Critical role for arginase II in osteoarthritis pathogenesis.

Osteoarthritis (OA) appears to be associated with various metabolic disorders, but the potential contribution of amino acid metabolism to OA pathogenesis has not been clearly elucidated. Here, we explored whether alterations in the amino acid metabolism of chondrocytes could regulate OA pathogenesis. Expression profiles of amino acid metabolism-regulating genes in primary-culture passage 0 mouse chondrocytes were examined by microarray analysis, and selected genes were further characterised in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The functional consequences of arginase II (Arg-II) were examined in Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg-II (Ad-Arg-II). The gene encoding Arg-II, an arginine-metabolising enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human patients with OA and various mouse models. Adenovirus-mediated overexpression of Arg-II in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2-/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg-II appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (matrix metalloproteinase 3 [MMP3] and MMP13) in chondrocytes via the nuclear factor (NF)-κB pathway. Our results indicate that Arg-II is a crucial regulator of OA pathogenesis in mice. Although chondrocytes of human and mouse do not identically, but similarly, respond to Arg-II, our results suggest that Arg-II could be a therapeutic target of OA pathogenesis.

Choi WS ，Yang JI ，Kim W ，Kim HE ，Kim SK ，Won Y ，Son YO ，Chun CH ，Chun JS ... -  《-》

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 ... -  《-》

Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT.

Oh H ，Kwak JS ，Yang S ，Gong MK ，Kim JH ，Rhee J ，Kim SK ，Kim HE ，Ryu JH ，Chun JS ... -  《-》

Gumiganghwal-tang ameliorates cartilage destruction via inhibition of matrix metalloproteinase.

Kyung-Bang Gumiganghwal-tang tablet (GMGHT) is a standardized Korean Medicine that could treat a cold, headache, arthralgia and fever. Although GMGHT has been used for arthritis-related diseases including a sprain, arthralgia, unspecified arthritis and knee arthritis, there is no pre-clinical evidence to treat osteoarthritis (OA). This study determined the drug dosage and the mechanisms of GMGHT for OA. OA was induced by intra-articular monoiodoacetic acid (MIA) injection in Sprague-Dawley rats. As calculated from the human equivalent dose formula, GMGHT was orally administered at the doses of 9.86, 98.6 and 986 mg/kg for 4 weeks. The arthritis score was performed by a blind test, and histological changes in articular cartilage were indicated by hematoxylin and eosin, Safranin O and toluidine blue staining. SW1353 chondrocytes were stimulated by interleukin (IL)-1β recombinant to analyze the expressions of Type II collagen, matrix metalloproteinases (MMPs) and nuclear factor (NF)-κB. Rough and punctate surfaces of the femoral condyle induced by MIA, were recovered by the GMGHT treatment. The arthritis score was significantly improved in the 968 mg/kg of GMGHT-treated cartilage. Loss of chondrocytes and proteoglycan were ameliorated at the deep zone of the subchondral bone plate by the GMGHT administration in OA rats. The expression of Type II collagen was increased, while MMP-1, -3 and -13 levels were decreased in the GMGHT-treated SW1353 chondrocytes. In addition, the GMGHT treatment regulated NF-κB activation along with IL-6, transforming growth factor-β and IL-12 production. GMGHT promoted the recovery of articular cartilage damage by inhibiting MMPs, accompanied with its anti-inflammatory effects in OA. GMGHT might be an alternative therapeutic treatment for OA.

Kim MH ，Choi Y ，Ahn KS ，Um JY ，Lee SG ，Hahm DH ，Yang WM ... -  《-》

Interleukin-6 plays an essential role in hypoxia-inducible factor 2α-induced experimental osteoarthritic cartilage destruction in mice.

Hypoxia-inducible factor 2α (HIF-2α) (encoded by Epas1) causes osteoarthritic (OA) cartilage destruction by regulating the expression of catabolic factor genes. We undertook this study to explore the role of interleukin-6 (IL-6) in HIF-2α-mediated OA cartilage destruction in mice. The expression of HIF-2α, IL-6, and catabolic factors was determined at the messenger RNA and protein levels in primary culture mouse chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild-type, HIF-2α-knockdown (Epas1+/-), and Il6-/- mice was caused by intraarticular injection of Epas1 adenovirus or destabilization of the medial meniscus. The role of IL-6 was determined by treating with recombinant IL-6 protein or by injecting HIF-2α adenovirus (AdEpas1) intraarticularly in mice with or without IL-6-neutralizing antibody. We found that Il6 is a direct target gene of HIF-2α in articular chondrocytes. Both Epas1 and Il6 were up-regulated in human and mouse OA cartilage, whereas HIF-2α knockdown in mice led to inhibition of both Il6 expression and cartilage destruction. Treatment with IL-6 enhanced Mmp3 and Mmp13 expression; conversely, Il6 knockdown inhibited HIF-2α-induced up-regulation of Mmp3 and Mmp13. Injection of IL-6 protein into mouse knee joints triggered OA cartilage destruction, whereas IL-6 neutralization led to blocking of HIF-2α-induced cartilage destruction with concomitant modulation of Mmp3 and Mmp13 expression. Moreover, Il6 knockout resulted in inhibition of AdEpas1-induced and destabilization of the medial meniscus-induced cartilage destruction as well as inhibition of Mmp3 and Mmp13 expression. Our findings indicate that IL-6 acts as a crucial mediator of HIF-2α-induced experimental OA cartilage destruction in mice via regulation of Mmp3 and Mmp13 levels.

Ryu JH ，Yang S ，Shin Y ，Rhee J ，Chun CH ，Chun JS ... -  《-》