15-Deoxy-Δ-12, 14-prostaglandin J2 acts cooperatively with prednisolone to reduce TGF-β-induced pro-fibrotic pathways in human osteoarthritis fibroblasts

[Abstract] Abstract Background/aims: 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. Materials: 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. Results: 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. Conclusions: 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

CEMIP (KIAA1199) induces a fibrosis-like process in osteoarthritic chondrocytes

peer reviewedCEMIP (for “Cell migration-inducing protein” also called KIAA1199 and Hybid for “Hyaluronan-binding protein”) expression is increased in cancers and described as a regulator of cell survival, growth and invasion. In rheumatoid arthritis, CEMIP is referred to as an angiogenic marker and participates in hyaluronic acid degradation. In this study, CEMIP expression is investigated in healthy and osteoarthritis (OA) cartilage from human and mouse. Its role in OA physiopathology is deciphered, specifically in chondrocytes proliferation and dedifferentiation and in the extracellular matrix remodeling. To this end, CEMIP, αSMA and types I and III collagen expressions were assessed in human OA and non-OA cartilage. CEMIP expression was also investigated in a mouse OA model. CEMIP expression was studied in vitro using a chondrocyte dedifferentiation model. High-throughput RNA sequencing was performed on chondrocytes after CEMIP silencing. Results showed that CEMIP was overexpressed in human and murine OA cartilage and along chondrocytes dedifferentiation. Most of genes deregulated in CEMIP-depleted cells were involved in cartilage turnover (e.g., collagens), mesenchymal transition and fibrosis. CEMIP regulated β-catenin protein level. Moreover, CEMIP was essential for chondrocytes proliferation and promoted αSMA expression, a fibrosis marker, and TGFβ signaling towards the p-Smad2/3 (Alk5/PAI-1) pathway. Interestingly, CEMIP was induced by the pSmad1/5 (Alk1) pathway. αSMA and type III collagen expressions were overexpressed in human OA cartilage and along chondrocytes dedifferentiation. Finally, CEMIP was co-expressed in situ with αSMA in all OA cartilage layers. In conclusion, CEMIP was sharply overexpressed in human and mouse OA cartilage and along chondrocytes dedifferentiation. CEMIP-regulated transdifferentiation of chondrocytes into “chondro-myo-fibroblasts” expressing α-SMA and type III collagen, two fibrosis markers. Moreover, these “chondro-myo-fibroblasts” were found in OA cartilage but not in healthy cartilage

Modulation of αVβ6 integrin in osteoarthritis-related synovitis and the interaction with VTN (381-397 a.a.) competing for TGF-β1 activation

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Etude protéique de l'insuffisance mitrale. Implication de l'autophagie dans la signalisation cellulaire.

Primary mitral regurgitation (MR), due to valve prolapse, is one of the most frequent valvular heart diseases affecting around 2.5% of the population. Primary mitral regurgitation is becoming increasingly more prevalent as a result of population aging. In its most severe form, it may lead to pressure overload and heart failure with poor outcomes and about 2% of sudden death. As there is no blood biomarker available in clinical laboratories for its diagnosis, observation of clinical manifestations and echocardiography measurements constitute the current management of patients. Moreover, the molecular deregulation leading to myxomatous degeneration and mitral valve prolapse remains largely unknown. In the present work, we used a targeted and a non-targeted proteomic approach in order to identify biomarkers for the diagnosis of primary mitral regurgitation. Moreover, we brought out potential biological processes altered in the pathology. We identified 7 differentially expressed proteins between patients with or without mitral regurgitation. Differential protein levels between groups indicated a decrease in the reverse cholesterol transport, a potential oxidative stress and a haemolytic state as well as impairment in cell proliferation and apoptosis. In addition, we found an increase of autophagy activity in myxomatous mitral valves compared to healthy mitral valves. In a second part of the work, we investigated the molecular regulation of the autophagy pathway. C16-ceramide treatment allowed us to induce autophagic cell death in HCT116 cells. We found that C16-ceramide induced the phosphorylation of emerin, a nuclear membrane protein, at its LEM domain. Moreover, we highlighted that this phosphorylation was required for the induction of autophagy by emerin. Moreover, we found a direct interaction between emerin and LC3, the marker of autophagosome formation. To go further in deciphering the role of emerin in the C16-ceramide autophagy pathway, we identified new binding partners through biochemical purification and mass spectrometry analysis

Le ranélate de strontium augmente la viabilité de chondrocytes humains stimulés par IL-1 bêta

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