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

Assignment of the rat genes coding for phenylalanine hydroxylase (PAH), tyrosine aminotransferase (TAT), and pyruvate kinase (PKL) to chromosomes 7, 19, 2, respectively.

A panel of hybrid clones segregating rat chromosomes in a mouse background was used to determine the chromosomal localization of three genes specifically expressed in hepatocytes. The phenylalanine hydroxylase, tyrosine aminotransferase, and pyruvate kinase genes were assigned to rat chromosomes 7, 19, and 2, respectively.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Assignment of the rat genes coding for alpha 1-antitrypsin (PI), phosphoenolpyruvate carboxykinase (PEPCK), alcohol dehydrogenase (ADH), and fructose-1,6-bisphosphatase (FDP).

The chromosomal localization of four genes which are expressed mainly in the liver has been undertaken for the rat. Using a panel of hybrid clones segregating rat chromosomes, and Southern blot analysis, alpha 1-AT (PI), PEPCK, ADH and FDP are assigned to rat Chromosomes (Chr) 6, 3, 2 and 17, respectively. Groups of synteny among rat, mouse and human species are discussed in relationship to the new assignments.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Haplotypic determinants of instability in the FRAX region: Concatenated mutation or founder effect?

The fragile X triplet repeat expansion at Xq27.3 has been shown to be associated with mutation or instability 600 kb distal at the FMR2 repeat locus. Concatenated mutation, whereby a mutation at one locus somehow interacts with mutation, recombination, deletion, or transposition at another locus, is a possible explanation. In this study we examine evidence from a sample of over 7,000 independent haplotypes from the FRAX region. We adopt the use of cladistic groups to more thoroughly define the properties of these haplotypes, and in doing so isolate one group of haplotypes which may be predisposed to the phenomenon of concatenated mutation. Distinguishing concatenated mutation from founder effects is difficult within a single population. We present our evidence for and against concatenated mutation, and in the process describe a previously undefined mutation at FRAXE. Hum Mutat 18:61-69, 2001

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