15-deoxyprostaglandin-j2 and Osteoarthritis

Synovial fibrosis is a pathological process that is observed in several musculoskeletal disorders and characterized by the excessive deposition of extracellular matrix, as well as cell migration and proliferation. Despite the fact that glucocorticoids are widely employed in the treatment of rheumatic pathologies such as osteoarthritis (OA) and rheumatoid arthritis, the mechanisms by which glucocorticoids act in the joint and their impacts on pro-fibrotic pathways are still unclear.. Human OA synovial fibroblasts were obtained from knee and hip joints. Cells were treated with prednisolone (1 mM) or transforming growth factor-beta 1 (TGF-β1) (10 ng/ml) for 1 and 7 days for quantification of RNA and protein expression (by real-time quantitative reverse transcription-PCR and western blot, respectively), 72 h for immunocytochemistry analysis, and 48 h for proliferation (by BrdU assay) and migration (by wound assay) studies. In addition, cells were preincubated with prednisolone and/or the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) for 6 h before adding TGF-β1. pSmad1/5, pSmad2 and β-catenin levels were analyzed by Western blot. The activin receptor-like kinase-5 (ALK-5) inhibitor (SB-431542) was employed for the mechanistic assays.. Prednisolone showed a predominant anti-fibrotic impact on fibroblast-like synoviocytes as it attenuated the spontaneous and TGF-β-induced gene expression of pro-fibrotic markers. Prednisolone also reduced α-sma protein and type III collagen levels, as well as cell proliferation and migration after TGF-β stimulation. However, prednisolone did not downregulate the gene expression of all the pro-fibrotic markers tested and did not restore the reduced PPAR-γ levels after TGF-β stimulation. Interestingly, anti-fibrotic actions of the glucocorticoid were reinforced in the presence of the PPAR-γ agonist 15d-PGJ2. Combined pretreatment modulated Smad2/3 levels and, similar to the ALK-5 inhibitor, blocked β-catenin accumulation elicited by TGF-β.. Prednisolone, along with 15d-PGJ2, modulates pro-fibrotic pathways activated by TGF-β in synovial fibroblasts at least partially through the inhibition of ALK5/Smad2 signaling and subsequent β-catenin accumulation. These findings shed light on the potential therapeutic effects of glucocorticoids treatment combined with a PPAR-γ agonist against synovial fibrosis, although future studies are warranted to further evaluate this concern.

Topics: Adult; Aged; beta Catenin; Cells, Cultured; Female; Fibroblasts; Fibrosis; Humans; Male; Middle Aged; Osteoarthritis; PPAR gamma; Prednisolone; Prostaglandin D2; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a central role in whole body metabolism by regulating adipocyte differentiation and energy storage. Recently, however, PPAR-gamma has also been demonstrated to affect proliferation, differentiation, and apoptosis of different cell types. As we have previously shown that BAY 11-7085-induced synovial fibroblast apoptosis is prevented by PPAR-gamma agonist 15d-PGJ2; the expression of PPAR-gamma in these cells was studied. Both PPAR-gamma1 and PPAR-gamma2 isoforms were cloned from synovial fibroblast RNA, but only PPAR-gamma1 was detected by Western blot, showing constitutive nuclear expression. Within minutes of BAY 11-7085 treatment, a PPAR-gamma1-specific band was shifted into a form of higher mobility, suggesting dephosphorylation, as confirmed by phosphatase treatment of cell extracts. Of interest, BAY 11-7085-induced PPAR-gamma1 dephosphorylation was followed by PARP and caspase-8 cleavage as well as by PPAR-gamma1 protein degradation. PPAR-gamma1 dephosphorylation was followed by the loss of PPAR-DNA binding activity ubiquitously present in synovial fibroblast nuclear extracts. Unlike the phosphorylated form, dephosphorylated PPAR-gamma1 was found in insoluble membrane cell fraction and was not ubiquitinated before degradation. PPAR-gamma1 dephosphorylation coincided with ERK1/2 phosphorylation that accompanies BAY 11-7085-induced synovial fibroblasts apoptosis. 15d-PGJ2, PGD2, and partially UO126, down-regulated ERK1/2 phosphorylation, protected cells from BAY 11-7085-induced apoptosis, and reversed both PPAR-gamma dephosphorylation and degradation. Furthermore, PPAR-gamma antagonist BADGE induced PPAR-gamma1 degradation, ERK1/2 phosphorylation, and synovial fibroblasts apoptosis. The results presented suggest an anti-apoptotic role for PPAR-gamma1 in synovial fibroblasts. Since apoptotic marker PARP is cleaved after PPAR-gamma1 dephosphorylation but before PPAR-gamma1 degradation, dephosphorylation event might be enough to mediate BAY 11-7085-induced apoptosis in synovial fibroblasts.

Topics: Apoptosis; Cell Line; Cell Nucleus; Fibroblasts; Gene Expression; Humans; Nitriles; Osteoarthritis; Phosphorylation; PPAR gamma; Prostaglandin D2; Sulfones; Synovial Membrane; Transfection