OPN Inhibits Autophagy through CD44, Integrin and the MAPK Pathway in Osteoarthritic Chondrocytes

Background: To investigate whether OPN has an effect on autophagy in human osteoarthritic chondrocytes and determine the roles of CD44, αvβ3 integrin and the MAPK pathway in this progress. Methods: First, we cultured human OA chondrocytes in vitro and then treated cells with rhOPN to determine autophagy changes. Next , the anti-CD44 and anti-CD51/61 monoclonal antibodies (Abs) or isotype IgG were used to determine the possible role of CD44 and αvβ3 integrin; subsequently, an inhibitor of the ERK MAPK pathway was used to investigate the role of ERK MAPK. Western blotting was used to measure the beclin1, LC3 II and MAPK protein expression, and mRFP-GFP-LC3 confocal imaging was used to detect the autophagy levels. CCK-8 was used to assay the proliferation and activity of chondrocytes. Results: Our results showed that the LC3 protein was greatly decreased in OA cartilage compared to normal cartilage ,and OPN suppressed the autophagy activity in chondrocytes in vitro. Blocking experiments with anti-CD44 and anti-CD51/61 Abs indicated that OPN could suppress the expression of LC3II and beclin1 through αvβ3 integrin and CD44. Our results also indicated that the ratio of p-ERK/ ERK but not p-P38/P38 and p-JNK/JNK was increased after the rhOPN treatment. The ERK inhibitor inhibited the activity of OPN in the suppression of autophagy, and the CCK-8 results showed that rhOPN could promote chondrocyte proliferation. Conclusions: OPN inhibited chondrocyte autophagy through CD44 and αvβ3 integrin receptors and via the ERK MAPK signaling pathway.

Effect of Micro-strain Stress on in Vitro Proliferation and Functional Expression of Human Osteoarthritic Chondrocytes

Background: This study aimed to analyze the in vitro effect of micro-strain stress on the proliferation and functional marker expression in chondrocytes isolated from human osteoarthritis cartilage samples.Methods: Chondrocytes isolated from human osteoarthritis cartilage samples were subjected to loading with different types of micro-strain stress. The proliferation activity was assessed by flow cytometry, and the functional expression of chondrocyte markers was detected by qRT-PCR and western blot. Results: Flow cytometry results showed stimulation of proliferation of human osteoarthritic chondrocytes when an adequate micro-strain stress was applied. qRT-PCR and western blot results showed that micro-strain stress promotes human osteoarthritic chondrocyte functional marker expression. These features coincide with the upregulation of multiple proteins and genes affecting cell proliferation and functional chondrocyte marker expression, including cyclin D1, collagen II, and Rock.Conclusion: Adequate micro-strain stress could activate the Rho/Rock signaling pathway in osteoarthritic chondrocytes, thus transmitting mechanical signals to the cytoskeleton. This process leads to cytoskeleton reorganization, and transmission of the mechanical signals to the downstream effectors to promote proliferation and functional marker expression of osteoarthritic chondrocytes.

Pleiotropic Roles of NOTCH1 Signaling in the Loss of Maturational Arrest of Human Osteoarthritic Chondrocytes

Notch signaling has been identified as a critical regulator of cartilage development and homeostasis. Its pivotal role was established by both several joint specific Notch signaling loss of function mouse models and transient or sustained overexpression. NOTCH1 is the most abundantly expressed NOTCH receptors in normal cartilage and its expression increases in osteoarthritis (OA), when chondrocytes exit from their healthy “maturation arrested state” and resume their natural route of proliferation, hypertrophy, and terminal differentiation. The latter are hallmarks of OA that are easily evaluated in vitro in 2-D or 3-D culture models. The aim of our study was to investigate the effect of NOTCH1 knockdown on proliferation (cell count and Picogreen mediated DNA quantification), cell cycle (flow cytometry), hypertrophy (gene and protein expression of key markers such as RUNX2 and MMP-13), and terminal differentiation (viability measured in 3-D cultures by luminescence assay) of human OA chondrocytes. NOTCH1 silencing of OA chondrocytes yielded a healthier phenotype in both 2-D (reduced proliferation) and 3-D with evidence of decreased hypertrophy (reduced expression of RUNX2 and MMP-13) and terminal differentiation (increased viability). This demonstrates that NOTCH1 is a convenient therapeutic target to attenuate OA progression.

Attenuative Effects of Platelet-Rich Plasma on 30 kDa Fibronectin Fragment-Induced MMP-13 Expression Associated with TLR2 Signaling in Osteoarthritic Chondrocytes and Synovial Fibroblasts

Proteolytic fragments of fibronectin can have catabolic effects on cartilage, menisci, and synovium. Previous studies have reported that Toll-like receptor (TLR) signaling pathways might be associated with joint inflammation and joint destruction. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions; however, it has yet to be determined whether PRP influences fibronectin fragment (FN-f) procatabolic activity and TLRs. In this study, human primary culture cells were treated with 30 kDa FN-f with/without PRP co-incubation, and then analyzed using real-time PCR to determine gene expression levels in articular chondrocytes, meniscal fibrochondrocytes, and synovial fibroblasts. Protein levels were evaluated by Western immunoblotting. This study observed an increase in the protein expression of matrix metalloproteinases (MMPs), Toll-like receptor 2 (TLR2), nitric oxide synthase 2 (NOS2), prostaglandin-endoperoxide synthase (PTGS2), and cyclooxygenase 2 (COX2) in articular chondrocytes, meniscal fibrochondrocytes, and synovial fibroblasts following insult with 30 kDa FN-f. Upregulation of these genes was significantly attenuated by PRP treatment. TLR2 and matrix metalloproteinase 13 (MMP-13) were also significantly attenuated by cotreatment with 30 kDa FN-f + PRP + TLR2 inhibitor. PRP treatment was shown to attenuate the 30 kDa FN-f-induced MMP-13 expression associated with the decreased expression of TLR2 in osteoarthritic chondrocytes and synovial fibroblasts. PRP treatment was also shown to attenuate procatabolic activity associated with MMP-13 expression via the TLR2 signaling pathway.

Hypoxia Inducible Factor-1α Is a Regulator of Autophagy in Osteoarthritic Chondrocytes

Objective To investigate the relationship between hypoxia inducible factor-1α (HIF-1α) and the autophagic response in osteoarthritic chondrocytes (OA), under inflammatory insult as represented by in vitro OA model. Methods Human chondrocyte cell line C28/I2 was cultured in both normoxic and hypoxic conditions and treated with interleukin-1β (IL1β) to emulate OA inflammatory insult in vitro. Cellular HIF-1α expression was silenced using siRNA transfection and cellular autophagic (P62/LC3II) response and OA chondrocyte damage (COL2A1/MMP13) related proteins were examined using western blotting. Cellular mitophagic (BNIP3/PINK1/Parkin) and apoptotic (Caspase/Cleaved Caspase 3) were also evaluated to assess mitophagy-mediated cell death due to HIF-1α silencing. Results Chondrocyte basal autophagy levels were higher in a HIF-1α elevated environment and was more resistant to IL1β-induced inflammatory insult. Increase in autophagic proteins showed better chondrocyte repair, which resulted a lower level of reactive oxygen species production, and lesser damage to chondrocyte integrity. Silencing HIF-1α activates cellular PINK1/Parkin and BNIP3 mitophagic proteins, which leads to the activation of Caspase/Cleaved Caspase 3 apoptotic cascade. Conclusion Our results show that chondrocyte autophagy is dependent on HIF-1α expression, showing the importance of HIF-1α in hypoxic chondrocyte function in OA. Dysregulation of HIF-1α expression results in the activation of mitophagy-mediated apoptosis.