Abstract
Mechanical loading and hydrostatic pressure (HP) regulate chondrocytes’ metabolism; however, how mechanical stimulation acts remain unclear. MicroRNAs (miRNAs) play an important role in cartilage homeostasis, mechanotransduction, and in the pathogenesis of osteoarthritis (OA). This study investigated the effects of a cyclic HP (1–5 MPa), in both normal and OA human chondrocytes, on the expression of miR-27a/b, miR-140, miR-146a/b, and miR-365, and of their target genes (MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4). Furthermore, we assessed the possible involvement of Wnt/β-catenin pathway in response to HP. Chondrocytes were exposed to HP for 3h and the evaluations were performed immediately after pressurization, and following 12, 24, and 48 h. Total RNA was extracted and used for real-time PCR. β-catenin was detected by Western blotting analysis and immunofluorescence. In OA chondrocytes, HP induced a significant increase (p < 0.01) of the expression levels of miR-27a/b, miR-140, and miR-146a, and a significant reduction (p < 0.01) of miR-365 at all analyzed time points. MMP-13, ADAMTS-5, and HDAC-4 were significantly downregulated following HP, while no significant modification was found for IGFBP-5. β-catenin levels were significantly increased (p < 0.001) in OA chondrocytes at basal conditions and significantly reduced (p < 0.01) by HP. Pressurization did not cause any significant modification in normal cells. In conclusion, in OA chondrocytes, HP restores the expression levels of some miRNAs, downregulates MMP-13, ADAMTS-5, and HDAC-4, and modulates the Wnt/β-catenin pathway activation.
Highlights
Osteoarthritis (OA) is a chronic degenerative joint disease characterized by degradation of articular cartilage, synovial inflammation, thickening of subchondral bone, and formation of osteophytes [1].The major feature of OA is the destruction of extracellular matrix components (ECM) due to the imbalance towards catabolic activities of cartilage-degrading enzymes, like matrix metalloproteinases (MMPs) and disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) [2]
Bioinformatics approaches and in vitro experiments demonstrated that dysregulated miRNAs in OA target several genes encoding extracellular matrix remodeling proteins and pro-inflammatory factors. miR-27a and miR-27b seem to be among the main studied miRNAs involved in OA; recent evidence has been reported that their lower expression in OA cartilage in comparison to normal cartilage was accompanied bydysregulation of both MMP-13 and insulin-like growth factor binding protein (IGFBP)-5 expression levels [17,18]. miR-140 could be considered a regulator of chondrocyte differentiation, bone development, and cartilage homeostasis
It has been reported that the Wnt/β-catenin pathway is associated with mechanical force application in joints, and that this signaling is activated after mechanical damage to cartilage [24,25].The Wnt/β-catenin pathway exerts a pivotal role in cartilage homeostasis and in OA pathogenesis [24]; in addition, it seems to be involved in the regulation of miRNA expression [26]
Summary
Osteoarthritis (OA) is a chronic degenerative joint disease characterized by degradation of articular cartilage, synovial inflammation, thickening of subchondral bone, and formation of osteophytes [1].The major feature of OA is the destruction of extracellular matrix components (ECM) due to the imbalance towards catabolic activities of cartilage-degrading enzymes, like matrix metalloproteinases (MMPs) and disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) [2]. It has been reported that the Wnt/β-catenin pathway is associated with mechanical force application in joints, and that this signaling is activated after mechanical damage to cartilage [24,25].The Wnt/β-catenin pathway exerts a pivotal role in cartilage homeostasis and in OA pathogenesis [24]; in addition, it seems to be involved in the regulation of miRNA expression [26]. The purpose of this study was to investigate the possible effect of cyclic hydrostatic pressure (HP) (1–5 MPa, 0.25 Hz) on miR-27a/b, miR-140, miR-146a/b, and miR-365 expression levels, as well as on the mRNA levels of their target genes, MMP-13, ADAMTS-5, IGFBP-5, and HDAC-4, in human normal and OA chondrocyte cultures. We evaluated the possible activation of the Wnt/β-catenin
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