IL-1β-induced miR-34a up-regulation inhibits Cyr61 to modulate osteoarthritis chondrocyte proliferation through ADAMTS-4.

Osteoarthritis (OA) is the most prevalent degenerative joint disease with multifactorial etiology caused by risk factors. The degradation of aggrecan by upregulated ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is the key event in the development of OA. ADAMTS-4 contributes to aggrecan degradation in human OA. Cysteine-rich angiogenic inducer 61 (Cyr61), which is associated with diseases related to chronic inflammation, is found in articular cartilage from patients with osteoarthritis and appears to suppress ADAMTS-4 activity, possibly leading to chondrocyte cloning. Herein, we first revealed that Cyr61 and ADAMTS-4 protein levels were remarkably increased in OA cartilage tissues and OA chondrocytes, and verified Cyr61 regulation of ADAMTS-4 in normal and OA chondrocyte. Further, we revealed that Cyr61 could promote OA chondrocyte proliferation through inhibiting ADAMTS-4. Overproduction of inflammatory cytokines plays a vital role in the pathological development of OA; herein, we demonstrated that IL-1β inhibited Cyr61, while promoted ADAMTS-4 expression. By using online tools and luciferase assays, we confirmed that miR-34a, a regulatory miRNA of chondrocyte proliferation, could directly bind to the 3'-UTR of Cyr61 to inhibit its expression; further, IL-1β regulated Cyr61 and ADAMTS-4 expression through miR-34a. In OA cartilage tissues, miR-34a, and IL-1β mRNA expression was up-regulated and positively correlated; miR-34a and Cyr61 mRNA was positively correlated, further indicating that suppressing miR-34a expression might rescue IL-1β-induced Cyr61 suppression, and promote OA chondrocyte proliferation. Taken together, we provided novel experimental basis for rescuing OA chondrocyte proliferation through miR-34a/Cyr61 axis.

Yang B ，Ni J ，Long H ，Huang J ，Yang C ，Huang X ... -  《-》

MicroRNA-92a-3p Regulates Aggrecanase-1 and Aggrecanase-2 Expression in Chondrogenesis and IL-1β-Induced Catabolism in Human Articular Chondrocytes.

Aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5) are secreted enzymes belonging to the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family that play significant roles in the progression of osteoarthritis (OA). Here, we aimed to determine whether the expression of ADAMTS-4/5 in chondrogenesis and inflammation is regulated by microRNA-92a-3p (miR-92a-3p). MiR-92a-3p and ADAMTS-4/5 expressions were determined by quantitative polymerase chain reaction (qPCR). To investigate the repressive effect of miR-92a-3p on ADAMTS-4/5 expression, chondrogenic human mesenchymal stem cells (hMSCs) and human chondrocytes were transfected with mature miR-92a-3p or an antisense inhibitor (anti-miR-92a-3p), respectively. ADAMTS-4/5 protein production was quantified by enzyme-linked immunosorbent assay (ELISA), and miR-92a-3p involvement in IL-1β-mediated catabolic effects was examined by immunoblotting. The roles of activated MAP kinases (MAPK) and nuclear factor (NF)-κB were evaluated by using specific inhibitors. Interaction between miR-92a-3p and its putative binding site in the 3'-untranslated region (3'-UTR) of ADAMTS-4/5 mRNA was confirmed by luciferase reporter assay. miR-92a-3p expression was elevated in chondrogenic hMSCs, with significantly lower expression in OA cartilage than in normal cartilage. Stimulation with IL-1β significantly reduced miR-92a-3p expression in primary human chondrocytes (PHCs). Transfection of chondrocytes with miR-92a-3p downregulated IL-1β-induced ADAMTS-4/5 expression, and the activity of a reporter construct containing the 3'-UTR of human ADAMTS-4/5 mRNA. MiR-92a-3p expression was suppressed upon IL-1β-induced activation of MAPK and NF-κB in chondrocytes. MiR-92a-3p is an important regulator of ADAMTS-4/5 in human chondrocytes and may contribute to the development of OA.

Mao G ，Wu P ，Zhang Z ，Zhang Z ，Liao W ，Li Y ，Kang Y ... -  《-》

CCN1 (Cyr61) Is Overexpressed in Human Osteoarthritic Cartilage and Inhibits ADAMTS-4 (Aggrecanase 1) Activity.

ADAMTS-4, also called aggrecanase 1, is considered to play a key role in aggrecan degradation in human osteoarthritic (OA) cartilage, but information about regulators of ADAMTS-4 aggrecanase activity remains limited. We undertook this study to search for molecules that modulate ADAMTS-4 activity. Molecules copurified with ADAMTS-4 from ADAMTS-4-transfected chondrocytic cells were sequenced by nanoscale liquid chromatography tandem mass spectrometry. Binding activity was determined by immunoprecipitation and solid-phase binding assay. Effects on ADAMTS-4 activity were examined by aggrecan digestion assay. Expression of the binding molecule in OA cartilage and chondrocytes was examined by immunohistochemistry and reverse transcription-polymerase chain reaction. We identified CCN1 (Cyr61) as an ADAMTS-4-binding protein and showed specific binding to the ADAMTS-4 cysteine-rich domain. Aggrecanase activity of ADAMTS-4 was inhibited by interaction with CCN1. Expression of messenger RNA for CCN1 was significantly higher in human OA cartilage than in normal cartilage. CCN1 was immunolocalized to chondrocytes in OA cartilage, showing direct correlations of immunoreactivity with the Mankin score of cartilage lesions and chondrocyte cloning. CCN1 and ADAMTS-4 were commonly coexpressed in clustered chondrocytes. CCN1 expression in OA chondrocytes was down-regulated by interleukin-1α (IL-1α) and up-regulated by transforming growth factor β (TGFβ). ADAMTS-4 expression was induced by treatment with IL-1α or TGFβ, but aggrecanase activity was detected only under stimulation with IL-1α. TGFβ-treated chondrocytes exhibited aggrecanase activity when CCN1 expression was knocked down. Our findings provide the first evidence that CCN1 suppresses ADAMTS-4 activity and that CCN1 overexpression is directly correlated with chondrocyte cloning in OA cartilage. Our results suggest that the TGFβ/CCN1 axis plays a role in chondrocyte cluster formation through inhibition of ADAMTS-4.

Chijiiwa M ，Mochizuki S ，Kimura T ，Abe H ，Tanaka Y ，Fujii Y ，Shimizu H ，Enomoto H ，Toyama Y ，Okada Y ... -  《-》

Downregulation of miR-221-3p contributes to IL-1β-induced cartilage degradation by directly targeting the SDF1/CXCR4 signaling pathway.

Zheng X ，Zhao FC ，Pang Y ，Li DY ，Yao SC ，Sun SS ，Guo KJ ... -  《-》

LncRNA MALAT1/MiR-145 Adjusts IL-1β-Induced Chondrocytes Viability and Cartilage Matrix Degradation by Regulating ADAMTS5 in Human Osteoarthritis.

Accumulating evidence suggests that microRNA-145 (miR-145) plays an important role in osteoarthritis (OA), which is a chronic progressive joint disease. Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes metastasis in cancers and functions as a sponge for miR-145. However, the role of MALAT1/miR-145 in OA pathogenesis has not yet been elucidated. The expression of MALAT1 and miR-145 was examined by quantitative real-time PCR; the interaction between miR-145, MALAT1 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was verified by luciferase reporter assay. Correlations among MALAT1, miR-145, and ADAMTS5 were analyzed by Spearman rank analysis. Chondrocytes viability and cartilage extracellular matrix (ECM) degradation were investigated with cell viability assay and Western blotting analyzing expression of ADAMTS5, collagen type 2 alpha 1 (COL2A1), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP). MALAT1 was upregulated, and miR-145 was downregulated in OA samples and IL-1β-induced chondrocytes. Mechanically, miR-145 could directly bind to MALAT1 and ADAMTS5. Moreover, miR-145 expression was negatively correlated with MALAT1 and ADAMTS5 expression in OA patients, whereas MALAT1 and ADAMTS5 expression was positively correlated. Functionally, overexpression of MALAT1 inhibited chondrocyte viability and promoted cartilage ECM degradation in IL-1β-induced chondrocytes. In support thereof, MALAT1 silencing and miR-145 upregulation exerted the opposite effect in IL-1β-induced chondrocytes. Moreover, the effect of MALAT1 was counteracted by miR-145 upregulation, and ADAMTS5 restoration could abate miR-145 effects. An MALAT1/miR-145 axis contributes to ECM degradation in IL-1β-induced chondrocytes through targeting ADAMTS5, suggesting that MALAT1/miR-145/ADAMTS5 signaling may underlie human OA pathogenesis.

Liu C ，Ren S ，Zhao S ，Wang Y ... -  《-》