PresentationsCOL10 expression in the limb cartilage.Under the OA induction in the wild-type joints, C/EBPβ was induced at the frontline of cartilage destruction, whereas in the C/EBPβ+/-joints, the destruction as well as chondrocyte hypertrophy and COL10 expression were significantly suppressed.In the ex vivo culture of C/EBPβ-/-costal chondrocytes, COL10 expression was significantly decreased compared to the wild-type culture.The mRNA level and promoter activity of COL10 were enhanced by the C/EBPβ transfection, and the core responsive region of the COL10 promoter was identified between -81 and -76 bp relative to the transcriptional start site.Since C/EBPβ and Runx2 are known to function as mutual transcriptional co-factors, we further generated C/EBPβ and Runx2 compound deficient (C/EBPβ-/-; Runx2+/-) mice.The C/EBPβ-/-; Runx2+/-mice exhibited severer dwarfism than the C/EBPβ-/-mice.Although chondrocyte hypertrophy and COL10 expression were comparable between the two genotypes, cartilage degradation and MMP13 expression were markedly suppressed by the Runx2 insufficiency.In the culture of SW1353 cells, co-transfection of C/EBPβ and Runx2 enhanced MMP13 expression, but not proliferation or COL10 expression, as compared to a single transfection of C/EBPβ or Runx2.The promoter activity of MMP13 was synergistically enhanced by the co-transfection, and the core responsive region was identified between -111 and -89 bp, which contains a C/EBP-binding motif, but not a Runx2-binding motif.Conclusions: C/EBPβ is a crucial transcription factor for chondrocyte hypertrophy and cartilage degradation.Runx2 contributes to the latter step as the co-factor, but not to the former step, indicating distinct transcriptional control of these sequential steps during endochondral ossification by C/EBPβ and Runx2.

Poster Presentations -Cartilage/Chondrocyte Biology S95 potent inducer of type II collagen synthesis in chondrocytes, but we have found that if the tubulin networks are disrupted, TGFb1-induced collagen mRNA and protein synthesis is prevented.However, expression levels of TGFb1 and TGFb1-receptor mRNA are significantly elevated in these cells.Our preliminary studies suggest the involvement of both the MAPK family (enhanced phosphorylation of both p38 and ERK1/2) and SMAD family members, but studies are ongoing to elucidate the mechanism(s) involved. Conclusions:We have shown that modulation of the cytoskeletal tubulin elements dramatically affects chondrocyte biosynthesis.We are currently characterising the mechanism(s) involved in these matrix changes.Clearly there is a strong link between the correct assembly/turnover of the microtubule network and tissue homeostasis.These studies will aid in elucidating the role of the chondrocyte cytoskeleton in signal transduction and provide an insight into how abnormal distribution/amounts of cytoskeletal proteins may contribute to pathologies such as osteoarthritis.

Objective: To provide a more complete picture of the effect of interleukin-1 beta (IL-1 beta) on adult human articular chondrocyte gene expression, in contrast to the candidate gene approach.Design: Chondrocytes from human knee cartilage were cultured in medium containing IL-1 beta. Changes in gene expression were analyzed by microarray and reverse transcriptase-polymerase chain reaction analysis. The ability of transforming growth factor beta-1 (TGF-beta 1), fibroblast growth factor (FGF)-18, and bone morphogenetic protein 2 (BMP-2) to alter the effects of IL-1 beta was analyzed. Computational analysis of the promoter regions of differentially expressed genes for transcription factor binding motifs was performed.Results: IL-1 beta-treated human chondrocytes showed significant increases in the expression of granulocyte colony stimulating factor-3, endothelial leukocyte adhesion molecule 1 and leukemia inhibitory factor as well as for a large group of chemokines that include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, CCL2, CCL3, CCL4, CCL5, CCL8, CCL20, CCL3L1, CX3CL1 and the cytokine IL-6. As expected, the mRNA for matrix metalloproteinase (MMP)-13 and BMP-2 also increased while mRNA for the matrix genes COL2A1 and aggrecan was down-regulated. A subset of chemokines increased rapidly at very low levels of IL-1 beta. The phenotype induced by IL-1 beta was partially reversed by TGF-beta 1, but not by BMP-2. In the presence of IL-1 beta, FGF-18 increased expression of ADAMTS-4, aggrecan, BMP-2, COL2A1, CCL3, CCL4, CCL20, CXCL1, CXCL3, CXCL6, IL-1 13, IL-6, and IL-8 and decreased ADAMTS-5, MMP-13, CCL2, and CCL8. Computational analysis revealed a high likelihood that the most up-regulated chemokines are regulated by the transcription factors myocyte enhancer binding factor-3 (MEF-3), CCAAT/enhancer binding protein (C/EBP) and nuclear factor-kappa B (NF-kappa B).Conclusion: IL-1 beta has a diverse effect on gene expression profile in human chondrocytes affecting matrix genes as well as chemokines and cytokines. TGF-beta 1 has the ability to antagonize some of the phenotype induced by IL-1 beta. (C) 2008 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Podium Presentationsexpression of WISP1 in synovium and cartilage.This signifies that wnt-signaling pathways are activated in preclinical species and in human disease, and also results in upregulation of matrix degrading enzymes in synovium and cartilage.We also demonstrated that high synovial WISP1 expression can cause enzyme-mediated cartilage damage, which may be independent of IL-1.These results demonstrate a potential role for WISP1 in experimental OA and in human OA clinical samples.

currence of new vertebral fractures and serious adverse events was not observed.Conclusions: Both RIS + VD3 (alfacalcidol) therapy and RIS + VK2 (menatetrenone) therapy increased the BMD and inhibited bone resorption similarly to RIS monotherapy in patients with primary osteoporosis.In this study, RIS + VK2 therapy was suggested to reduce the inhibition of bone formation compared with RIS monotherapy, and was thus considered to be more effective for improving bone strength.

Purpose: Matrix metalloproteinases (MMPs) have long been considered as excellent targets for the treatment of osteoarthritis (OA).However, realization of this concept has been hampered by dose-limiting musculoskeletal side effects observed in humans with broad spectrum inhibitors.MMP-13, the major type II collagenase, is highly up-regulated in OA/RA and therefore strongly implicated in the pathogenesis of OA.Selective inhibition of MMP-13 may provide the desired cartilage degradation protection while overcoming the musculoskeletal toxicity seen by non-selective inhibition of MMPs.Methods: Selectivity determination.Kinetic studies.Culture of bovine and human cartilage explants.Results: We have identified a new class of very potent and highly selective MMP-13 inhibitors which showed no activity against other MMPs, TACE or members of the ADAMTS family at concentrations up to 20μM.Kinetic studies specified this class of compounds as mixed-type, non-zinc binding inhibitors.Excessive cleavage of type II collagen is a fundamental pathological feature of OA.In cultured explants from OA patients, collagen degradation was significantly reduced by the selective MMP-13 inhibitor.Inhibition of collagen degradation was studied in bovine and human articular cartilage systems, where the cartilage degradation was induced by the addition of pro-inflammatory cytokines IL-1a and oncostatin M. Our highly selective MMP-13 inhibitors completely blocked type II collagen degradation in bovine explants and showed up to 80% inhibition in human OA cartilage, while proteoglycan degradation remained unchanged.These results were confirmed with different biomarkers (C1,2C, C2C, CTX-II) and hydroxyproline determination which validates MMP-13 as the primary collagenase in the IL-1/OSM-induced cartilage degradation process.Conclusions: Our results emphasize the role of MMP-13 in pathologic cartilage degradation and verify the value of selective MMP-13 inhibitors for potential treatment of OA and RA.