Elisabeth A. Morris

Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis

Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific ‘aggrecanase’ site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary ‘aggrecanase’ responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.

Protein Kinase Cζ Is Up-regulated in Osteoarthritic Cartilage and Is Required for Activation of NF-κB by Tumor Necrosis Factor and Interleukin-1 in Articular Chondrocytes

Protein kinase Cζ (PKCζ) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCζ is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCζ may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCζ, implicating PKCζ activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCζ activity in NF-κB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-α caused an increase in NF-κB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKCζ-sparing inhibitor calphostin C. A cell-permeable PKCζ pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-κB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCζ protein effectively prevented cytokine-mediated NF-κB activation in primary chondrocytes. These data implicate PKCζ as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

Identification of a Novel HtrA1-susceptible Cleavage Site in Human Aggrecan: EVIDENCE FOR THE INVOLVEMENT OF HtrA1 IN AGGRECAN PROTEOLYSIS IN VIVO

Mass spectrometry-based proteomic analyses performed on cartilage tissue extracts identified the serine protease HtrA1/PRSS11 as a major protein component of human articular cartilage, with elevated levels occurring in association with osteoarthritis. Overexpression of a catalytically active form of HtrA1, but not an active site mutant (S328A), caused a marked reduction in proteoglycan content in chondrocyte-seeded alginate cultures. Aggrecan degradation fragments were detected in conditioned media from the alginate cultures overexpressing active HtrA1. Incubation of native or recombinant aggrecan with wild type HtrA1 resulted in distinct cleavage of these substrates. Cleavage of aggrecan by HtrA1 was strongly enhanced by HtrA1 agonists such as CPII, a C-terminal hexapeptide derived from the C-propeptide of procollagen IIα1 (i.e. chondrocalcin). A novel HtrA1-susceptible cleavage site within the interglobular domain (IGD) of aggrecan was identified, and an antibody that specifically recognizes the neoepitope sequence (VQTV356) generated at the HtrA1 cleavage site was developed. Western blot analysis demonstrated that HtrA1-generated aggrecan fragments containing the VQTV356 neoepitope were significantly more abundant in osteoarthritic cartilage compared with cartilage from healthy joints, implicating HtrA1 as a critical protease involved in proteoglycan turnover and cartilage degradation during degenerative joint disease.

LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model.

Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E2 (PGE2) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRα and -β) are expressed in cartilage, with LXRβ being the predominant isoform. Here we show that genetic disruption of Lxrβ gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE2 production in articular cartilage treated with IL-1β, indicating a protective role of LXRβ in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE2 production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE2 synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.

N-((8-hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2).

N-((8-Hydroxy-5-substituted-quinolin-7-yl)(phenyl)methyl)-2-phenyloxy/amino-acetamide inhibitors of ADAMTS-5 (Aggrecanase-2) have been prepared. Selected compounds 10, 14, 25, and 53 show sub-microM ADAMTS-5 potency and good selectivity over the related metalloproteases ADAMTS-4 (Aggrecanase-1), MMP-13, and MMP-12. Compound 53 shows a good balance of potent ADAMTS-5 inhibition, moderate CYP3A4 inhibition and good rat liver microsome stability. This series of compounds represents progress towards selective ADAMTS-5 inhibitors as disease modifying osteoarthritis agents.

Global analysis of nuclear receptor expression and dysregulation in human osteoarthritic articular cartilage: Reduced LXR signaling contributes to catabolic metabolism typical of osteoarthritis

OBJECTIVE Compare the expression and regulation of nuclear receptors (NRs) in osteoarthritic and normal human articular cartilage. METHOD The transcriptional levels of 48 NRs and additional related proteins were measured in mRNA from human articular cartilage from subjects with osteoarthritis (OA) and compared to samples from subjects without OA, using microarrays, individual quantitative reverse transcriptase polymerase chain reaction assays, and a custom human NR TaqMan Low Density Array (TLDA). The functional effect of liver X receptor (LXR) activity in cartilage was studied by measuring proteoglycan (PG) synthesis and degradation in articular cartilage explant cultures following treatment with the synthetic LXR agonist T0901317. RESULTS Thirty-one of 48 NRs analyzed by TLDA were found to be measurably expressed in human articular cartilage; 23 of these 31 NRs showed significantly altered expression in OA vs unaffected cartilage. Among these, LXRalpha and LXRbeta, and their heterodimeric partners retinoid X receptor (RXR)alpha and RXRbeta were all expressed at significantly lower levels in OA cartilage, as were LXR target genes ABCG1 and apolipoproteins D and E. Addition of LXR agonist to human OA articular chondrocytes and to cartilage explant cultures resulted in activation of LXR-mediated transcription and significant reduction of both basal and interleukin (IL)-1-mediated PG degradation. CONCLUSIONS Articular cartilage expresses a substantial number of NRs, and a large proportion of the expressed NRs are dysregulated in OA. In particular, LXR signaling in OA articular cartilage is impaired, and stimulation of LXR transcriptional activity can counteract the catabolic effects of IL-1. We conclude that LXR agonism may be a possible therapeutic option for OA.

Synthesis and biological evaluation of ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides: a class of potent aggrecanase-1 inhibitors.

The prevention of aggrecan (a key component of cartilage) cleavage via the inhibition of aggrecanase-1 may provide a unique opportunity to stop the progression of cartilage degradation in osteoarthritis. The evaluation of a series of biphenylsulfonamides resulted in the identification of the ((4-keto)-phenoxy)methyl biphenyl-4-sulfonamides analogs (19-21 and 24) with improved Agg-1 inhibition and MMP-2, MMP-13 activity.

Immortalized mouse articular cartilage cell lines retain chondrocyte phenotype and respond to both anabolic factor BMP‐2 and pro‐inflammatory factor IL‐1

Articular cartilage chondrocytes help in the maintenance of tissue homeostasis and function of the articular joint. Study of primary chondrocytes in culture provides information closely related to in vivo functions of these cells. Limitations in the primary culture of chondrocytes have lead to the development of cells lines that serve as good surrogate models for the study of chondrocyte biology. In this study, we report the establishment and characterization of chondrocyte cell lines, MM‐Sv/HP and MM‐Sv/HP‐2 from mouse articular cartilage. Cells were isolated from mouse femoral head articular cartilage, immortalized and maintained in culture through numerous passages. The morphology of the cells was from fibroblastic to polygonal in nature. Gene expression studies using quantitative PCR (Q‐PCR) were performed on cells in monolayer culture and cells embedded in a three‐dimensional alginate matrix. Stimulation of cells in monolayer culture with anabolic factor, BMP‐2, resulted in increased gene expression of the extracellular matrix molecules, aggrecan and type II collagen and their regulator transcription factor, Sox9. Treatment by pro‐inflammatory IL‐1 resulted in increased gene expression of catabolic effectors including Aggrecanases (ADAMTS4, ADAMTS5), MMP‐13 and nitric oxide synthase (Nos2). Cells in alginate treated with BMP‐2 resulted in increased synthesis of proteoglycan which was released into the conditioned media on IL‐1 stimulation. Western analysis of conditioned media showed the presence of Aggrecanase‐cleaved aggrecan fragments. In summary, MM‐Sv/HP and MM‐Sv/HP‐2 show preservation of important characteristics of articular chondrocytes as examined under multiple culture conditions and would provide a useful reagent in the study of chondrocyte biology. J. Cell. Physiol. 215: 68–76, 2008.

Immortalized cell lines from mouse xiphisternum preserve chondrocyte phenotype

Chondrocytes are unique to cartilage and the study of these cells in vitro is important for advancing our understanding of the role of these cells in normal homeostasis and disease including osteoarthritis (OA). As there are limitations to the culture of primary chondrocytes, cell lines have been developed to overcome some of these obstacles. In this study, we developed a procedure to immortalize and characterize chondrocyte cell lines from mouse xiphisternum. The cells displayed a polygonal to fibroblastic morphology in monolayer culture. Gene expression studies using quantitative PCR showed that the cell lines responded to bone morphogenetic protein 2 (BMP‐2) by increased expression of matrix molecules, aggrecan, and type II collagen together with transcriptional factor, Sox9. Stimulation by IL‐1 results in the increased expression of catabolic effectors including MMP‐13, nitric oxide synthase, ADAMTS4, and ADAMTS5. Cells cultured in alginate responded to BMP‐2 by increased synthesis of proteoglycan (PG), a major matrix molecule of cartilage. IL‐1 treatment of cells in alginate results in increased release of PG into the conditioned media. Further analysis of the media showed the presence of Aggrecanase‐cleaved aggrecan fragments, a signature of matrix degradation. These results show that the xiphisternum chondrocyte cell lines preserve their chondrocyte phenotype cultured in either monolayer or 3‐dimensional alginate bead culture systems. In summary, this study describes the establishment of chondrocyte cell lines from the mouse xiphisternum that may be useful as a surrogate model system to understand chondrocyte biology and to shed light on the underlying mechanism of pathogenesis in OA. J. Cell. Physiol. 209: 551–559, 2006.

143 BENEFICIAL EFFECTS OF LIVER X RECEPTOR (LXR) MODULATION ON MATRIX METABOLISM AND PROSTAGLANDIN E2 (PGE2) PRODUCTION IN CARTILAGE

were treated with H-89, a PKA inhibitor, W-7, a calmodulin inhibitor or LY294002, a PI3K inhibitor prior to 4a-PDD stimulation in SOX9dependent reporter assay. To examine the chondrogenic differentiation, ATDC5 cells were co-stimulated with 0 to 120 nM 4a-PDD and 10mg/ml insulin for 10 day, then cells were stained with alcian blue. The amount of SOX9 protein was estimated by Western Blot analysis using anti-SOX9 antibody. Results: We have screened 120,000 cDNA clones and identified 46 genes that activated SOX9-dependent reporter activity. In cDNA microarray analysis, the mRNA levels of Sox5, Sox6, Ifitm5, Myd116, Mef2c and TRPV4 genes were elevated during chondrogenic differentiation of ATDC5 cells treated with insulin. TRPV4, a cation channel molecule was further investigated in this study since it had a strong effect on SOX9dependent transcription. mRNA expression of TRPV4 gene was observed in ATDC5, C3H10T1/2, murine primary chondrocytes prepared from the rib cages and hind limb buds in embryonic day12 embryos, but not in NIH3T3. When ATDC5 cells or C3H10T1/2 cells were treated with various concentration of 4a-PDD, SOX9-dependent transcription was elevated in dose dependent manner and this effect was abolished by the addition of ruthenium red, a TRPV antagonist. In ATDC5 cells, H-89 and W-7 inhibited SOX9-dependent reporter activity caused by the stimulation with 4a-PDD while LY294002 did not. When ATDC5 cells were co-stimulated with 4a-PDD and insulin, GAG accumulation was significantly increased as compared with insulin alone whereas 4a-PDD alone showed no effect. Similar result was obtained in C3H10T1/2 cells co-stimulated with 4aPDD and BMP-2. Co-stimulation with 4a-PDD demonstrated further elevations of mRNAs for type II collagen and aggrecan in ATDC5 cells when compared to insulin alone. 4a-PDD stimulation increased the amount of SOX9 protein in both ATDC5 and C3H10T1/2 cells. Conclusions: We have identified TRPV4 by its ability to activate SOX9dependent transcription. Activation of TRPV4 promoted chondrogenic differentiation of ATDC5 cells in cooperation with insulin and C3H10T1/2 cells in corporation with BMP-2 in vitro. The protein level of SOX9 was increased by the stimulation with 4a-PDD. These observations suggest that TRPV4 may correlate the process of chondrogenesis.