Takumi Nakagawa

Involvement of receptor activator of nuclear factor κB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis

OBJECTIVE To clarify the mechanism by which osteoclasts are formed in culture of rheumatoid synoviocytes by exploring the involvement of receptor activator of nuclear factor kappaB ligand (RANKL)/osteoclast differentiation factor (ODF). METHODS Osteoclast formation was evaluated in cocultures of rheumatoid synovial fibroblasts and peripheral blood mononuclear cells (PBMC) in the presence of macrophage colony stimulating factor and 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) utilizing separating membrane filters. RANKL/ODF expression was examined by Northern blotting in synovial tissues from 5 rheumatoid arthritis (RA) patients and tissues from patients with giant cell tumor (GCT), osteosarcoma (OS), and osteoarthritis (OA). RANKL/ODF expression and the ability of synovial fibroblasts to support osteoclastogenesis were investigated in coculture with PBMC in the presence or absence of 1,25(OH)2D3, and soluble RANKL/ODF and osteoprotegerin (OPG)/osteoclastogenesis inhibitory factor (OCIF) were measured by enzyme-linked immunosorbent assay. The effects of OPG/OCIF on the osteoclastogenesis in the primary culture of rheumatoid synoviocytes and the coculture system were determined. RESULTS Synovial fibroblasts did not induce osteoclastogenesis when separately cocultured with PBMC. Northern blotting revealed that RANKL/ODF was highly expressed in all tissues from RA and GCT patients, but not from OA or OS patients. Cultured rheumatoid synovial fibroblasts efficiently induced osteoclastogenesis in the presence of 1,25(OH)2D3, which was accompanied by up-regulated expression of RANKL/ODF and decreased production of OPG/OCIF. Osteoclastogenesis from synoviocytes was dose-dependently inhibited by OPG/OCIF. CONCLUSION RANKL/ODF expressed on synovial fibroblasts is involved in rheumatoid bone destruction by inducing osteoclastogenesis and would therefore be a good therapeutic target.

Transcriptional regulation of endochondral ossification by HIF-2α during skeletal growth and osteoarthritis development

Chondrocyte hypertrophy followed by cartilage matrix degradation and vascular invasion, characterized by expression of type X collagen (COL10A1), matrix metalloproteinase-13 (MMP-13) and vascular endothelial growth factor (VEGF), respectively, are central steps of endochondral ossification during normal skeletal growth and osteoarthritis development. A COL10A1 promoter assay identified hypoxia-inducible factor-2α (HIF-2α, encoded by EPAS1) as the most potent transactivator of COL10A1. HIF-2α enhanced promoter activities of COL10A1, MMP13 and VEGFA through specific binding to the respective hypoxia-responsive elements. HIF-2α, independently of oxygen-dependent hydroxylation, was essential for endochondral ossification of cultured chondrocytes and embryonic skeletal growth in mice. HIF-2α expression was higher in osteoarthritic cartilages versus nondiseased cartilages of mice and humans. Epas1-heterozygous deficient mice showed resistance to osteoarthritis development, and a functional single nucleotide polymorphism (SNP) in the human EPAS1 gene was associated with knee osteoarthritis in a Japanese population. The EPAS1 promoter assay identified RELA, a nuclear factor-κB (NF-κB) family member, as a potent inducer of HIF-2α expression. Hence, HIF-2α is a central transactivator that targets several crucial genes for endochondral ossification and may represent a therapeutic target for osteoarthritis.

C/EBPβ and RUNX2 cooperate to degrade cartilage with MMP-13 as the target and HIF-2α as the inducer in chondrocytes

To elucidate the molecular mechanism underlying the endochondral ossification process during the skeletal growth and osteoarthritis (OA) development, we examined the signal network around CCAAT/enhancer-binding protein-β (C/EBPβ, encoded by CEBPB), a potent regulator of this process. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBPβ in chondrocytes. C/EBPβ and RUNX2 were induced and co-localized in highly differentiated chondrocytes during the skeletal growth and OA development of mice and humans. The compound knockout of Cebpb and Runx2 in mice caused growth retardation and resistance to OA with decreases in cartilage degradation and matrix metalloproteinase-13 (Mmp-13) expression. C/EBPβ and RUNX2 cooperatively enhanced promoter activity of MMP13 through specific binding to a C/EBP-binding motif and an osteoblast-specific cis-acting element 2 motif as a protein complex. Human genetic studies failed to show the association of human CEBPB gene polymorphisms with knee OA, nor was there a genetic variation around the identified responsive region in the human MMP13 promoter. However, hypoxia-inducible factor-2α (HIF-2α), a functional and genetic regulator of knee OA through promoting endochondral ossification, was identified as a potent and functional inducer of C/EBPβ expression in chondrocytes by the CEBPB promoter assay. Hence, C/EBPβ and RUNX2, with MMP-13 as the target and HIF-2α as the inducer, control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.

Influence of surface microstructure on the reaction of the active ceramics in vivo.

When porosity and macro-pore size differ in the same ceramic, the mode of bone regeneration and the degradation of ceramics in vivo is said to be different. However, the reactions in vivo of ceramics that have a different microstructure with the same porosity and the same macro-pore size, are not so far known. In this study, two kinds of β-tricalcium phosphate (TCP) that had different microstructures but the same porosity and macro-pore size, were manufactured. These TCP were implanted in the distal femurs of 20 mature male rabbits, and their respective areas of ceramics and of regenerated bone were measured after 4, 12 and 24 wk. In both TCPs, the regenerated bone similarly decreased from 4–24 wk in a different way. The area of ceramics in one of these TCPs significantly decreased gradually throughout the observation period. On the other hand, the other TCP showed no marked decrease during the same period. This suggested a possibility that the difference in micro-structure has a large effect on the reaction of the ceramics in the bone.

Prevention and management of knee osteoarthritis and knee cartilage injury in sports

Articular cartilage defects in the knee of young or active individuals remain a problem in orthopaedic practice. These defects have limited ability to heal and may progress to osteoarthritis. The prevalence of knee osteoarthritis among athletes is higher than in the non-athletic population. The clinical symptoms of osteoarthritis are joint pain, limitation of range of motion and joint stiffness. The diagnosis of osteoarthritis is confirmed by the symptoms and the radiological findings (narrowing joint space, osteophyte formation and subchondral sclerosis). There is no strong correlation between symptoms and radiographic findings. The aetiology of knee osteoarthritis is multifactorial. Excessive musculoskeletal loading (at work or in sports), high body mass index, previous knee injury, female gender and muscle weakness are well-known risk factors. The high-level athlete with a major knee injury has a high incidence of knee osteoarthritis. Cartilage injuries are frequently observed in young and middle-aged active athletes. Often this injury precedes osteoarthritis. Reducing risk factors can decrease the prevalence of knee osteoarthritis. The prevention of knee injury, especially anterior cruciate ligament and meniscus injury in sports, is important to avoid progression of knee osteoarthritis.

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.

Overexpression of the csk gene suppresses tumor metastasis in vivo

The non‐receptor tyrosine kinase c‐Src has been implicated in the development of numerous human cancers. c‐Src is activated in colon cancers, particularly in highly metastatic cells, and its overexpression strongly correlates with tumor progression. C‐terminal Src kinase (Csk) has been demonstrated to negatively regulate Src family tyrosine kinases through tyrosine phosphorylation at the C‐terminal regulatory site (Tyr‐527). We report herein that down‐regulation of Src kinase activity by adenovirus‐mediated csk gene transfer abrogated the highly metastatic phenotype of colon cancer cells. Overexpression of Csk decreased Src tyrosine kinase activity in NL‐17 cells, the highly metastatic clone of mouse colon adenocarcinoma 26. Importantly, Csk overexpression in NL‐17 cells resulted in significant suppression of in vivo metastasis, without affecting its tumorgenicity. Csk overexpression decreased the invasiveness of NL‐17 cells through Matrigel, in vitro reconstituted basement membrane. Gelatin zymography confirmed the decreased protein levels of MMP‐2 (gelatinase A) in the supernatants of Csk‐overexpressed NL‐17 cells. These results provide a therapeutic basis for interfering with metastasis of colon cancer by csk gene‐mediated down‐regulation of Src kinase activity. Int. J. Cancer 88:384–391, 2000.

Production of interleukin-11 in bone-derived endothelial cells and its role in the formation of osteolytic bone metastasis

The interactions of the cells in the bone microenvironment play important roles in bone remodeling. Osteoblasts are involved in the bone remodeling through the production of soluble factors that regulate proliferation and differentiation of osteoclasts and through cell–cell interactions. Histological studies have suggested that endothelial cells are also associated with some osteolytic bone diseases. However, it is still unclear how endothelial cells contribute to bone resorption. We established bone-derived endothelial cells (BDECs) to study their roles in bone remodeling. The established BDECs promoted bone resorption in a murine neonatal calvaria organ culture system by secreting a soluble bone resorption-inducing factor(s) when stimulated by several inflammatory cytokines. This bone resorption-inducing factor was identified as interleukin-11 (IL-11). IL-11 is known to enhance bone resorption by promoting osteoclastogenesis and by suppressing the activity of osteoblasts. The production of IL-11 in BDECs was also promoted by conditioned medium of human melanoma A375M cells. Because A375M cells formed osteolytic bone metastasis in vivo, BDECs might be involved in pathological osteolysis by producing IL-11. These results suggest that endothelial cells in bone play important roles in the promotion of bone resorption by secreting IL-11 in physiological and pathological conditions.

Intraoperative 3-Dimensional Imaging-Based Navigation-Assisted Anatomic Double-Bundle Anterior Cruciate Ligament Reconstruction

In anatomic double-bundle anterior cruciate ligament (ACL) reconstruction, it is more technically demanding, even for experienced surgeons, to place 2 femoral tunnels within the ACL attachment than to place 2 tibial tunnels. We describe a technique using a three-dimensional (3-D) fluoroscopy-based navigation system to place 2 femoral tunnels accurately. After a reference frame is rigidly attached to the femur, an intraoperative image of the distal femur is obtained. The image is transferred to a navigation system and reconstructed into a 3-D image. During the placement of guidewires for the femoral tunnels through an accessory medial portal, a femoral guide with a tracker feeds back to the surgeons the direction of the guidewire on the 3-D femur bone surface image in real-time. The femoral guide is placed at the center of the footprint with the aid of visual guidance of the navigation and an arthroscopic view. The flexion angle of the knee is then adjusted to prevent posterior blowout on the computer screen during insertion of the guidewire. The length of the femoral tunnel can also be estimated before overdrilling the guidewire. This technology allows surgeons to place 2 femoral tunnels precisely without any complication during anatomic double-bundle ACL reconstruction.

SOX11 contributes to the regulation of GDF5 in joint maintenance

BackgroundIndividual skeletal elements of the vertebrate limbs arise through a segmentation process introducing joints in specific locations. However, the molecular pathways controlling joint formation and subsequent joint maintenance are largely unknown. In this study, we focused on SOX11, and its contribution to the regulation of GDF5, a secreted signal necessary for proper joint formation and postnatal joint homeostasis.ResultsSox11 is initially expressed broadly in the murine cartilage condensations at early stages of skeletal development, but its expression is specifically increased in the forming joint interzone as is forms. SOX11 overexpression can directly activate GDF5 expression both in vitro and in micromass cell cultures prepared from chick limb buds. Conserved SOX family binding sites are present in the 5’ UTR region of the GDF5 gene and we show SOX11 can specifically bind to one of them. While misexpression of Sox11 in developing chick limbs through RCAS virus infection does not induce Gdf5 expression in ectopic locations, it does enhance its expression. To explore the roles of Sox11 in joint homeostasis, we analyzed adult knee joints in an osteoarthritis mouse model where the medial meniscus and the medial collateral ligament were removed. We also analyzed knee joints from human subjects who underwent total knee replacement surgery. We find that SOX11 is mainly expressed in the weight-bearing areas of knee joints, and its expression is decreased in degraded cartilage during progression of knee osteoarthritis in both mice and humans.ConclusionsThis work implicates SOX11 as a potential regulator of GDF5 expression in joint maintenance and suggests a possible role in the pathogenesis of osteoarthritis.