Akira Nifuji

Establishment of tendon-derived cell lines exhibiting pluripotent mesenchymal stem cell-like property.

Development of the musculoskeletal system requires coordinated formation of distinct types of tissues, including bone, cartilage, muscle, and tendon. Compared to muscle, cartilage, and bone, cellular and molecular bases of tendon development have not been well understood due to the lack of tendon cell lines. The purpose of this study was to establish and characterize tendon cell lines. Three clonal tendon cell lines (TT-E4, TT-G11, and TT-D6) were established using transgenic mice harboring a temperature-sensitive mutant of SV40 large T antigen. Proliferation of these cells was significantly enhanced by treatment with bFGF and TGF-beta but not BMP2. Tendon phenotype-related genes such as those encoding scleraxis, Six1, EphA4, COMP, and type I collagen were expressed in these tendon cell clones. In addition to tendon phenotype-related genes, expression of osteopontin and Cbfal was observed. These clonal cell lines formed hard fibrous connective tissue when implanted onto chorioallantoic membrane in ovo. Furthermore, these cells also formed tendon-like tissues when they were implanted into defects made in patella tendon in mice. As these tendon cell lines also produced fibrocartilaginous tissues in tendon defect implantation experiments, mesenchymal stem cell properties were examined. Interestingly, these cells expressed genes related to osteogenic, chondrogenic, and adipogenic lineages at low levels when examined by RT-PCR. TT-G11 and TT-E4 cells differentiated into either osteoblasts or adipocytes, respectively, when they were cultured in cognate differentiation medium. These observations indicated that the established tendon cell line possesses mesenchymal stem cell-like properties, suggesting the existence of mesenchymal stem cell in tendon tissue.

Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice

Rheumatoid arthritis is one of the most critical diseases that impair the quality of life of patients, but its pathogenesis has not yet been fully understood. Osteopontin (OPN) is an extracellular matrix protein containing Arg-Gly-Asp (RGD) sequence, which interacts with αvβ3 integrins, promotes cell attachment, and cell migration and is expressed in both synovial cells and chondrocytes in rheumatoid arthritis; however, its functional relationship to arthritis has not been known. Therefore, we investigated the roles of OPN in the pathogenesis of inflammatory process in a rheumatoid arthritis model induced by a mixture of anti-type II collagen mAbs and lipopolysaccharide (mAbs/LPS). mAbs/LPS injection induced OPN expression in synovia as well as cartilage, and this expression was associated with joint swelling, destruction of the surface structures of the joint based on scanning electron microscopy, and loss of toluidine blue-positive proteoglycan content in the articular cartilage in wild-type mice. In contrast, OPN deficiency prevented the mice from such surface destruction, loss of proteoglycan in the articular joint cartilage, and swelling of the joints even when the mice were subjected to mAbs/LPS injection. Furthermore, mAbs/LPS injection in wild-type mice enhanced the levels of CD31-positive vessels in synovia and terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive chondrocytes in the articular cartilage, whereas such angiogenesis as well as chondrocyte apoptosis was suppressed significantly in OPN-deficient mice. These results indicated that OPN plays a critical role in the destruction of joint cartilage in the rheumatoid arthritis model in mice via promotion of angiogenesis and induction of chondrocyte apoptosis.

Unloading Induces Osteoblastic Cell Suppression and Osteoclastic Cell Activation to Lead to Bone Loss via Sympathetic Nervous System

Osteoporosis is one of the major health problems in our modern world. Especially, disuse (unloading) osteoporosis occurs commonly in bedridden patients, a population that is rapidly increasing due to aging-associated diseases. However, the mechanisms underlying such unloading-induced pathological bone loss have not yet been fully understood. Since sympathetic nervous system could control bone mass, we examined whether unloading-induced bone loss is controlled by sympathetic nervous tone. Treatment with β-blocker, propranolol, suppressed the unloading-induced reduction in bone mass. Conversely, β-agonist, isoproterenol, reduced bone mass in loaded mice, and under such conditions, unloading no longer further reduced bone mass. Analyses on the cellular bases indicated that unloading-induced reduction in the levels of osteoblastic cell activities, including mineral apposition rate, mineralizing surface, and bone formation rate, was suppressed by propranolol treatment and that isoproterenol-induced reduction in these levels of bone formation parameters was no longer suppressed by unloading. Unloading-induced reduction in the levels of mineralized nodule formation in bone marrow cell cultures was suppressed by propranolol treatment in vivo. In addition, loss of a half-dosage in the dopamine β-hydroxylase gene suppressed the unloading-induced bone loss and reduction in mineralized nodule formation. Unloading-induced increase in the levels of osteoclastic activities such as osteoclast number and surface as well as urinary deoxypyridinoline was all suppressed by the treatment with propranolol. These observations indicated that sympathetic nervous tone mediates unloading-induced bone loss through suppression of bone formation by osteoblasts and enhancement of resorption by osteoclasts.

Osteopontin Expression in Osteoblasts and Osteocytes During Bone Formation Under Mechanical Stress in the Calvarial Suture In Vivo

To clarify the role of OPN in bone formation under mechanical stress, we examined the expression and the function of OPN in bone using an expansion force‐induced osteogenesis model. Our results indicated that OPN expression was enhanced during the bone formation and that OPN would be one of the positive factors for the bone formation under mechanical stress.

Resistance to Unloading-Induced Three-Dimensional Bone Loss in Osteopontin-Deficient Mice†

Recent development in three‐dimensional (3D) imaging of cancellous bone has made possible true 3D quantification of trabecular architecture. This provides a significant improvement in the measures available to study and understand the mechanical functions of cancellous bone. We recently reported that the presence of osteopontin (OPN) was required for the effects of mechanical stress on bone as OPN‐null (OPN−/−) mice showed neither enhancement of bone resorption nor suppression of bone formation when they were subjected to unloading by tail suspension. However, in this previous study, morphological analyses were limited to two‐dimensional (2D) evaluation. Although bone structure is 3D and thus stress effect should be evaluated based on 3D parameters, no such 3D morphological features underlying the phenomenon have been known. To elucidate the role of OPN in mediating mechanical stress effect based on true quantitative examination of bone, we evaluated 3D trabecular structures of hindlimb bones of OPN−/− mice after tail suspension. Tail suspension significantly reduced 3D parameters of bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and anisotropy and increased 3D parameters on trabecular separation (Tb.Sp) in wild‐type mice. In contrast, these 3D parameters were not altered after tail suspension in OPN−/− mice. These data provided evidence that OPN is required for unloading‐induced 3D bone loss.

Negative Regulation of Bone Morphogenetic Protein/Smad Signaling by Cas-interacting Zinc Finger Protein in Osteoblasts*

Bone morphogenetic protein (BMP) signaling regulates body axis determination, apoptosis, and differentiation of various types of cells including neuron, gut, and bone cells. However, the molecules involved in such BMP regulation of biological events have not been fully understood. Here, we examined the involvement of Cas-interacting zinc finger protein (CIZ) in the modulation of BMP2-induced osteoblastic cell differentiation. CIZ overexpression in osteoblastic MC3T3E1 cells suppressed BMP2-enhanced expression of alkaline phosphatase, osteocalcin, and type I collagen genes. Upstream analyses revealed that CIZ overexpression also suppressed BMP2-induced enhancement of the mRNA expression of Cbfa1, which is a critical transcription factor for osteoblastic differentiation. BMP-induced Smad1 and Smad5 activation of GCCG-mediated transcription was blocked in the presence of CIZ overexpression. CIZ overexpression alone in the absence of BMP2 moderately enhanced basal levels of Cbfa1 mRNA expression. CIZ overexpression also enhanced 1.8-kb Cbfa1 promoter activity in the absence of BMP2, whereas it suppressed the promoter activity in the presence of BMP2. Finally, CIZ overexpression suppressed the formation of mineralized nodules in osteoblastic cell cultures. These data indicate that CIZ is a novel type inhibitor of BMP/Smad signaling.

The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass by inhibiting bone morphogenetic protein–induced bone formation

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)–induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2–induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.

Bone morphogenetic protein‐2 enhances osterix gene expression in chondrocytes

Osterix is a recently identified zinc‐finger‐containing transcription factor, which is required for skeletogenesis as no bone formation was observed in osterix‐deficient mice. Osterix was first cloned as a gene whose expression was enhanced by BMP in C2C12 cells. As BMP induces ectopic bone formation in vivo via a pathway reminiscent to endochondral bone formation, BMP may also regulate osterix gene expression in chondrocytes. However, no information was available regarding the BMP actions on osterix gene expression in chondrocytes. We therefore examined the effects of BMP‐2 on osterix gene expression in chondrocytes in culture. RT‐PCR analysis indicated that osterix mRNA was expressed in the primary cultures of chondrocytes derived from mouse rib cartilage. The treatment with BMP‐2 enhanced the levels of osterix transcripts within 24 h and the enhancement was still observed at 48 h based on RT‐PCR analysis. This BMP effect was specific to this cytokine, as TGF‐β did not alter osterix gene expression. BMP effects on the osterix mRNA levels were also confirmed by Northern blot analysis. The enhancing effect of BMP on osterix gene expression was observed in a dose‐dependent manner starting at 200 ng/ml. The BMP enhancement of the osterix gene expression in chondrocytes was blocked in the presence of a protein synthesis inhibitor, cycloheximide, while it was still observed in the presence of 5,6‐dichloro‐1‐β D‐ribofuranosylbenzimidazol (DRB) suggesting the involvement of post‐transcriptional events, which require new protein synthesis. These results indicated that osterix gene is expressed in the primary cultures of chondrocytes and its expression is under the control of BMP‐2. J. Cell. Biochem. 88: 1077–1083, 2003.

Inhibitory helix‐loop‐helix transcription factors Id1/Id3 promote bone formation in vivo

Bone formation is under the control of a set of transcription factors. Ids are inhibitory helix‐loop‐helix (HLH) transcription factors and expression of Id genes in osteoblasts is under the control of calciotropic agents such as BMP and vitamin D. However, the function of Ids during bone formation in vivo has not yet been elucidated. We, therefore, examined the role of Id1 and Id3 in the regulation of bone metabolism in vivo. Using wild type and Id1/Id3 heterozygous knockout mice, we analyzed calvarial bone formation in the suture by X‐ray picture, proliferation, and mineralization activities of primary calvarial osteoblasts by MTT assay and alizarin red staining and onthotopic in vivo bone formation by BMP injection onto calvaria and micro CT analysis. The width of calvarial sutures was reduced by more than 50% in Id1/Id3 heterozygous knock out mice. Analyses on the cellular basis for the mechanism underlying the defects in the mutant mice revealed suppression of proliferation and mineralization in osteoblasts derived from Id1/Id3 heterozygous knock out mice. Furthermore, Id1/Id3 heterozygous knock out mice suppressed BMP‐induced bone formation in vivo. These results indicated that Id1 and Id3 are positive factors to promote bone formation in vivo.

Osteopontin‐Deficiency Suppresses Growth of B16 Melanoma Cells Implanted in Bone and Osteoclastogenesis in Co‐Cultures

Tumor metastasis and invasion to bone is one of major medical issues in our modern societies. Osteopontin deficiency decreased tumor invasion in bone based on knockout mouse study. In bone, osteopontin is a positive factor to increase tumor invasion.