAR-{gamma} in this effect. MMP1 gene promoter analysis revealed the involvement of a cis-acting element located at -83 to -77, shown to be a composite PPRE/AP1 site. Gel mobility and supershift assays demonstrated that PPAR-{gamma} and c-Fos/c-Jun proteins bind this cis-acting element in a mutually exclusive way. Our data highlight a new PPAR-{gamma}-dependent inhibitory mechanism on IL-1{beta}-mediated cartilage degradation occurring through DNA binding competition on the composite PPRE/AP1 site in the MMP1 promoter

Basic fibroblast growth factor as a selective inducer of matrix Gla protein gene expression in proliferative chondrocytes.

Matrix Gla protein (MGP) is a member of the vitamin K-dependent gamma carboxylase protein family expressed in cartilage. Insulin-like growth factor I (IGF1) stimulates chondrocyte differentiation, whereas basic fibroblast growth factor (FGF2) acts in an opposite manner. We explored the differential expression and regulation by IGF1 and FGF2 of the MGP gene during chondrocyte differentiation. We used a primary culture system of rabbit epiphyseal chondrocytes to show that MGP mRNA is mainly expressed during serum-induced proliferation. Much lower MGP mRNA content is observed in post-mitotic chondrocytes, which newly express alpha 1X procollagen mRNA, a marker of late-differentiated cells. From studies of a series of growth factors, it was shown that IGF1 decreased chondrocyte MGP transcripts, whereas FGF2 had the opposite effect. FGF2 stimulated chondrocyte MGP production in a dose- and time-dependent manner at the mRNA and protein levels. FGF2 acted in a dose- and time-dependent manner, reaching a maximum at 10 ng/ml at 20 h. The protein synthesis inhibitor cycloheximide did not modify FGF2 action, in agreement with a direct effect. Actinomycin D abolished FGF2-induced stimulation, strongly suggesting that FGF2 modulated MGP gene transcription. We transiently transfected chondrocytes with a construct containing the mouse MGP promoter from -5000 to -168 base pairs, relative to the transcription start site of the gene linked to the luciferase gene (MGP-Luc). In transfected cells, FGF2 stimulated luciferase activity up to sevenfold while IGF1 had no effect. Hence, FGF2 induces transcription of the MGP gene via the 5'-flanking region of the gene. Using a series of deleted MGP-Luc constructs, we identified a sequence of 748 base pairs which was sufficient for transcriptional activation by FGF2. These results led us to postulate that the inhibitory chondrogenic action of FGF2 involves a mechanism whereby MGP gene transcription and protein are induced

Effect of intra-uterine growth retardation on cellular proliferation and differentiation in developing rat

International audienc

Study of two ectopeptidases in the susceptibility to celiac disease: Two newly identified polymorphisms of dipeptidylpeptidase IV

No abstract availabl

Hypoxia down-regulates CCAAT/enhancer binding protein-alpha expression in breast cancer cells.

International audienceThe transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) is involved in the control of cell differentiation and proliferation, and has been suggested to act as a tumor suppressor in several cancers. By using microarray analysis, we have previously shown that hypoxia and estrogen down-regulate C/EBP alpha mRNA in T-47D breast cancer cells. Here, we have examined the mechanism by which the down-regulation by hypoxia takes place. Using the specific RNA polymerase II inhibitor 5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside, the mRNA stability was analyzed under normoxia or hypoxia by quantitative reverse transcription-PCR. Hypoxia reduced the half-life of C/EBP alpha mRNA by approximately 30%. C/EBP alpha gene promoter studies indicated that hypoxia also repressed the transcription of the gene and identified a hypoxia-responsive element (-522; -527 bp), which binds to hypoxia-inducible factor (HIF)-1 alpha, as essential for down-regulation of C/EBP alpha transcription in hypoxia. Immunocytochemical analysis showed that C/EBP alpha was localized in the nucleus at 21% O(2), but was mostly cytoplasmic under 1% O(2). Knockdown of HIF-1 alpha by RNAi restored C/EBP alpha to normal levels under hypoxic conditions. Immunohistochemical studies of 10 tumor samples did not show any colocalization of C/EBP alpha and glucose transporter 1 (used as a marker for hypoxia). Taken together, these results show that hypoxia down-regulates C/EBP alpha expression in breast cancer cells by several mechanisms, including transcriptional and posttranscriptional effects. The down-regulation of C/EBP alpha in hypoxia is mediated by HIF-1

Genome Search in Celiac Disease

SummaryCeliac disease (CD), a malabsorption disorder of the small intestine, results from ingestion of gluten. The HLA risk factors involved in CD are well known but do not explain the entire genetic susceptibility. To determine the localization of other genetic risk factors, a systematic screening of the genome has been undertaken. The typing information of 281 markers on 110 affected sib pairs and their parents was used to test linkage. Systematic linkage analysis was first performed on 39 pairs in which both sibs had a symptomatic form of CD. Replication of the regions of interest was then carried out on 71 pairs in which one sib had a symptomatic form and the other a silent form of CD. In addition to the HLA loci, our study suggests that a risk factor in 5qter is involved in both forms of CD (symptomatic and silent). Furthermore, a factor on 11qter possibly differentiates the two forms. In contrast, none of the regions recently published was confirmed by the present screening