OPN inhibits autophagy through CD44, integrin and the MAPK pathway in osteoarthritic chondrocytes
Research Square (Research Square)(2022)
Abstract
BackgroundTo investigate whether osteopontin (OPN) affects autophagy in human osteoarthritic chondrocytes and determine the roles of CD44, alpha v beta 3 integrin and the Mitogen-activated protein kinase (MAPK) pathway in this progress. MethodsFirst, we compared the autophagy levels in the human osteoarthritis (OA) and normal cartilage, then, we cultured human OA chondrocytes in vitro and treated cells with recombinant human OPN (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 alpha v beta 3 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 proteins expressions, mRFP-GFP-LC3 confocal imaging and transmission electron microscopy were also used to detect the autophagy levels. Cell Counting Kit-8 (CCK-8) was used to assay the proliferation and activity of chondrocytes. ResultsThe 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 alpha v beta 3 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. ConclusionOPN inhibited chondrocyte autophagy through CD44 and alpha v beta 3 integrin receptors and via the ERK MAPK signaling pathway.
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Key words
autophagy,chondrocyte,integrin,CD44,osteoarthritis,osteopontin
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