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 (10 ng/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 100 μM of Wy14643, CPT-1 by 100 nM of GW501516 and adiponectin by 10 μM 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.