Naoshi Fukui

Stimulation of BMP-2 Expression by Pro-Inflammatory Cytokines IL-1 and TNF-α in Normal and Osteoarthritic Chondrocytes

Background: Destruction of cartilage in osteoarthritis is a direct effect of an imbalance between catabolic and anabolic activities in the tissue. While a great deal is known about catabolism, we sought to determine the biochemical basis of the anabolic activity.Methods: Cartilage was isolated from normal and osteoarthritic patients and subjected to both cell and explant culture. mRNA expression levels of the growth and differentiation factors bone morphogenetic protein-2 (BMP-2), BMP-4, BMP-6, cartilage-derived morphogenetic protein-1 (CDMP-1), connective tissue growth factor (CTGF), and activin were determined. BMP-2 was localized in osteoarthritic cartilage by immunohistochemistry. To determine the mechanism of BMP-2 stimulation, chondrocytes were cultured with TGF-&bgr; (transforming growth factor-&bgr;), insulin-like growth factor-1 (IGF-1), interleukin-1&bgr; (IL-1&bgr;), and tumor necrosis factor-&agr; (TNF-&agr;). The BMP-2 response was monitored by quantitative real-time polymerase chain reaction to ascertain mRNA levels and by Western blot analysis, BMP-2 protein quantitation, and immunohistochemistry to determine protein levels.Results: BMP-2 was found to be up-regulated in osteoarthritic chondrocytes and cartilage. In cell culture, IL-1&bgr; and TNF-&agr; increased BMP-2 mRNA and protein levels by eightfold and fifteenfold, respectively, whereas IGF-1 and TGF-&bgr;1 had no effect. In cartilage explant cultures, IL-1&bgr; and TNF-&agr; increased BMP-2 levels both intracellularly and extracellularly. Functional relevance was suggested by co-localization of BMP-2 and newly synthesized type-II procollagen within the same cells.Conclusions: BMP-2 acts as a stimulus of anabolic activities in normal and osteoarthritic chondrocytes. Furthermore, the pro-inflammatory cytokines IL-1&bgr; and TNF-&agr;, known to be present in synovium and cartilage of patients with osteoarthritis, stimulate the production of active BMP-2.Clinical Relevance: These results suggest that, in addition to their role in catabolism of cartilage in osteoarthritis, the pro-inflammatory cytokines IL-1&bgr; and TNF-&agr; may stimulate anabolic events through production of BMP-2. BMP-2, in turn, increases the synthesis of chondrocyte extracellular molecules, aggrecan and type-II collagen. Consequently, local inflammatory events may contribute to the remodeling or repair of cartilage.

Common variants in DVWA on chromosome 3p24.3 are associated with susceptibility to knee osteoarthritis

Susceptibility to osteoarthritis, the most common human arthritis, is known to be influenced by genetic factors. Through a genome-wide association study using ∼100,000 SNPs, we have identified a previously unknown gene on chromosome 3p24.3, DVWA, which is associated with susceptibility to knee osteoarthritis. Expressed specifically in cartilage, DVWA encodes a 276-amino-acid protein with two regions corresponding to the von Willebrand factor type A domain (VWA domain). Several DVWA SNPs are significantly associated with knee osteoarthritis in two independent Japanese case-control cohorts. This association was replicated in a Japanese population cohort and a Han Chinese case-control cohort (combined P = 7.3 × 10−11). DVWA protein binds to β-tubulin, and the binding is influenced by two highly associated missense SNPs (rs11718863 and rs7639618) located in the VWA domain. The Tyr169-Cys260 isoform of DVWA, which is overrepresented in knee osteoarthritis, showed weaker interaction. Our findings reveal a new paradigm for study of osteoarthritis etiology and pathogenesis.

Predictors of place of death for Japanese patients with advanced-stage malignant disease in home care settings: A nationwide survey

Although the place of death for patients with advanced malignancy is influenced by multiple factors, few studies have systematically investigated the determinants of place of death. The objective of the current retrospective study was to clarify the predictors of home death throughout the duration of home palliative care for Japanese patients with advanced malignant disease.

Roles of chondrocytes in the pathogenesis of osteoarthritis.

Recent investigations into the striking heterogeneity of cellular reaction patterns that are characteristic for, and puzzling in, osteoarthritic cartilage degeneration addressed apoptotic cell death of the chondrocytes and its underlying mechanisms. Also, chondrocyte senescence in terms of telomere reduction was reported to correlate with cartilage degeneration. Pathways of anabolic and catabolic activity of the cells as well as mechanisms for phenotypic instability of chondrocytes were further characterized. The mechanoregulation of the cartilage cells appears to be centrally involved in all these processes, in particular cell death and anabolic activity. Methodologically, the past year has seen the first steps of functional genomics in osteoarthritis research, providing new insights into the cell biology of this condition.

New Sequence Variants in HLA Class II/III Region Associated with Susceptibility to Knee Osteoarthritis Identified by Genome-Wide Association Study

Osteoarthritis (OA) is a common disease that has a definite genetic component. Only a few OA susceptibility genes that have definite functional evidence and replication of association have been reported, however. Through a genome-wide association study and a replication using a total of ∼4,800 Japanese subjects, we identified two single nucleotide polymorphisms (SNPs) (rs7775228 and rs10947262) associated with susceptibility to knee OA. The two SNPs were in a region containing HLA class II/III genes and their association reached genome-wide significance (combined P = 2.43×10−8 for rs7775228 and 6.73×10−8 for rs10947262). Our results suggest that immunologic mechanism is implicated in the etiology of OA.

Tumour budding at invasive margins and outcome in colorectal cancer

Objective  Tumour budding, defined as small clusters of undifferentiated cancer cells at invasive margins, has been shown to reflect biologic aggressiveness of colorectal cancers. We therefore examined the prognostic significance of tumour budding in patients with colorectal carcinoma, particularly focusing on comparisons with other clinicopathological findings.

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.

Proinflammatory role of amphiregulin, an epidermal growth factor family member whose expression is augmented in rheumatoid arthritis patients

BackgroundThe epidermal growth factor (EGF) and EGF receptor (EGFR) families play important roles in the hyperplastic growth of several tissues as well as tumor growth. Since synovial hyperplasia in rheumatoid arthritis (RA) resembles a tumor, involvement of the EGF/EGFR families in RA pathology has been implied. Although several reports have suggested that ErbB2 is the most important member of the EGFR family for the synovitis in RA, it remains unclear which members of the EGF family are involved. To clarify the EGF-like growth factors involved in the pathology of RA, we investigated the expression levels of seven major EGF-like growth factors in RA patients compared with those in osteoarthritis (OA) patients and healthy control subjects.MethodsThe expression levels of seven EGF-like growth factors and four EGFR-like receptors were measured in mononuclear cells isolated from bone marrow and venous blood, as well as in synovial tissues, using quantitative RT-PCR. Further evidence of gene expression was obtained by ELISAs. The proinflammatory roles were assessed by the growth-promoting and cytokine-inducing effects of the corresponding recombinant proteins on cultured fibroblast-like synoviocytes (FLS).ResultsAmong the seven EGF-like ligands examined, only amphiregulin (AREG) was expressed at higher levels in all three RA tissues tested compared with the levels in OA tissues. The AREG protein concentration in RA synovial fluid was also higher than that in OA synovial fluid. Furthermore, recombinant human AREG stimulated FLS to proliferate and produce several proinflammatory cytokines, including angiogenic cytokines such as interleukin-8 and vascular endothelial growth factor (VEGF), in a dose-dependent manner. The VEGF mRNA levels in RA synovia and VEGF protein concentrations in RA synovial fluid were significantly higher than those in the corresponding OA samples and highly correlated with the levels of AREG.ConclusionThe present findings suggest that AREG functions to stimulate synovial cells and that elevated levels of AREG may be involved in the pathogenesis of RA.

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.

Addition of an arch support improves the biomechanical effect of a laterally wedged insole

In order to examine if the addition of an arch support could improve the biomechanical effect of the laterally wedged insole, three-dimensional gait analysis was performed on 20 healthy volunteers. Kinetic and kinematic parameters at the knee and subtalar joints were compared among the following four types of insoles; a 5-mm thick flat insole, a flat insole with an arch support (AS), a 6 degrees inclined laterally wedged insole (LW), and a laterally wedged insole with an arch support (LWAS). The knee adduction moment averaged for the entire stance phase was reduced by the use of LW and LWAS by 7.7% and 13.3%, respectively, from that with FLAT. The difference in knee adduction moment between LW and LWAS was most obvious in the late stance, which was ascribed to the difference in the progression angle between those insoles. The analyses also revealed that LW tended to increase step width, and that such an increase was completely eliminated by the addition of an arch support to LW. This reduction of step width could be another mechanism for the further reduction of the moment with LWAS. The analyses of biomechanical parameters at the subtalar joints suggested that LWAS allowed the subject to walk in a more natural manner, while exerting greater biomechanical effects than LW. Thus, the addition of an arch support to the laterally wedged insole reduced knee adduction moment more efficiently, possibly through the elimination of potential negative effects of the laterally wedged insole.