Yasuko Ikeda

Pro-inflammatory Cytokine Tumor Necrosis Factor-α Induces Bone Morphogenetic Protein-2 in Chondrocytes via mRNA Stabilization and Transcriptional Up-regulation

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-α (TNF-α) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-α induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3′-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3′-untranslated regions, and transient transfection experiments demonstrated that TNF-α increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-α modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-κB.

Regional differences in chondrocyte metabolism in osteoarthritis: A detailed analysis by laser capture microdissection

OBJECTIVE To determine the change in metabolic activity of chondrocytes in osteoarthritic (OA) cartilage, considering regional difference and degree of cartilage degeneration. METHODS OA cartilage was obtained from knee joints with end-stage OA, at both macroscopically intact areas and areas with various degrees of cartilage degeneration. Control cartilage was obtained from age-matched donors. Using laser capture microdissection, cartilage samples were separated into superficial, middle, and deep zones, and gene expression was compared quantitatively in the respective zones between OA and control cartilage. RESULTS In OA cartilage, gene expression changed markedly with the site. The expression of cartilage matrix genes was highly enhanced in macroscopically intact areas, but the enhancement was less obvious in the degenerated areas, especially in the upper regions. In contrast, in those regions, the expression of type III collagen and fibronectin was most enhanced, suggesting that chondrocytes underwent a phenotypic change there. Within OA cartilage, the expression of cartilage matrix genes was significantly correlated with SOX9 expression, but not with SOX5 or SOX6 expression. In OA cartilage, the strongest correlation was observed between the expression of type III collagen and fibronectin, suggesting the presence of a certain link(s) between their expression. CONCLUSION The results of this study revealed a comprehensive view of the metabolic change of the chondrocytes in OA cartilage. The change of gene expression profile was most obvious in the upper region of the degenerated cartilage. The altered gene expression at that region may be responsible for the loss of cartilage matrix associated with OA.

Zonal Gene Expression of Chondrocytes in Osteoarthritic Cartilage

OBJECTIVE To determine the chondrocyte metabolism in respective zones of osteoarthritic (OA) cartilage. METHODS OA cartilage was obtained from macroscopically intact areas of 4 knee joints with end-stage OA. The cartilage was divided into 3 zones, and gene expression profiles were determined in the respective zones by a custom-designed microarray that focused on chondrocyte-related genes. For the genes whose expression was significantly different among the zones, the expression was compared between OA and control cartilage in the respective zones by an analysis using laser capture microdissection and real-time polymerase chain reaction (PCR). For some genes, the correlation of expression was investigated in specific cartilage zones. RESULTS A total of 198 genes (approximately 40% of those investigated) were found to be expressed at significantly different levels among the zones. Expression of 26 of those genes was evaluated by laser capture microdissection and real-time PCR, which confirmed the validity of microarray analysis. The expression of cartilage matrix genes was mostly enhanced in OA cartilage, at similar levels across the zones but at different magnitudes among the genes. The expression of bone-related genes was induced either in the superficial zone or in the deep zone, and positive correlations were found among their expression in the respective zones. The expression of 5 proteinase genes was most enhanced in the superficial zone, where their expression was correlated, suggesting the presence of a common regulatory mechanism(s) for their expression. CONCLUSION In OA cartilage, the metabolic activity of chondrocytes differed considerably among zones. Characteristic changes were observed in the superficial and deep zones.

Changes of human menisci in osteoarthritic knee joints

OBJECTIVE To investigate the changes of knee menisci in osteoarthritis (OA) in human. METHODS OA and control menisci were obtained from 42 end-stage OA knees with medial involvement and 28 non-arthritic knees of age-matched donors, respectively. The change of menisci in OA was evaluated by histology, and gene expression of major matrix components and anabolic factors was analyzed in the anterior horn segments by quantitative PCR (qPCR). In those regions of menisci, the rate of collagen neo-synthesis was evaluated by [(3)H]proline incorporation, and the change of matrix was investigated by ultrastructural observation and biomechanical measurement. RESULTS In OA menisci, the change in histology was rather moderate in the anterior horn segments. However, despite the modest change in histology, the expression of type I, II, III procollagens was dramatically increased in those regions. The expression of insulin-like growth factor 1 (IGF-1) was markedly enhanced in OA menisci, which was considered to be responsible, at least partly, for the increase in procollagen gene expression. Interestingly, in spite of marked increase in procollagen gene expression, incorporation of [(3)H]proline increased only modestly in OA menisci, and impaired collagen synthesis was suggested. This finding was consistent with the results of ultrastructural observation and biomechanical measurement, which indicated that the change of meniscal matrix was modest in the macroscopically preserved areas of OA menisci. CONCLUSION Although the expression of major matrix components was markedly enhanced, matrix synthesis was enhanced only modestly, and the changes of matrix in human OA menisci were rather modest in the non-degenerated areas.

Up-regulation of interleukin-6 and vascular endothelial growth factor-A in the synovial fluid of temporomandibular joints affected by synovial chondromatosis

Our aim was to explore important inflammatory mediators for synovial chondromatosis in the temporomandibular joints (TMJs) by analysing synovial fluid. Samples were collected from 10 patients with unilateral synovial chondromatosis of the TMJ. Control samples were obtained from 11 subjects with no symptoms in the TMJ. Concentrations of aggrecan, interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)-A were measured in the samples of synovial fluid, and the results in the two groups compared. The tissues from the affected TMJ were examined histologically and immunohistochemically. Of the proteins evaluated, the concentrations of aggrecan, IL-6, and VEGF-A were significantly higher in the group with synovial chondromatosis. The immunohistochemical analysis showed that the synovial cells around the osteocartilaginous nodules were vigorously expressing VEGF-A. IL-6 and VEGF-A are thought to have important roles in the pathology of synovial chondromatosis of the TMJ.

αvβ5 Integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes

OBJECTIVE When cultured in monolayers, articular chondrocytes undergo an obvious phenotypic change. Although the involvement of integrins has been suggested, the exact mechanisms of the change have not been determined. This study was undertaken to clarify the mechanisms underlying the loss of chondrocyte phenotype early after plating. METHODS Primary cultured human articular chondrocytes were used for the experiments. Involvement of respective integrins in the phenotypic change was investigated in RNA interference (RNAi) experiments. A signaling pathway involved in the change was identified in experiments using specific inhibitors and adenoviruses encoding mutated genes involved in the pathway. Adenoviruses carrying mutated GTPases were used to determine the involvement of small GTPases in the process. RESULTS In monolayer-cultured chondrocytes, suppression of αv or β5 integrin expression by RNAi inhibited morphologic changes in the cells and increased (or prevented a reduction in) the expression of various cartilage matrix genes. Consistent results were obtained in experiments using a blocking antibody and a synthetic inhibitor of αvβ5 integrin. The decrease in cartilage matrix gene expression in chondrocytes after plating was mediated by ERK signaling, which was promoted primarily by αvβ5 integrin. In articular chondrocytes, the affinity of αvβ5 integrin for ligands was regulated by the small GTPase R-Ras. R-Ras was gradually activated in monolayer-cultured chondrocytes after plating, which caused a gradual decline in cartilage matrix gene expression through enhanced αvβ5 integrin activation and the subsequent increase in ERK signaling. CONCLUSION Our findings indicate that αvβ5 integrin may be involved in the change that occurs in monolayer-cultured chondrocytes after plating.

α5β1 integrin induces the expression of noncartilaginous procollagen gene expression in articular chondrocytes cultured in monolayers

IntroductionArticular chondrocytes undergo an obvious phenotypic change when cultured in monolayers. During this change, or dedifferentiation, the expression of type I and type III procollagen is induced where normal chondrocytes express little type I and type III procollagen. In this study, we attempted to determine the mechanism(s) for the induction of such procollagen expression in dedifferentiating chondrocytes.MethodsAll experiments were performed using primary-cultured human articular chondrocytes under approval of institutional review boards. Integrin(s) responsible for the induction of type I and type III procollagen expression were specified by RNAi experiments. The signal pathway(s) involved in the induction were determined by specific inhibitors and RNAi experiments. Adenovirus-mediated experiments were performed to identify a small GTPase regulating the activity of integrins in dedifferentiating chondrocytes. The effect of inhibition of integrins on dedifferentiation was investigated by experiments using echistatin, a potent disintegrin. The effect of echistatin was investigated first with monolayer-cultured chondrocytes, and then with pellet-cultured chondrocytes.ResultsIn dedifferentiating chondrocytes, α5β1 integrin was found to be involved in the induction of type I and type III procollagen expression. The induction was known to be mediated by v-akt murine thymoma viral oncogene homolog (AKT) signaling. Among the three AKT isoforms, AKT1 seemed to be most involved in the signaling. Elated RAS viral (r-ras) oncogene homolog (RRAS) was considered to regulate the progression of dedifferentiation by modulating the affinity and avidity of α5β1 integrin to ligands. Echistatin inhibited dedifferentiation of monolayer-cultured chondrocytes. Furthermore, the matrix formed by pellet-cultured chondrocytes more closely resembled that of normal cartilage compared with the controls.ConclusionsThe result of this study has shown, for the first time, that α5β1 integrin may be responsible for the induction of non-cartilaginous collagen expression in chondrocytes undergoing dedifferentiation. Again, this study has shown that the inhibition of ligand ligation to integrins may be an effective strategy to inhibit phenotypic change of cultured chondrocytes, and to improve the quality of matrix synthesized by primary cultured chondrocytes.

The Use of Laser Capture Microdissection on Adult Human Articular Cartilage for Gene Expression Analysis

The integrity of articular cartilage is maintained by chondrocytes, the sole type of cell that resides within the tissue. The noncalcified region of articular cartilage can be divided into three zones based on histological features, in which the chondrocyte metabolism is known to differ obviously among the zones. In pathological cartilage, the chondrocyte metabolism may change dramatically, which could play a pivotal role in the progression of the disease. Since such change in metabolism differs obviously from site to site within cartilage, it is crucial to determine the chondrocyte metabolism in respective regions. To this end, we have employed laser-capture microdissection (LCM) to analyze chondrocyte metabolism in various regions of pathological and control cartilage. In this report, we describe our protocol for LCM on adult human cartilage tissue. With this protocol, a specific site of cartilage tissue was successfully obtained by LCM for gene expression analysis.

192 ZONAL GENE EXPRESSION OF CHONDROCYTES IN OSTEOARTHRITIC CARTILAGE

Objective. To determine the chondrocyte metabolism in respective zones of osteoarthritic (OA) cartilage. Methods. OA cartilage was obtained from macroscopically intact areas of 4 knee joints with end-stage OA. The cartilage was divided into 3 zones, and gene expression profiles were determined in the respective zones by a custom-designed microarray that focused on chondrocyte-related genes. For the genes whose expression was significantly different among the zones, the expression was compared between OA and control cartilage in the respective zones by an analysis using laser capture microdissection and real-time polymerase chain reaction (PCR). For some genes, the correlation of expression was investigated in specific cartilage zones. Results. A total of 198 genes (40% of those investigated) were found to be expressed at significantly different levels among the zones. Expression of 26 of those genes was evaluated by laser capture microdissection and real-time PCR, which confirmed the validity of microarray analysis. The expression of cartilage matrix genes was mostly enhanced in OA cartilage, at similar levels across the zones but at different magnitudes among the genes. The expression of bone-related genes was induced either in the superficial zone or in the deep zone, and positive correlations were found among their expression in the respective zones. The expression of 5 proteinase genes was most enhanced in the superficial zone, where their expression was correlated, suggesting the presence of a common regulatory mechanism(s) for their expression. Conclusion. In OA cartilage, the metabolic activity of chondrocytes differed considerably among zones. Characteristic changes were observed in the superficial and deep zones.

A11 INTEGRIN αVβ5 MAY BE RESPONSIBLE FOR THE SUPPRESSION OF CARTILAGE MATRIX GENES AND FORMATION OF CYTOSKELETONS DURING DEDIFFERENTIATION IN THE MONOLAYER CULTURED ADULT HUMAN ARTICULAR CHONDROCYTES

of F-spondin by 1.5 to 3 fold by 24h. Similarly, chondrocytes exposed to retinol increased F-spondin expression by 2.5 fold as compared to control cells. We next assessed the effects of F-spondin on chondrocyte functions. Chondrocytes treated with purified F-spondin exhibited increased expression of BMP2, type II collagen, aggrecan and TGFB1. Finally, we hypothesized that F-spondin, like thrombospondin, activates latent TGF-beta, since both have conserved WSxW and KRFK motif sequence in TSR domains, which are known to bind to latent TGF-b1 and release active dimer TGF-b1. Unstimulated OA cartilage explants spontaneously released active TGFbeta1 (200pg/g cartilage) in culture supernatants. The addition of recombinant F-spondin (R&D systems) increased the active TGF-b1 by 225% (550pg/g cartilage) but not total (latent and active) TGF-b1. Conclusions: These studies demonstrate that F-spondin is overexpressed in OA cartilage. The data also suggest that one role of F-spondin in OA is to upregulate anabolic factors, perhaps indirectly via its capacity to activate latent TGF-b1.