Agonists of peroxisome proliferators-activated receptors (PPAR) α, β/δ or γ reduce transforming growth factor (TGF)-β-induced proteoglycans' production in chondrocytes

SummaryObjectiveTo investigate the potency of selective agonists of peroxisome proliferators-activated receptors' (PPAR) isotypes (α, β/δ or γ) to modulate the stimulating effect of transforming growth factor-β1 (TGF-β1) on proteoglycans' (PGs) synthesis in chondrocytes.MethodRat chondrocytes embedded in alginate beads and cultured under low serum conditions were exposed to TGF-β1 (10ng/ml), alone or in combination with the following agonists: Wy14643 for PPARα, GW501516 for PPARβ/δ, rosiglitazone (ROSI) for PPARγ, in the presence or absence of PPAR antagonists (GW6471 for PPARα, GW9662 for PPARγ). PGs' synthesis was evaluated by radiolabelled sulphate incorporation and glycosaminoglycans' (GAGs) content by Alcian blue staining of beads and colorimetric 1.9 dimethyl-methylene blue assay after beads' solubilization. Phosphorylation of Extracellular Signal-related Kinase1/2 (ERK1/2), Smad2/3 and p38-MAPK was assessed by Western Blot and production of prostaglandin E2 (PGE2) by Enzyme immuno-assay (EIA). Levels of mRNA for PPAR target genes [acyl-CoA oxidase (ACO) for PPARα; mitochondrial carnitin palmitoyl transferase-1 (CPT-1) for PPARβ/δ and adiponectin for PPARγ], aggrecan, TGF-β1 and genes controlling GAGs' side chains' synthesis were quantified by real time polymerase chain reaction and normalized over RP29 housekeeping gene.ResultsACO was selectively up-regulated by 100μM of Wy14643, CPT-1 by 100nM of GW501516 and adiponectin by 10μM of ROSI without cell toxicity. TGF-β1 increased PGs' synthesis by four-fold, GAGs' content and deposition by 3.5-fold and six-fold, respectively, while inducing aggrecan expression around 10-fold without modifying mRNA levels of GAGs' controlling enzymes. PPAR agonists inhibited the stimulating effect of TGF-β1 by 24–44% on PGs' synthesis and over 75% on aggrecan, GAGs' content and deposition with the following rank order of potency: ROSI>GW501516≥Wy14643. TGF-β1-induced phosphorylation of Smad2/3 and ERK1/2 was reduced by ROSI over GW501516 but not by Wy14643 whereas stimulated PGE2 production was inhibited by Wy14643 over GW501516 but not by ROSI. The effect of PPAR agonists on PPAR target genes and TGF-β1-induced aggrecan expression was reversed selectively by PPAR antagonists.ConclusionIn chondrocytes' beads, PPAR agonists reduced the stimulating effect of TGF-β1 on PGs by inhibiting TGF-β1-induced aggrecan expression in an isotype-selective manner. Thus, PPAR agonists could be deleterious in situation of cartilage repair although being protective in situation of cartilage degradation

Evidence for species differences in the regulation of MMPs by all-trans retinoic acid in cytokine-stimulated chondrocytes.

International audienceIn inflammatory conditions, chondrocytes produce large amounts of matrix metalloproteases (MMP) and nitric oxide (NO) thought to contribute to joint degradation. We tested the ability of all-trans retinoic acid (ATRA, a retinoic acid receptor (RAR) agonist) to modulate these inflammatory genes in chondrocytes from humans or rats, chosen as representative of animal models of arthritis. All RAR subtypes and RXR-alpha or -beta were expressed at the mRNA level in both species, although IL-1beta (10 ng/ml) inhibited RAR subtypes more markedly in rat than in human cells. ATRA (300 or 1000 nM) inhibited IL-1-induced expression of iNOS and nitrites level in both species, although the NO pathway was induced maximally in rat cells. ATRA displayed controversial effects on MMPs between rat and human chondrocytes, especially for MMP-9 expression. The effects of ATRA were irrelevant to the nuclear translocation of AP-1. The present data underlines that retinoids have a species-dependent impact on IL-1-induced responses in chondrocytes, suggesting that extrapolation of their pharmacological properties from animal cells has a poor relevance to clinical situation