Retinoid X receptor and peroxisome proliferator-activated receptor-gamma agonists cooperate to inhibit matrix metalloproteinase gene expression

Introduction We recently described the ability of retinoid X receptor (RXR) ligand LG100268 (LG268) to inhibit interleukin1-beta (IL-1-)-driven matrix metalloproteinase-1 (MMP-1) and MMP-13 gene expression in SW-1353 chondrosarcoma cells. Other investigators have demonstrated similar effects in chondrocytes treated with rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPAR), for which RXR is an obligate dimerization partner. The goals of this study were to evaluate the inhibition of IL-1--induced expression of MMP1 and MMP-13 by combinatorial treatment with RXR and PPAR ligands and to investigate the molecular mechanisms of this inhibition. Methods We used real-time reverse transcription-polymerase chain reaction to measure LG268- and rosiglitazone-mediated inhibition of MMP gene transcription in IL-1--treated SW-1353 chondrosarcoma cells. An in vitro collagen destruction assay was a functional readout of MMP collagenolytic activity. Luciferase reporter assays tested the function of a putative regulatory element in the promoters of MMP-1 and MMP-13, and chromatin immunoprecipitation (ChIP) assays detected PPAR and changes in histone acetylation at this site. Posttranslational modification of RXR and PPAR by small ubiquitinlike modifier (SUMO) was assayed with immunoprecipitation and Western blot. Results Rosiglitazone inhibited MMP-1 and MMP-13 expression in IL-1--treated SW-1353 cells at the mRNA and heterogeneous nuclear RNA levels and blunted IL-1--induced collagen destruction in vitro. Combining LG268 and rosiglitazone had an additive inhibitory effect on MMP-1 and MMP-13 transcription and collagenolysis. IL-1- inhibited luciferase expression in the MMP reporter assay, but rosiglitazone and LG268 had no effect. ChIP indicated that treatment with IL-1-, but not LG268 and rosiglitazone, increased PPAR at the proximal promoters of both MMPs. Finally, rosiglitazone or LG268 induced 'cross-SUMOylation' of both the target receptor and its binding partner, and IL-1--alone had no effect on SUMOylation of RXR and PPAR but antagonized the ligand-induced SUMOylation of both receptors. Conclusions The PPAR and RXR ligands rosiglitazone and LG268 may act through similar mechanisms, inhibiting MMP-1 and MMP-13 transcription. Combinatorial treatment activates each partner of the RXR:PPAR heterodimer and inhibits IL-1-induced expression of MMP-1 and MMP-13 more effectively than either compound alone. We conclude that the efficacy of combined treatment with lower doses of each drug may minimize potential side effects of treatment with these compounds. AP-1: activator protein-1; ChIP: chromatin immunoprecipitation; DMEM: Dulbecco's modified Eagle's medium; DR-1: direct repeat-1; ECM: extracellular matrix; FBS: fetal bovine serum; FXR: farnesoid X receptor; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HA: hemagglutinin; HAPPAR: hemagglutinin-tagged peroxisome proliferator-activated receptor-gamma; HAT: histone acetyltransferase; HBSS: Hanks' balanced salt solution; HDAC: histone deacetylase; hnRNA: heterogeneous nuclear RNA; IL-1: interleukin-1-beta; IP: immunoprecipitation; LG268: LG100268; LH: lactalbumin hydrosylate; LXR: liver X receptor; MMP: matrix metalloproteinase; MMPI: matrix metalloproteinase inhibitor; MSS: musculoskeletal syndrome; NHR: nuclear hormone receptor; OA: osteoarthritis; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PPAR: peroxisome proliferator-activated receptor-gamma; PPRE: peroxisome proliferator-activated receptor-gamma response element; RA: rheumatoid arthritis; RT: reverse transcription; RXR: retinoid X receptor; SUMO: small ubiquitin-like modifier; TCA: trichloracetic acid.