Je-Hwang Ryu

Regulation of the Catabolic Cascade in Osteoarthritis by the Zinc-ZIP8-MTF1 Axis

Osteoarthritis (OA), primarily characterized by cartilage degeneration, is caused by an imbalance between anabolic and catabolic factors. Here, we investigated the role of zinc (Zn2+) homeostasis, Zn2+ transporters, and Zn(2+)-dependent transcription factors in OA pathogenesis. Among Zn2+ transporters, the Zn2+ importer ZIP8 was specifically upregulated in OA cartilage of humans and mice, resulting in increased levels of intracellular Zn2+ in chondrocytes. ZIP8-mediated Zn2+ influx upregulated the expression of matrix-degrading enzymes (MMP3, MMP9, MMP12, MMP13, and ADAMTS5) in chondrocytes. Ectopic expression of ZIP8 in mouse cartilage tissue caused OA cartilage destruction, whereas Zip8 knockout suppressed surgically induced OA pathogenesis, with concomitant modulation of Zn2+ influx and matrix-degrading enzymes. Furthermore, MTF1 was identified as an essential transcription factor in mediating Zn2+/ZIP8-induced catabolic factor expression, and genetic modulation of Mtf1 in mice altered OA pathogenesis. We propose that the zinc-ZIP8-MTF1 axis is an essential catabolic regulator of OA pathogenesis.

Wnt-7a Causes Loss of Differentiated Phenotype and Inhibits Apoptosis of Articular Chondrocytes via Different Mechanisms

Although regulation of chondrogenesis and cartilage development by Wnt signaling is well established, the function of Wnt in the maintenance and destruction of cartilage remains largely unknown. Here we investigated the involvement and regulatory mechanisms of Wnt signaling in cartilage destruction. We found that interleukin-1β, the primary pro-inflammatory cytokine involved in cartilage destruction, induces expression of Wnt-5a and -7a in primary culture articular chondrocytes. The level of β-catenin was also increased in chondrocytes of arthritic cartilage, suggesting the association of Wnt/β-catenin signaling with arthritic cartilage destruction. In addition, our results show that Wnt-7a induces dedifferentiation and inhibits NO-induced apoptosis of primary culture articular chondrocytes. Wnt-7a induces dedifferentiation of articular chondrocytes by stimulating transcriptional activity of β-catenin, whereas NO-induced apoptosis is inhibited via the activation of cell survival signaling, such as phosphatidylinositol 3-kinase and Akt, which block apoptotic signaling cascade. Our results collectively suggest that Wnt-7a is associated with cartilage destruction by regulating the maintenance of differentiation status and the apoptosis of articular chondrocytes via different mechanisms.

Opposing Roles of WNT-5A and WNT-11 in Interleukin-1β Regulation of Type II Collagen Expression in Articular Chondrocytes

Interleukin (IL)-1β is a major catabolic cytokine that plays a pivotal role in cartilage destruction. This study examined the possible involvement and regulatory mechanisms of Wnt signaling in IL-1β-induced inhibition of type II collagen expression in chondrocytes. Treatment of chondrocytes with IL-1β up-regulated Wnt-5a and down-regulated Wnt-11 expression. Conditioned medium from Wnt-5a-expressing cells inhibited type II collagen expression, whereas knockdown of Wnt-5a by siRNA blocked the inhibitory effects of IL-1β on type II collagen expression. In contrast to the inhibitory effects of Wnt-5a, Wnt-11 stimulated type II collagen expression. Wnt-5a and Wnt-11 did not cause accumulation of β-catenin or activation of the β-catenin-Tcf/Lef transcriptional complex. Instead, we found that Wnt-5a activated c-Jun N-terminal kinase and that an inhibitor of this kinase blocked Wnt-5a inhibition of type II collagen expression. In contrast, Wnt-11 activated protein kinase C and an inhibitor of this kinase blocked Wnt-11 stimulation of type II collagen expression. Collectively, these results indicate that Wnt-5a and Wnt-11 signaling through distinct non-canonical Wnt pathways have opposing effects on type II collagen expression by chondrocytes.

β-Catenin regulates expression of cyclooxygenase-2 in articular chondrocytes ☆

Pro-inflammatory cytokine such as interleukin (IL)-1beta causes inflammation of articular cartilage via induction of cyclooxygenase (COX)-2 expression. We investigated in this study the role of beta-catenin in the IL-1beta regulation of COX-2 expression in articular chondrocytes. IL-1beta increased expression of COX-2 and induced accumulation and nuclear translocation of transcriptionally competent beta-catenin. Inhibition of beta-catenin degradation by the treatment of cells with LiCl or proteasome inhibitor stimulated expression of COX-2, indicating that transcriptionally active beta-catenin is sufficient to induce COX-2 expression. This was demonstrated further by the observation that ectopic expression of transcriptionally competent beta-catenin stimulated expression of COX-2. Levels of beta-catenin and COX-2 protein were increased in osteoarthritic and rheumatoid arthritic cartilage, suggesting that beta-catenin may play a role in the inflammatory responses of arthritic cartilage. Taken together, our data suggest that accumulation of transcriptionally active beta-catenin contributes to the expression of COX-2 in articular chondrocytes.

REGULATION OF TYPE II COLLAGEN EXPRESSION BY HISTONE DEACETYLASE IN ARTICULAR CHONDROCYTES

Histone deacetylase (HDAC) regulates various cellular processes by modulating gene expression. Here, we investigated the role of HDAC in the expression of type II collagen, a marker of differentiated chondrocytes. We found that HDAC activity in primary articular chondrocytes decreases during dedifferentiation induced by serial monolayer culture and that the activity recovered during redifferentiation induced by three-dimensional culture in a cell pellet. Inhibition of HDAC with trichostatin A or PXD101 was sufficient to block type II collagen expression in primary culture chondrocytes. HDAC inhibition also blocked the redifferentiation of dedifferentiated chondrocytes by suppressing the synthesis and accumulation of type II collagen. HDAC inhibition promoted the expression of Wnt-5a, which is known to inhibit type II collagen expression, and knockdown of Wnt-5a blocked the ability of HDAC inhibitors to suppress type II collagen expression. In addition, the induction of Wnt-5a expression by HDAC inhibitors was associated with acetylation of the Wnt-5a promoter. Taken together, our results suggest that HDAC promotes type II collagen expression by suppressing the transcription of Wnt-5a.

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

Objective 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. Methods 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+/−, Epas1fl/fl;Col2a1-Cre and Col2a1-Nampt transgenic (TG) mice. Primary cultured mouse chondrocytes were treated with recombinant NAMPT protein or were infected with adenoviruses. Results 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. Conclusions 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.

Cytokine-like 1 Knock-out Mice (Cytl1−/−) Show Normal Cartilage and Bone Development but Exhibit Augmented Osteoarthritic Cartilage Destruction

We have shown that cytokine-like 1 (Cytl1) is a novel autocrine regulatory factor that regulates chondrogenesis of mouse mesenchymal cells (Kim, J. S., Ryoo, Z. Y., and Chun, J. S. (2007) J. Biol. Chem. 282, 29359–29367). In this previous work, we found that Cytl1 expression was very low in mesenchymal cells, increased dramatically during chondrogenesis, and decreased during hypertrophic maturation, both in vivo and in vitro. Moreover, exogenous addition or ectopic expression of Cytl1 caused chondrogenic differentiation of mouse limb bud mesenchymal cells. In the current study, we generated a Cytl1 knock-out (Cytl1−/−) mouse to investigate the in vivo role of Cytl1. Deletion of the Cytl1 gene did not affect chondrogenesis or cartilage development. Cytl1−/− mice also showed normal endochondral ossification and long bone development. Additionally, ultrastructural features of articular cartilage, such as matrix organization and chondrocyte morphology, were similar in wild-type and Cytl1−/− mice. However, Cytl1−/− mice were more sensitive to osteoarthritic (OA) cartilage destruction. Compared with wild-type littermates, Cytl1−/− mice showed more severe OA cartilage destruction upon destabilization of the medial meniscus of mouse knee joints. In addition, expression levels of Cytl1 were markedly decreased in OA cartilage of humans and experimental mice. Taken together, our results suggest that, rather than regulating cartilage and bone development, Cytl1 is required for the maintenance of cartilage homeostasis, and loss of Cytl1 function is associated with experimental OA cartilage destruction in mice.

Hypoxia-inducible factor-2α is an essential catabolic regulator of inflammatory rheumatoid arthritis.

Hypoxia-inducible factor-2α (HIF-2α) is sufficient to cause experimental rheumatoid arthritis and acts to regulate the functions of fibroblast-like cells from tissue surrounding joints, independent of HIF-1α.

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

OBJECTIVE 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. METHODS 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. RESULTS 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. CONCLUSION 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.

Low-density lipoprotein receptor–related protein 5 governs Wnt-mediated osteoarthritic cartilage destruction

IntroductionWnt ligands bind to low-density lipoprotein receptor–related protein (LRP) 5 or 6, triggering a cascade of downstream events that include β-catenin signaling. Here we explored the roles of LRP5 in interleukin 1β (IL-1β)- or Wnt-mediated osteoarthritic (OA) cartilage destruction in mice.MethodsThe expression levels of LRP5, type II collagen, and catabolic factors were determined in mouse articular chondrocytes, human OA cartilage, and mouse experimental OA cartilage. Experimental OA in wild-type, Lrp5 total knockout (Lrp5-/-) and chondrocyte-specific knockout (Lrp5fl/fl;Col2a1-cre) mice was caused by aging, destabilization of the medial meniscus (DMM), or intra-articular injection of collagenase. The role of LRP5 was confirmed in vitro by small interfering RNA–mediated knockdown of Lrp5 or in Lrp5-/- cells treated with IL-1β or Wnt proteins.ResultsIL-1β treatment increased the expression of LRP5 (but not LRP6) via JNK and NF-κB signaling. LRP5 was upregulated in human and mouse OA cartilage, and Lrp5 deficiency in mice inhibited cartilage destruction. Treatment with IL-1β or Wnt decreased the level of Col2a1 and increased those of Mmp3 or Mmp13, whereas Lrp5 knockdown ameliorated these effects. In addition, we found that the functions of LRP5 in arthritic cartilage were subject to transcriptional activation by β-catenin. Moreover, Lrp5-/- and Lrp5fl/fl;Col2a1-cre mice exhibited decreased cartilage destruction (and related changes in gene expression) in response to experimental OA.ConclusionsOur findings indicate that LRP5 (but not LRP6) plays an essential role in Wnt/β-catenin-signaling-mediated OA cartilage destruction in part by regulating the expression levels of type II collagen, MMP3, and MMP13.