Takahide Kurokawa

Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.

The mechanism of action of macrophage colony-stimulating factor (M-CSF) in osteoclast development was examined in a co-culture system of mouse osteoblastic cells and spleen cells. In this co-culture, osteoclast-like multinucleated cells (MNCs) were formed within 6 d in response to 10 nM 1 alpha,25(OH)2D3 added only for the final 2 d of culture. Simultaneously adding hydroxyurea for the final 2 d completely inhibited proliferation of cultured cells without affecting 1 alpha,25(OH)2D3-stimulated MNC formation. Autoradiographic examination using [3H]-thymidine revealed that osteoclast progenitors primarily proliferated during the first 4 d, whereas their differentiation into MNCs occurred predominantly during the final 2 d of culture in response to 1 alpha,25(OH)2D3. When anti-M-CSF antibody or anti-M-CSF receptor antibody was added either for the first 4 d or for the final 2 d, the MNC formation was similarly inhibited. In co-cultures of normal spleen cells and osteoblastic cells obtained from op/op mice, which cannot produce functionally active M-CSF, the lack of M-CSF either for the first 4 d or for the final 2 d failed to form MNCs in response to 1 alpha,25(OH)2D3 added for the last 2 d. These results clearly indicate that M-CSF is indispensable for both proliferation of osteoclast progenitors and their differentiation into mature osteoclasts.

Wortmannin, a specific inhibitor of phosphatidylinositol-3 kinase, blocks osteoclastic bone resorption

The biological role of phosphatidylinositol (PI)‐3 kinase was examined in osteoclast‐like multinucleated cells (OCLs) formed in co‐cultures of mouse osteoblastic cells and bone marrow cells. The expression of PI‐3 kinase in OCLs was confirmed by Western blot analysis. Wortmannin (WT), a specific inhibitor of PI‐3 kinase, inhibited PI‐3 kinase activity in OCLs both in vitro and in vivo. WT also inhibited pit‐forming activity on dentine slices and disrupted a ringed structure of F‐actin‐containing dots (an actin ring) in OCLs in a dose‐dependent manner. The inhibitory profiles of WT for pit and actin ring formation were similar to that for PI‐3 kinase activity in OCLs. Electron microscopic analysis revealed that OCLs treated with WT did not form ruffled borders. Instead, numerous electron lucent vacuoles of differing sizes were found throughout the cytoplasm. These results suggest that PI‐3 kinase is important in osteoclastic bone resorption.

Suppression of arthritic bone destruction by adenovirus-mediated csk gene transfer to synoviocytes and osteoclasts

Rheumatoid arthritis (RA) is characterized by a chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages, and plasma cells, all of which manifest signs of activation. Recent studies have revealed the essential role of osteoclasts in joint destruction in RA. Src family tyrosine kinases are implicated in various intracellular signaling pathways, including mitogenic response to growth factors in fibroblasts, activation of lymphocytes, and osteoclastic bone resorption. Therefore, inhibiting Src activity can be a good therapeutic strategy to prevent joint inflammation and destruction in RA. We constructed an adenovirus vector carrying the csk gene, which negatively regulates Src family tyrosine kinases. Csk overexpression in cultured rheumatoid synoviocytes remarkably suppressed Src kinase activity and reduced their proliferation rate and IL-6 production. Bone-resorbing activity of osteoclasts was strongly inhibited by Csk overexpression. Furthermore, local injection of the virus into rat ankle joints with adjuvant arthritis not only ameliorated inflammation but suppressed bone destruction. In conclusion, adenovirus-mediated direct transfer of the csk gene is useful in repressing bone destruction and inflammatory reactions, suggesting the involvement of Src family tyrosine kinases in arthritic joint breakdown and demonstrating the feasibility of intervention in the kinases for gene therapy in RA. off

Stimulation of Bone Formation by Recombinant Fibroblast Growth Factor-2 in Callotasis Bone Lengthening of Rabbits

Bone lengthening by callotasis is one of the most useful methods not only for the treatment of short extremities but also for extensive bone defects; however, the procedure takes a long time especially for the consolidation of the distracted callus. In this study, effects of a single local injection of recombinant human fibroblast growth factor-2 (FGF-2 or basic FGF) on callotasis bone lengthening were examined in rabbits. Ten days after the osteotomy at the middle of the tibia and the installment of an external fixator, the osteotomized site was distracted at a rate of 1.4 mm/day for 7 days, resulting in 9.8 mm lengthening. On the final day of distraction, 200 μg of FGF-2 in 150 μl of saline solution or vehicle alone was injected into the center of the distracted callus. Injection of FGF-2 increased bone formation at the distracted callus radiologically and histologically. A significant effect on bone mineral content (BMC) at the callus was observed as early as 2 weeks, and FGF-2 increased the BMC about twofold at 5 weeks after a normal remodeling process. We conclude that the callotasis method in combination with FGF-2 injection at the consolidation step could be clinically beneficial to shorten the bone lengthening period.

Immunohistochemical demonstration of bone morphogenetic protein-2 and transforming growth factor-β in the ossification of the posterior longitudinal ligament of the cervical spine

To clarify the mechanism of ossification of the posterior longitudinal ligament, immunohistochemical localization of bone morphogenetic protein-2 and transforming growth factor-β was examined using surgical specimens of ligament tissues from an affected patient. Two poly-clonal antibone morphogenetic protein-2 antibodies and an anti-human transforming growth factor-β antibody were used as primary antibodies. Bone morphogenetic protein-2 and transforming growth factor-β were present in ossified matrix and chondrocytes of adjacent cartilaginous areas of ossification of the posterior longitudinal ligament. Although immunostaining with antibone morphogenetic protein-2 antibodies also was observed in mesenchymal cells with fibroblastic features in the immediate vicinity of the cartilaginous areas, no staining could be detected with anti-human transforming growth factor-β antibody in these cells. The presence of these factors were specific for the ossified ligament because no immunostaining was observed after using antibodies in the posterior longitudinal ligament at unossified levels from the same patient. It is suggested that bone morphogenetic protein-2 and transforming growth factor-β play important roles in the development of ossification of the posterior longitudinal ligament and that bone morphogenetic protein-2 may act as an initiating factor in the development of ossification of the posterior longitudinal ligament by stimulating differentiation of mesenchymal progenitor cells. Transforming growth factor-β may stimulate bone formation at a later stage of the process of ectopic ossification.

Osteoclasts express high levels of p60c-src, preferentially on ruffled border membranes.

Expression of p60c‐src , the normal cellular counterpart of the transforming protein of Rous sarcoma virus (RSV), p60c‐src , was examined in mouse and rat authentic osteoclasts and mouse osteoclast‐like multinucleated cells (MNCs) formed in vitro. In co‐cultures of mouse osteoblastic cells and spleen cells, the expression of p60c‐src strikingly increased on day 5 in parallel with the appearance of MNCs in the presence of 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3). Immunohistochemical examination confirmed the high level expression of p60c‐src in both mouse authentic osteoclasts and MNCs. Electron microscopic examination revealed that p60c‐src was primarily localized on ruffled border membranes and vacuoles, but not on the clear zone in rat authentic osteoclasts. These results suggest that p60 c‐src is important in osteoclastic bone resorption.

Fluid Shear Stress Increases the Production of Granulocyte-Macrophage Colony-Stimulating Factor by Endothelial Cells via mRNA Stabilization

To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm2 markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.5 times, respectively, the amount released by the static controls at 24 hours, but it had no significant influence on the release of granulocyte CSF or macrophage CSF. The results of reverse transcriptase-polymerase chain reaction demonstrated that GM-CSF mRNA began to increase as early as 2 hours after initiation of 15 dyne/cm2 shear stress and continued to increase with time, reaching a peak of about four times the control levels at 24 hours. This increase in GM-CSF mRNA levels in response to shear stress depended on protein synthesis, because it was blocked by cycloheximide. Neither nuclear run-on assay or luciferase assay using a reporter gene containing GM-CSF gene promoter showed any significant change in transcription of the GM-CSF gene even after 24-hour exposure to a shear stress of 15 dyne/cm2. Actinomycin D chase experiments using a competitive polymerase chain reaction showed that shear stress extended the half-life of GM-CSF mRNA from approximately 23 to 42 minutes in HUVECs. These findings suggest that fluid shear stress increases the production of GM-CSF in HUVECs via mRNA stabilization.

Reduced expression of interleukin-11 in bone marrow stromal cells of senescence-accelerated mice (SAMP6) : relationship to osteopenia with enhanced adipogenesis

Aging is associated with an increase in bone marrow adipose tissue and a reduction in bone turnover. The P6 strain of senescence‐accelerated mice (SAM) exhibit an early decrease in bone mass with a reduction in bone remodeling. In the bone marrow, suppressed osteoblastogenesis and osteoclastogenesis with enhanced adipogenesis are observed. The present study was undertaken to clarify the mechanism of age‐related changes in bone turnover using bone marrow cells from SAMP6 mice. Because interleukin (IL)‐11 has been shown to potently inhibit adipogenesis and to stimulate osteoclast formation, the effect of IL‐11 on the differentiation of bone marrow cells was examined. The impaired formation of both osteoblasts and osteoclasts was restored and the enhanced formation of adipocytes was suppressed by the addition of 10 pM recombinant human IL‐11. Other cytokines that activate gp130 as a common signal transducer, IL‐6 and leukemia inhibitory factor, did not have such effects. Sequence analysis of the entire coding region of IL‐11 cDNA obtained from SAMP6 stromal cells revealed no mutations. Constitutively secreted IL‐11 protein into culture media, and its mRNA expression stimulated by transforming growth factor β were reduced in stromal cells from SAMP6 compared with those in control mice. These results demonstrate that the expression of IL‐11 is reduced in bone marrow cells of SAMP6 and suggest that the reduction in IL‐11 actions is involved in the impairment of both osteoblastogenesis and osteoclastogenesis in these mice. There is a possibility that alterations in IL‐11 actions may be associated with the age‐related impairment in bone metabolism.

Inhibition of Bone Resorption by Pamidronate Cannot Restore Normal Gain in Cortical Bone Mass and Strength in Tail‐Suspended Rapidly Growing Rats

To clarify how the changes in bone formation and resorption affect bone volume and strength after mechanical unloading, the effect of inhibition of bone resorption by a potent bisphosphonate, pamidronate, on bone mineral density (BMD), histology, and strength of hind limb bones was examined using tail‐suspended growing rats. Tail suspension for 14 days reduced the gain in the BMD of the femur at both the metaphysis rich in trabecular bone and the diaphysis rich in cortical bone. Treatment with pamidronate increased the total BMD as well as that of the metaphysis of the femur but had almost no effect on the BMD of the diaphysis in both control and tail‐suspended rats. Histological examinations revealed that 14‐day tail suspension caused a loss of secondary cancellous bone with a reduction in the trabecular number and thickness in comparison with control rats. In the femoral diaphysis, the diameter and cortical bone thickness increased to a lesser degree in tail‐suspended rats when compared with rats without tail suspension, and a marked reduction in bone formation and the layers of alkaline phosphatase–positive cells was observed at the periosteal side. Pamidronate treatment increased secondary cancellous bone but could not restore normal growth‐induced periosteal bone apposition and bone strength. Because the material strength of the femoral diaphysis at the tissue level was not affected by pamidronate treatment, the inability of pamidronate to prevent the reduction in physical strength of the femoral diaphysis does not appear to be due to a change in the quality of newly formed bone. These results demonstrate that tail suspension reduces the growth‐induced periosteal modelling drift and that the antiresorptive agent pamidronate is unable to restore normal periosteal bone apposition.

Fibroblasts of spinal ligaments pathologically differentiate into chondrocytes induced by recombinant human bone morphogenetic protein-2: morphological examinations for ossification of spinal ligaments.

To elucidate the process of ossification in spinal ligaments, an aqueous solution containing recombinant human bone morphogenetic protein (BMP)-2 (40 micrograms/100 microL) was injected into murine ligamenta flava, and the ossification process was analyzed morphologically. In the control group, the solution administered lacked the protein; these flattened ligamentous fibroblasts possessing BMP receptors type IA and type II existed among type I collagen bundles. In the week immediately following the injection of BMP-2, ligamentous fibroblasts began to proliferate, differentiating into alkaline phosphatase-positive chondrocytes surrounded by an extracellular matrix composed of type I and II collagen. By the second week, differentiated chondrocytes of various stages were observed in type II collagen-rich matrix. These chondrocytes showed an abundance of BMP receptors type IA and II. The pathologically induced cartilage was resorbed by chondroclasts, permitting migration of blood vessels and osteogenic cells, as well as providing a site for endochondral ossification. By the third week, BMP-induced ossification had compressed the spinal cord, and by the sixth week, the ligamentous tissue had been almost completely replaced by bone. Ligamentous fibroblasts appeared to possess BMP receptors, as well as the potentiality to differentiate into chondrocytes. BMP receptors were upregulated during chondrification of ligamentous fibroblasts induced by exogenous BMP-2, suggesting that BMPs may play an important role in ossification of spinal ligaments.