Inhibition of Notch1 promotes hedgehog signalling in a HES1-dependent manner in chondrocytes and exacerbates experimental osteoarthritis.

Notch ligands and receptors have recently been shown to be differentially expressed in osteoarthritis (OA). We aim to further elucidate the functional role of Notch signalling in OA using Notch1 antisense transgenic (Notch1 AS) mice. Notch and hedgehog signalling were analysed by real-time PCR and immunohistochemistry. Notch-1 AS mice were employed as a model of impaired Notch signalling in vivo. Experimental OA was induced by destabilisation of the medial meniscus (DMM). The extent of cartilage destruction and osteophyte formation was analysed by safranin-O staining with subsequent assessment of the Osteoarthritis Research Society International (OARSI) and Mankin scores and µCT scanning. Collagen X staining was used as a marker of chondrocyte hypertrophy. The role of hairy/enhancer of split 1 (Hes-1) was investigated with knockdown and overexpression experiments. Notch signalling was activated in human and murine OA with increased expression of Jagged1, Notch-1, accumulation of the Notch intracellular domain 1 and increased transcription of Hes-1. Notch1 AS mice showed exacerbated OA with increases in OARSI scores, osteophyte formation, increased subchondral bone plate density, collagen X and osteocalcin expression and elevated levels of Epas1 and ADAM-TS5 mRNA. Inhibition of the Notch pathway induced activation of hedgehog signalling with induction of Gli-1 and Gli-2 and increased transcription of hedgehog target genes. The regulatory effects of Notch signalling on Gli-expression were mimicked by Hes-1. Inhibition of Notch signalling activates hedgehog signalling, enhances chondrocyte hypertrophy and exacerbates experimental OA including osteophyte formation. These data suggest that the activation of the Notch pathway may limit aberrant hedgehog signalling in OA.

Lin NY ，Distler A ，Beyer C ，Philipi-Schöbinger A ，Breda S ，Dees C ，Stock M ，Tomcik M ，Niemeier A ，Dell'Accio F ，Gelse K ，Mattson MP ，Schett G ，Distler JH ... -  《-》

Cartilage-specific Sirt6 deficiency represses IGF-1 and enhances osteoarthritis severity in mice.

Collins JA ，Kim CJ ，Coleman A ，Little A ，Perez MM ，Clarke EJ ，Diekman B ，Peffers MJ ，Chubinskaya S ，Tomlinson RE ，Freeman TA ，Loeser RF ... -  《-》

Inhibition of Rac1 activity by controlled release of NSC23766 from chitosan microspheres effectively ameliorates osteoarthritis development in vivo.

Osteoarthritis (OA) is a degenerative joint disease characterised by cartilage degradation and chondrocyte hypertrophy. A recent study showed that Rac1 promoted expression of MMP13 and chondrocyte hypertrophy within the growth plate. These findings warrant further investigations on the roles of Rac1 in OA development and therapy in animal models. To investigate the role and mechanistic pathway of Rac1 involvement in pathological changes of OA chondrocytes in vitro and OA development in vivo, as well as to develop a strategy of modulating Rac1 activity for OA treatment. OA and normal cartilage from human or mice were used for immunohistochemical study and Rac1 activity assay. Chondrocytes treated with IL1β and the untreated control were subjected to the Rac1 activity assay. Chondrocytes transfected with CA-Rac1, DN-Rac1 or GFP were cultured under conditions for inducing calcification. To evaluate the effect of Rac1 in OA development, an OA model was created by anterior cruciate ligament transection in mice. CA-Rac1, DN-Rac1 and GFP lentivirus, or NSC23766, were injected intra-articularly. Joints were subjected to histological analysis. It was found that there is aberrant Rac1 activation in human OA cartilage. Rac1 activity could also be elevated by IL1β. Additionally, activated Rac1 promoted expression of MMP13, ADAMTS-5 and COLX by chondrocytes, partially through the β-catenin pathway. Moreover, activation of Rac1 in knee joints by CA-Rac1 lentivirus accelerated OA progression, while inhibition of Rac1 activity by DN-Rac1 lentivirus or Rac1 inhibitor NSC23766 delayed OA development. Therefore, we developed a strategy of controlled release of NSC23766 from chitosan microspheres to OA joints, which effectively protected cartilage from destruction. These findings demonstrated that Rac1 activity is implicated in OA development. Also, controlled release of Rac1 inhibitor is a promising strategy for OA treatment.

Zhu S ，Lu P ，Liu H ，Chen P ，Wu Y ，Wang Y ，Sun H ，Zhang X ，Xia Q ，Heng BC ，Zhou Y ，Ouyang HW ... -  《-》

NAMPT (visfatin), a direct target of hypoxia-inducible factor-2α, is an essential catabolic regulator of osteoarthritis.

Hypoxia-inducible factor 2α (HIF-2α), encoded by Epas1, causes osteoarthritic cartilage destruction by regulating the expression of matrix-degrading enzymes. We undertook this study to explore the role of nicotinamide phosphoribosyltransferase (NAMPT or visfatin) in HIF-2α-mediated osteoarthritic cartilage destruction. The expression of HIF-2α, NAMPT and matrix-degrading enzymes was determined at the mRNA and protein levels in human osteoarthritis (OA) cartilage, mouse experimental OA cartilage and primary cultured mouse chondrocytes. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) surgery or intra-articular injection of Ad-Epas1 or Ad-Nampt in wild-type, Epas1(+/-), Epas1(fl/fl);Col2a1-Cre and Col2a1-Nampt transgenic (TG) mice. Primary cultured mouse chondrocytes were treated with recombinant NAMPT protein or were infected with adenoviruses. We found that the Nampt gene is a direct target of HIF-2α in articular chondrocytes and OA cartilage. NAMPT protein, in turn, increased mRNA levels and activities of MMP3, MMP12 and MMP13 in chondrocytes, an action that was necessary for HIF-2α-induced expression of catabolic enzymes. Gain-of-function studies (intra-articular injection of Ad-Nampt; Col2a1-Nampt TG mice) and loss-of-function studies (intra-articular injection of the NAMPT inhibitor FK866) demonstrated that NAMPT is an essential catabolic regulator of osteoarthritic cartilage destruction caused by HIF-2α or DMM surgery. Our findings indicate that NAMPT, whose corresponding gene is a direct target of HIF-2α, plays an essential catabolic role in OA pathogenesis and acts as a crucial mediator of osteoarthritic cartilage destruction caused by HIF-2α or DMM surgery.

Yang S ，Ryu JH ，Oh H ，Jeon J ，Kwak JS ，Kim JH ，Kim HA ，Chun CH ，Chun JS ... -  《-》

Inducible chondrocyte-specific overexpression of BMP2 in young mice results in severe aggravation of osteophyte formation in experimental OA without altering cartilage damage.

In osteoarthritis (OA) chondrocytes surrounding lesions express elevated bone morphogenetic protein 2 (BMP2) levels. To investigate the functional consequence of chondrocyte-specific BMP2 expression, we made a collagen type II dependent, doxycycline (dox)-inducible BMP2 transgenic mouse and studied the effect of elevated BMP2 expression on healthy joints and joints with experimental OA. We cloned a lentivirus with BMP2 controlled by a tet-responsive element and transfected embryos of mice containing a collagen type II driven cre-recombinase and floxed rtTA to gain a mouse expressing BMP2 solely in chondrocytes and only upon dox exposure (Col2-rtTA-TRE-BMP2). Mice were treated with dox to induce elevated BMP2 expression. In addition, experimental OA was induced (destabilisation of the medial meniscus model) with or without dox supplementation and knee joints were isolated for histology. Dox treatment resulted in chondrocyte-specific upregulation of BMP2 and severely aggravated formation of osteophytes in experimental OA but not in control mice. Moreover, elevated BMP2 levels did not result in alterations in articular cartilage of young healthy mice, although BMP2-exposure did increase VDIPEN expression in the articular cartilage. Strikingly, despite apparent changes in knee joint morphology due to formation of large osteophytes there were no detectible differences in articular cartilage: none with regard to structural damage nor in Safranin O staining intensity when comparing destabilisation of the medial meniscus with or without dox exposure. Our data show that chondrocyte-specific elevation of BMP2 levels does not alter the course of cartilage damage in an OA model in young mice but results in severe aggravation of osteophyte formation.

Blaney Davidson EN ，Vitters EL ，Bennink MB ，van Lent PL ，van Caam AP ，Blom AB ，van den Berg WB ，van de Loo FA ，van der Kraan PM ... -  《-》