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Natsuo Yasui

Publications and source records attributed to Natsuo Yasui.

At least 19 recordsLinked to original sources
PubMed2007

Distinct expression of mast cell tryptase and protease activated receptor-2 in synovia of rheumatoid arthritis and osteoarthritis.

The objective of this study is to examine the differential expression of mast cell tryptase and its receptor, protease-activated receptor-2 (PAR-2), in the synovium and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Biochemical and immunohistochemical analyses were performed to determine whether the trypsin-like protease in the synovium is identical to mast cell tryptase. The effects of mast cell tryptase on the proliferation of synovial fibroblast-like cells (SFCs) and the release of IL-8 thereof were evaluated by the [3H]-thymidine incorporation and ELISA, respectively. The trypsin-like protease in the synovium of RA patients was identical to human mast cell tryptase, which was composed of two subunits: 33 and 34 kDa. The 33- and 34-kDa proteins are different glycosylated forms of the 31-kDa protein, which was unglycosylated. Mast cell tryptase activity in RA synovial fluid was significantly higher than that in OA synovial fluid, while their activities and expression in the synovium were similar. Expression of PAR-2 mRNA in the synovium was higher in RA than in OA. Mast cell tryptase containing the unglycosylated 31-kDa subunit was the predominant form in synovial fluid. RA patients had higher amounts of this subunit in their synovial fluid than OA patients. Mast cell tryptase and PAR-2 activating peptide stimulated the proliferation of SFCs and release of IL-8 from these cells. Mast cell tryptase secretion into RA synovial fluid is higher than OA synovial fluid. Mast cell tryptase in synovial fluid stimulates the proliferation of SFCs and the release of pro-inflammatory cytokines via PAR-2, which may contribute to exacerbation of synovitis in RA.

Adult↗
PubMed2007

Genetic association between the PRKCH gene encoding protein kinase Ceta isozyme and rheumatoid arthritis in the Japanese population.

OBJECTIVE: Analyses of families with rheumatoid arthritis (RA) have suggested the presence of a putative susceptibility locus on chromosome 14q21-23. This large population-based genetic association study was undertaken to examine this region. METHODS: A 2-stage case-control association study of 950 unrelated Japanese patients with RA and 950 healthy controls was performed using >400 gene-based common single-nucleotide polymorphisms (SNPs). RESULTS: Multiple SNPs in the PRKCH gene encoding the eta isozyme of protein kinase C (PKCeta) showed significant single-locus disease associations, the most significant being SNP c.427+8134C>T (odds ratio 0.72, 95% confidence interval 0.62-0.83, P = 5.9 x 10(-5)). Each RA-associated SNP was consistently mapped to 3 distinct regions of strong linkage disequilibrium (i.e., linkage disequilibrium or haplotype blocks) in the PRKCH gene locus, suggesting that multiple causal variants influence disease susceptibility. Significant SNPs included a novel common missense polymorphism of the PRKCH gene, V374I (rs2230500), which lies within the ATP-binding site that is highly conserved among PKC superfamily members. In circulating lymphocytes, PRKCH messenger RNA was expressed at higher levels in resting T cells (CD4(+) or CD8(+)) than in B cells (CD19(+)) or monocytes (CD14(+)) and was significantly down-regulated through immune responses. CONCLUSION: Our results provide evidence of the involvement of PRKCH as a susceptibility gene for RA in the Japanese population. Dysregulation of PKCeta signal transduction pathway(s) may confer increased risk of RA through aberrant T cell-mediated autoimmune responses.

Adult↗
PubMed2006

A novel type of EWS-CHOP fusion gene in myxoid liposarcoma.

The cytogenetic hallmark of myxoid type and round cell type liposarcoma consists of reciprocal translocation of t(12;16)(q13;p11) and t(12;22)(q13;q12), which results in fusion of TLS/FUS and CHOP, and EWS and CHOP, respectively. Nine structural variations of the TLS/FUS-CHOP chimeric transcript have been reported, however, only two types of EWS-CHOP have been described. We describe here a case of myxoid liposarcoma containing a novel EWS-CHOP chimeric transcript and identified the breakpoint occurring in intron 13 of EWS. Reverse transcription-polymerase chain reaction and direct sequence showed that exon 13 of EWS was in-frame fused to exon 2 of CHOP. Genomic analysis revealed that the breaks were located in intron 13 of EWS and intron 1 of CHOP.

Adult↗
PubMed2006

High-grade slippage of the lumbar spine in a rat model of spondylolisthesis: effects of cyclooxygenase-2 inhibitor on its deformity.

STUDY DESIGN: Radiographic and histologic evaluation of spondylolisthesis in a rat model. OBJECTIVES: To investigate the effects of etodolac, a cox-2 inhibitor, on the severity of spondylolisthesis in a 4-week-old rat model. SUMMARY OF BACKGROUND DATA: Spondylolisthesis occurs associated with spondylolysis in some pediatric patients. In such patients, the percent of forward slippage varies individually ranging between 0% and 100%. The factors determining the severity of forward slippage have not been clarified as yet. In earlier studies, we found that growth plate stress fracture was the basic lesion and that slippage was a consequence of the stress fracture. Hence, we hypothesized that the capacity of bone healing might be an important determinant of the degree of forward slippage. METHODS: A lumbar spine slippage model was prepared in 4-week-old rats with vertebral physis fracture. To disrupt the fracture healing, the cyclooxygenase-2 (cox-2) inhibitor etodolac was used, and its effects on slippage and deformity were evaluated radiologically and histologically. RESULTS: In the etodolac group, forward slip significantly increased (P < 0.05) to the Meyerding Grade III while in the control rats it was Grade I or II. Bone remodeling of the vertebral body was suppressed by etodolac. Histologically, epiphyseal separation with slippage was observed in all the control and etodolac-treated rats. However, in the etodolac-treated group, the epiphyseal plate was greatly separated and did not present periosteal thickening at the physis fracture site. CONCLUSION: Vertebral forward slippage occurred in young rats after epiphyseal separation. In the etodolac group, slippage increased as deterioration of the bone healing capacity increased. Poor bony healing is suggested as one of the determinants of high-grade spondylolisthesis in children and adolescents.

Animals↗
PubMed2006

High tibial osteotomy for osteoarthritis of the knee with varus deformity utilizing the hemicallotasis method.

INTRODUCTION: A hemicallotasis method has been developed utilizing an external fixator as high tibial osteotomy (HTO), and satisfactory results of this method with the external fixator have been reported. This external fixator has a universal joint that moves in all directions. We have recently designed a hemicallotasis device for this operation. METHODS: HTO for the knee with varus deformity utilizing the hemicallotasis method was performed on 44 knees. The patients had a mean age at operation of 65 years (range 49-82 years), a mean follow-up period of 68 months (range 36-119 months), and a mean preoperative knee score of 66 points (range 27-90 points). RESULTS: The operated knees had a mean knee score at the final follow-up of 86 points (range 51-98 points), but the mean range of knee motion was not changed as follows. Before surgery, the mean flexion was 129 degrees (range 90-150 degrees) and the mean extension was -5 degrees (range -30 to 0 degrees), whereas at the final follow-up, the corresponding values were 127 degrees (range 85-150 degrees) and -4 degrees (range -25 to 0 degrees), respectively. Radiographically, the femorotibial joint was classified as grade 2 in 9 knees, grade 3 in 21 knees, and grade 4 in 14 knees according to the classification of osteoarthritis (Kellgren and Laurence). The patellofemoral joint was also classified as grade 1 in 39 knees, grade 2 in 2 knees, and grade 3 in 3 knees. The mean femorotibial angle was 184 degrees (4 degrees varus) before surgery, 169 degrees (11 degrees valgus) after pin extraction, and was maintained at the final follow-up. The complications of this method were relatively few and consisted of pin-tract infection (8 knees), deep vein thrombosis (3 knees), and delayed union (2 knees). No peroneal nerve palsy or compartment syndrome was encountered. No knee was converted to total arthroplasty. However, administration of analgesics was necessary in ten knees at the final follow-up. CONCLUSION: The hemicallotasis method easily determined the angle of correction even in the knees with ligamentous laxity. Nevertheless, one of the major demerits of this method was a longer period of application of the external fixator. The level of evidence was level IV (case series).

Aged↗
PubMed2006

Metaphyseal dysplasia of Braun-Tinschert type: report of a Japanese girl.

We report on a 7-year-old Japanese girl with metaphyseal dysplasia (MD) of Braun-Tinschert type, a recently recognized, autosomal dominant sclerosing bone dysplasia. All individuals with the disorder from four families in the literature originated from a small town in Bohemia or its vicinity. The occurrence of the disorder in a Japanese girl indicates that it is not restricted to Germans. The radiographic hallmarks of the disorder include metaphyseal undermodeling (Erlenmeyer-flask deformity); osteosclerosis of the chondroosseous junctions, metaphyseal cortices, and epiphyseal margins; and exostosis-like bone excrescences at the metaphyseal-diaphyseal junctions. In the girl we described, the latter two findings were conspicuous at age 4 years, but became less prominent with increasing age. The metaphyseal trabeculae were somewhat coarse. The humeri exhibited varus deformity, and the ulnae and fibulae mild bowing. The mean bone mineral density of the lumbar spine was lower than that of age-matched controls. The patient exhibited premature loss of primary teeth, likely to be a sign of increased periodontal bone resorption. Markers of bone formation and resorption were both increased, an indication of a high rate of bone turnover.

Abnormalities, Multiple↗
PubMed2006

Bisphosphonate modulates morphological and mechanical properties in distraction osteogenesis through inhibition of bone resorption.

Despite the general clinical acceptance of distraction osteogenesis and much attention to bone formation in this method, little is recognized about activated bone resorption in the regenerated bone. The purpose of this study was to demonstrate the simultaneously activated bone resorption with activated bone formation and to investigate the role and efficacy of bisphosphonate in distraction osteogenesis. Left tibiae of 54 immature rabbits were lengthened for 3 weeks at a rate of 0.7 mm/day after a 1-week lag. Regenerated bone was quantitatively investigated by radiographic bone density, bone histomorphometry, and three-point bending testing. Animals received either vehicle or nitrogen-containing bisphosphonate (N-BP), YM529/ONO5920 at doses of 0.4 mg/kg/w or 0.004 mg/kg/w for 6 weeks. Regenerated bone of the vehicle group showed a radiologically characteristic zone structure containing the osteopenic zones adjacent to the sclerotic zones. The regenerated bone of the 0.4-mg/kg/w group showed no osteopenic zones during the course and eventually became homogeneously radiodense. The bone volume corresponding to the osteopenic zone of this group was 5.6-fold greater compared with that of the vehicle group. The lengthened bone strength of this group was 3.3-fold greater in ultimate force than that of the vehicle group and equivalent to the contralateral tibia. The 0.004-mg/kg/w group had no substantial differences compared with the vehicle group, despite radiological enhancement of the mineralized front as well as somewhat delayed bone resorption. These results demonstrate that not only bone formation but also bone resorption is highly activated in the regenerated bone, implying high bone turnover. Sufficient N-BP caused a notable modulation in morphological properties of the regenerated bone through inhibition of highly activated bone resorption and eventually increased mechanical properties.

Absorptiometry, Photon↗
PubMed2006

No evidence for association of the ENPP1 (PC-1) K121Q variant with risk of type 2 diabetes in a Japanese population.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1, also known as PC-1) inhibits insulin signal transduction pathway(s). Previous studies have demonstrated the K121Q variant of the ENPP1 gene to have a significant functional role in determining susceptibility to insulin resistance and type 2 diabetes (T2D). To assess whether the K121Q variant has any impact on T2D in Japanese, we undertook an extensive case-control association study using a total of 911 unrelated Japanese T2D patients and 876 control subjects. No significant difference was observed in either genotype distribution (P=0.95) or allele frequency (P=0.83) between T2D and control groups. Notably, the frequency of the ancestral Q121 allele, which is also present in other primates, was quite high in African-Americans, and showed a marked ethnic variation (77.3% in African-Americans, 16.7% in European Americans, 10.5% in Japanese and 4.2% in Han Chinese). Consequently, the pairwise F(ST )value (a classic measure of genetic distance between pairs of population) showed highly significant differentiations between African-American and non-African-American populations (F(ST)>0.3). Our results indicated that the K121Q variant of the ENPP1 gene has very little, if any, impact on T2D susceptibility in Japanese, but may play a role in the inter-ethnic variability in insulin resistance and T2D.

Black or African American↗
PubMed2006

JRAB/MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic recycling of occludin.

The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Ca2+ switch assay further revealed that MICAL-L2-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rab13-binding protein).

Actin Cytoskeleton↗
PubMed2006

Intrafamilial phenotypic diversity in multiple epiphyseal dysplasia associated with a COL9A2 mutation (EDM2).

We describe a Japanese family with an autosomal dominant multiple epiphyseal dysplasia (MED EDM2) showing significant phenotypic diversity among the five affected members. Genomic analysis for COL9A2 identified an Ex3-1A>G heterozygous mutation, which has been proved to result in skipping of exon 3. The proband was a 9-year-old boy, who presented with ulnar club hands due to severe epiphyseal dysplasia in the distal ulnae. Radiological examination showed multiple epiphyseal dysplasias, predominantly involving the knee and the wrist. The hip appeared almost normal. The malalignment of the wrist was successfully treated with a limb lengthening procedure. The phenotype of the asymptomatic 12-year-old brother was similar to, but milder than, that of the proband. The asymptomatic 39-year-old mother, the 35-year-old uncle, and the 65-year-old grandmother with bilateral painful knees showed radiographically mild and severe osteoarthritis of the knee, respectively, and none of them had wrist deformity.

Base Sequence↗
PubMed2006

MRI signal changes of the pedicle as an indicator for early diagnosis of spondylolysis in children and adolescents: a clinical and biomechanical study.

STUDY DESIGN: Clinical review of pediatric patients with lumbar spondylolysis and biomechanical analysis using finite-element lumbar spine model. OBJECTIVES: To evaluate the usefulness of the signal changes observed on MR images of the pedicle for the early diagnosis of spondylolysis, and to investigate the pathomechanism of the signal changes based on the stresses in pedicles, as predicted using finite-element analyses. Furthermore, to evaluate the usefulness of the signal change to predict the bony healing following conservative treatment. SUMMARY OF BACKGROUND DATA: Since early-stage spondylolysis can achieve osseous healing conservatively, it is important to diagnose this disorder as early as possible. Presently, there is no well-established, noninvasive, and reliable diagnostic tool for the early diagnosis. METHODS: Thirty-seven pediatric patients with spondylolysis were included. Sixty-eight defects were examined and their stages as revealed on CT scans were recorded. High signal changes (HSC) of the pedicles on axial T2-weighted MRI were compared with the CT-based stages of the defect. Among them, 16 patients, including 15 boys and 1 girl, were treated conservatively for at least a 3-month period. Bony healing of the fracture site was evaluated on CT, and the results were compared between two groups with or without HSC at the initial consultation. Using a three-dimensional nonlinear finite-element model of the L3-L5 segment, stress distributions in the pars and pedicle regions were analyzed in response to 400 N compression and 10.6 Nm moment. RESULTS: Based on CTs, 68 pars defects were classified as follows: 8 very early, 24 late-early, 16 progressive, and 20 terminal stages. All defects in very early and late-early stages (100%) showed HSC on T2-weighted MRI at the ipsilateral pedicle. Among 16 progressive stages, eight (50%) showed HSC, while no defects of the terminal stage (0%) were found to have HSC. In total, 29 pars defects were treated conservatively out of 16 patients. In 19 of the HSC positive defects, 15 (79%) showed bony healing after the conservative treatment, whereas none of the 10 HSC negative defects (0%) showed any healing. The results were statistically significant at P < 0.05 (chi). Stress results from the finite-element model indicated that pars interarticularis showed the highest value in all loading modes, and the pedicle showed the second highest. CONCLUSIONS: The correlation between the high stresses in the pedicle and the corresponding HSC suggest that signal changes in MRI could be used as an indicator for early diagnosis of spondylolysis. The HSC of the pedicle provided useful information to diagnose early stage spondylolysis. Furthermore, the HSC may be a good indicator as to whether a bony union will result from conservative treatment.

Adolescent↗
PubMed2006

Association of single-nucleotide polymorphisms in the suppressor of cytokine signaling 2 (SOCS2) gene with type 2 diabetes in the Japanese.

Several previous linkage scans in type 2 diabetes (T2D) families indicated a putative susceptibility locus on chromosome 12q15-q22, while the underlying gene for T2D has not yet been identified. We performed a region-wide association analysis on 12q15-q22, using a dense set of >500 single-nucleotide polymorphisms (SNPs), in 1492 unrelated Japanese individuals enrolled in this study. We identified an association between T2D and a haplotype block spanning 13.6 kb of genomic DNA that includes the entire SOCS2 gene. Evolutionary-based haplotype analysis of haplotype-tagging SNPs followed by a "sliding window" haplotypic analysis indicated SNPs that mapped to the 5' region of the SOCS2gene to be associated with T2D with high statistical significance. The SOCS2 gene was expressed ubiquitously in human and murine tissues, including pancreatic beta-cell lines. Adenovirus-mediated expression of the SOCS2 gene in MIN6 cells or isolated rat islets significantly suppressed glucose-stimulated insulin secretion. Our data indicate that SOCS2 may play a role in susceptibility to T2D in the Japanese.

Adenoviridae↗
PubMed2006

Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest.

In animal models, several ubiquitin ligases play an important role in skeletal muscle atrophy caused by unloading. In this study we examined protein ubiquitination and ubiquitin ligase gene expression in quadriceps femoris muscle from healthy volunteers after 20-day bedrest to clarify ubiquitin-dependent proteolysis in human muscles after unloading. During bedrest, thickness and cross-sectional area of the quadriceps femoris muscle decreased significantly by 4.6% and 3.7%, respectively. Ubiquitinated proteins accumulated in these atrophied human muscles. A real-time reverse transcription-polymerase chain reaction system showed that bedrest significantly upregulated expression of two ubiquitin ligase genes, Cbl-b and atrogin-1. We also performed DNA microarray analysis to examine comprehensive gene expression in the atrophied muscle. Bedrest mainly suppressed the expression of muscle genes associated with control of gene expression in skeletal muscle. Our results suggest that, in humans, Cbl-b- or atrogin-1-mediated ubiquitination plays an important role in unloading-induced muscle atrophy, and that unloading stress may preferentially inhibit transcriptional responses in skeletal muscle.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Effects of monofilament nylon coated with basic fibroblast growth factor on endogenous intrasynovial flexor tendon healing.

PURPOSE: We developed a monofilament nylon thread that can release various growth factors to enhance intrinsic reparative processes after flexor tendon injury. We evaluated the properties of this thread in vitro and in vivo. METHODS: Nylon threads were coated with gelatin that subsequently was cross-linked in glutaraldehyde. The thread was soaked in basic fibroblast growth factor (bFGF) solution (400 microg/mL). Exogenous bFGF in the thread was released constantly over the course of 1 week. The biologic activity of bFGF and the biomechanical strength of the thread were examined in vitro and its efficacy was investigated in an in vivo rabbit tendon repair model after early flexion exercises. The sutured sites were examined histologically (hematoxylin-eosin, immunohistochemistry, in situ hybridization), biochemically (Western blot test), and biomechanically (ultimate load) after surgery. RESULTS: This gelatin-coated thread absorbed iodine 125-labeled bFGF in a time-dependent manner. The total amount of bFGF absorbed by the thread within the tendon tissue was between 3 and 15 mug depending on the concentration of bFGF solution. Basic fibroblast growth factor protein was delivered selectively-not in the surrounding scar but in the repaired tendon-for 3 weeks. Histologic analysis showed that the cellular density at the repaired site increased in accordance with the expression of bFGF messenger RNA and protein in the tendon. Endogenous bFGF expression seemed to be enhanced transiently by exogenous bFGF during the first few weeks. The epitenon showed a vigorous fibroblastic response to the coated thread and the ultimate load also was increased significantly at 3 weeks after surgery. CONCLUSIONS: This bFGF-coated nylon suture gave excellent results in delivering a drug selectively to tendon; it also induced an increase of biomechanical strength and a thickening of the epitenon layer in vivo during a 3-week period, thereby accelerating cellular proliferation, initially peripherally and later centrally. This system may become a therapeutic tool to be used in hand surgery.

Animals↗
PubMed2006

Sp1 family of transcription factors regulates the human alpha2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells.

UNLABELLED: Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation. INTRODUCTION: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts. MATERIALS AND METHODS: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for alpha2(XI) protein and COL11A2 regulatory mechanisms. RESULTS AND CONCLUSIONS: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and alpha2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.

Base Sequence↗
PubMed2006

Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.

UNLABELLED: Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl-b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl-b may mediate denervation-induced osteopenia by inhibiting IGF-I signaling in osteoblasts. INTRODUCTION: Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading-mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING-type ubiquitin ligase, Cbl-b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl-b played an important role in the resistance of osteoblasts to IGF-I. MATERIALS AND METHODS: Cbl-b-deficient (Cbl-b(-/-)) or wildtype (Cbl-b(+/+)) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF-I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl-b-overexpressing or -deficient osteoblastic cells to various growth factors. RESULTS: In Cbl-b(+/+) mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl-b protein in osteoprogenitor cells and in osteocalcin-positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl-b(-/-) mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF-I. Therefore, we focused on Cbl-b-mediated modification of IGF-I signaling. Denervation decreased the amounts of insulin receptor substrate-1 (IRS-1), phosphatidly inositol 3-phosphate kinase (PI3K), and Akt-1 proteins in femora of Cbl-b(+/+) mice, whereas the amounts of these IGF-I signaling molecules in femora of Cbl-b(-/-) mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl-b(-/-) mice were more stimulated to proliferate by IGF-I treatment compared with those from Cbl-b(+/+) mice. Furthermore, overexpression of Cbl-b increased ubiquitination and degradation of IRS-1 in primary Cbl-b(-/-) osteoblastic cells, leading to their impaired mitogenic response to IGF-I. CONCLUSIONS: These results suggest that Cbl-b induces resistance of osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.

Animals↗