Hirotaka Chikuda

Regulation of osteoclast apoptosis by ubiquitylation of proapoptotic BH3-only Bcl-2 family member Bim

Osteoclasts (OCs) undergo rapid apoptosis without trophic factors, such as macrophage colony‐stimulating factor (M‐CSF). Their apoptosis was associated with a rapid and sustained increase in the pro‐apoptotic BH3‐only Bcl‐2 family member Bim. This was caused by the reduced ubiquitylation and proteasomal degradation of Bim that is mediated by c‐Cbl. Although the number of OCs was increased in the skeletal tissues of bim−/− mice, the mice exhibited mild osteosclerosis due to reduced bone resorption. OCs differentiated from bone marrow cells of bim−/− animals showed a marked prolongation of survival in the absence of M‐CSF, compared with bim+/+ OCs, but the bone‐resorbing activity of bim−/− OCs was significantly reduced. Overexpression of a degradation‐resistant lysine‐free Bim mutant in bim−/− cells abrogated the anti‐apoptotic effect of M‐CSF, while wild‐type Bim did not. These results demonstrate that ubiquitylation‐dependent regulation of Bim levels is critical for controlling apoptosis and activation of OCs.

Connection Between B Lymphocyte and Osteoclast Differentiation Pathways

Osteoclasts differentiate from the hemopoietic monocyte/macrophage cell lineage in bone marrow through cell-cell interactions between osteoclast progenitors and stromal/osteoblastic cells. Here we show another osteoclast differentiation pathway closely connected with B lymphocyte differentiation. Recently the TNF family molecule osteoclast differentiation factor/receptor activator of NF-κB ligand (ODF/RANKL) was identified as a key membrane-associated factor regulating osteoclast differentiation. We demonstrate that B-lymphoid lineage cells are a major source of endogenous ODF/RANKL in bone marrow and support osteoclast differentiation in vitro. In addition, B-lymphoid lineage cells in earlier developmental stages may hold a potential to differentiate into osteoclasts when stimulated with M-CSF and soluble ODF/RANKL in vitro. B-lymphoid lineage cells may participate in osteoclastogenesis in two ways: they 1) express ODF/RANKL to support osteoclast differentiation, and 2) serve themselves as osteoclast progenitors. Consistent with these observations in vitro, a decrease in osteoclasts is associated with a decrease in B-lymphoid cells in klotho mutant mice (KL−/−), a mouse model for human aging that exhibits reduced turnover during bone metabolism, rather than a decrease in the differentiation potential of osteoclast progenitors. Taken together, B-lymphoid lineage cells may affect the pathophysiology of bone disorders through regulating osteoclastogenesis.

Independent impairment of osteoblast and osteoclast differentiation in klotho mouse exhibiting low-turnover osteopenia

We recently identified a new gene, klotho, which is involved in the suppression of multiple aging phenotypes. The mouse homozygous for a disruption of the klotho locus (kl/kl) exhibited multiple pathological conditions resembling human aging. Histomorphometric analysis revealed low-turnover osteopenia in kl/kl mice. The decrease in bone formation exceeded that of bone resorption, resulting in a net bone loss. The number of osteoblast progenitors determined by ex vivo bone marrow cultures was reduced in kl/kl mice. In addition, cultured osteoblastic cells derived from kl/kl mice showed lower alkaline phosphatase activity and matrix nodule formation than those from wild-type mice. Osteoclastogenesis in the coculture of marrow cells and osteoblastic cells was decreased only when marrow cells originated from kl/kl mice independently of the origin of osteoblastic cells. We also found that the expression of osteoprotegerin, an osteoclastogenesis inhibitor, was significantly upregulated in kl/kl mice. We conclude that a defect in the klotho gene expression causes the independent impairment of both osteoblast and osteoclast differentiation, leading to low-turnover osteopenia. Because this state represents a characteristic feature of senile osteoporosis in humans, kl/kl mice can be regarded as a useful model for investigating cellular and molecular mechanisms of age-related bone loss.

GSK-3β Controls Osteogenesis through Regulating Runx2 Activity

Despite accumulated knowledge of various signalings regulating bone formation, the molecular network has not been clarified sufficiently to lead to clinical application. Here we show that heterozygous glycogen synthase kinase-3β (GSK-3β)-deficient mice displayed an increased bone formation due to an enhanced transcriptional activity of Runx2 by suppressing the inhibitory phosphorylation at a specific site. The cleidocranial dysplasia in heterozygous Runx2-deficient mice was significantly rescued by the genetic insufficiency of GSK-3β or the oral administration of lithium chloride, a selective inhibitor of GSK-3β. These results establish GSK-3β as a key attenuator of Runx2 activity in bone formation and as a potential molecular target for clinical treatment of bone catabolic disorders like cleidocranial dysplasia.

Regulation of multiple ageing-like phenotypes by inducible klotho gene expression in klotho mutant mice

Mice carrying a loss-of-function mutation in the klotho gene (KL(-/-) mice) develop ageing-like symptoms around 4 weeks after birth and suffer from multiple age-related disorders observed in humans, including osteoporosis, arteriosclerosis, and pulmonary emphysema. The klotho gene encodes a single-pass transmembrane protein that may function in signaling pathways that suppress ageing. To investigate the ability of Klotho to regulate the development of ageing-related disorders, we established an inducible Klotho expression system using KL(-/-) mice carrying an exogenous klotho gene fused to the mouse metallothionein-I promoter, in which Klotho expression was dependent on zinc water feeding. We demonstrate that many advanced ageing-like KL(-/-) phenotypes were restored to normal whenever Klotho expression was induced. Conversely, decreasing Klotho expression in these rescued KL(-/-) mice induced several ageing-like KL(-/-) phenotypes. Our data indicate that Klotho may be effective in the treatment of multiple age-related disorders and is essential for maintaining animals free of these disorders.

Phosphorylation of GSK-3β by cGMP-dependent protein kinase II promotes hypertrophic differentiation of murine chondrocytes

cGMP-dependent protein kinase II (cGKII; encoded by PRKG2) is a serine/threonine kinase that is critical for skeletal growth in mammals; in mice, cGKII deficiency results in dwarfism. Using radiographic analysis, we determined that this growth defect was a consequence of an elongated growth plate and impaired chondrocyte hypertrophy. To investigate the mechanism of cGKII-mediated chondrocyte hypertrophy, we performed a kinase substrate array and identified glycogen synthase kinase-3beta (GSK-3beta; encoded by Gsk3b) as a principal phosphorylation target of cGKII. In cultured mouse chondrocytes, phosphorylation-mediated inhibition of GSK-3beta was associated with enhanced hypertrophic differentiation. Furthermore, cGKII induction of chondrocyte hypertrophy was suppressed by cotransfection with a phosphorylation-deficient mutant of GSK-3beta. Analyses of mice with compound deficiencies in both protein kinases (Prkg2(-/-)Gsk3b(+/-)) demonstrated that the growth retardation and elongated growth plate associated with cGKII deficiency were partially rescued by haploinsufficiency of Gsk3b. We found that beta-catenin levels decreased in Prkg2(-/-) mice, while overexpression of cGKII increased the accumulation and transactivation function of beta-catenin in mouse chondroprogenitor ATDC5 cells. This effect was blocked by coexpression of phosphorylation-deficient GSK-3beta. These data indicate that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3beta by cGKII.

Incidence and risk factors for mortality of vertebral osteomyelitis: a retrospective analysis using the Japanese diagnosis procedure combination database

Objective To examine the incidence of vertebral osteomyelitis (VO) and the clinical features of VO focusing on risk factors for death using a Japanese nationwide administrative database. Design Retrospective observational study. Setting Hospitals adopting the Diagnosis Procedure Combination system during 2007–2010. Participants We identified 7118 patients who were diagnosed with VO (International Classification of Diseases, 10th Revision codes: A18.0, M46.4, M46.5, M46.8, M46.9, M48.9 and M49.3, checked with the detailed diagnoses in each case and all other codes indicating the presence of a specific infection) and hospitalised between July and December, 2007–2010, using the Japanese Diagnosis Procedure Combination database. Main outcome measures The annual incidence of VO was estimated. Logistic regression analysis was performed to analyse factors affecting in-hospital mortality in the VO patients. Dependent variables included patient characteristics (age, sex and comorbidities), procedures (haemodialysis and surgery) and hospital factors (type of hospital and hospital volume). Results Overall, 58.9% of eligible patients were men and the average age was 69.2 years. The estimated incidence of VO increased from 5.3/100 000 population per year in 2007 to 7.4/100 000 population per year in 2010. In-hospital mortality was 6%. There was a linear trend between higher rates of in-hospital mortality and greater age. A higher rate of in-hospital mortality was significantly associated with haemodialysis use (ORs, 10.56 (95% CI 8.12 to 13.74)), diabetes (2.37 (1.89 to 2.98)), liver cirrhosis (2.63 (1.49 to 4.63)), malignancy (2.68, (2.10 to 3.42)) and infective endocarditis (3.19 (1.80 to 5.65)). Conclusions Our study demonstrates an increasing incidence of VO, and defines risk factors for death with a nationwide database. Several comorbidities were significantly associated with higher rates of in-hospital death in VO patients.

Natural Course and Prognostic Factors in Patients With Mild Cervical Spondylotic Myelopathy With Increased Signal Intensity on T2-Weighted Magnetic Resonance Imaging

Study Design. A retrospective comparative study. Objective. To investigate natural course and prognostic factors in patients with mild forms of cervical spondylotic myelopathy (CSM), focusing on intramedullary increased signal intensity (ISI) on T2-weighted magnetic resonance imaging. Summary of Background Data. Long-term natural course of mild forms of CSM, especially with ISI on magnetic resonance imaging, remains uncertain. Methods. Patients with CSM who visited our institution between 1992 and 2004 and did not undergo surgery at first visit were retrospectively reviewed. The inclusion criteria were as follows: (1) motor function Japanese Orthopedic Association scores of 3 or more in both upper and lower extremities and (2) cervical spinal cord compression with ISI on T2-weighted magnetic resonance imaging. There were 45 patients, with a mean follow-up period of 78 months (range, 24–208). We investigated long-term natural history by setting the timing of conversion to surgery due to neurological deterioration as an end point. We further compared prognostic parameters between patients who converted to surgery and those who continued to be followed up nonsurgically. Results. Sixteen patients gradually deteriorated and underwent decompression surgery, whereas 27 patients did not. Apart from these, 2 patients with acute spinal cord injury after minor trauma underwent surgery. Kaplan-Meier survival analysis revealed that 82% or 56% of patients did not require surgery 5 or 10 years after the initial treatment, respectively. As for prognostic factors, Cox proportional hazard analysis revealed that total cervical range of motion (hazard ratio: 3.25), segmental kyphosis in the maximum compression segment (hazard ratio: 4.51), and local slip (hazard ratio: 4.67) were statistically significant. Conclusion. Fifty-six percent of patients with clinically mild CSM with ISI had not deteriorated or undergone surgery at 10 years. Large range of motion, segmental kyphosis, and instability at the narrowest canal were considered to be adverse prognostic factors.

Radiographic analysis of the cervical spine in patients with retro-odontoid pseudotumors.

Study Design. A retrospective review of 10 consecutive patients with a noninflammatory retro-odontoid pseudotumor. Objective. To examine the radiographic characteristics in patients with a retro-odontoid pseudotumor and to evaluate the efficacy of posterior fusion. Summary of Background Data. A retro-odontoid pseudotumor, a reactive fibrocartilaginous mass, is known to develop after chronic atlantoaxial instability; however, one-third of the reported cases showed no overt atlantoaxial instability. The pathomechanism for such “atypical” cases remains unclear, although altered cervical motion secondary to ossification of the anterior longitudinal ligament (OALL) or severe spondylosis has been implicated. Methods. We reviewed the charts and radiographs of 10 patients with a retro-odontoid pseudotumor who underwent surgery. Preoperative radiographs were evaluated for atlas-dens interval (ADI), presence of OALL, range of motion, and segmental motion adjacent to the atlantoaxial joint. Computed tomography was evaluated for degenerative changes of zygapophysial joints. Results. There were 6 men and 4 women. Atlantoaxial instability (ADI >4 mm) was observed in 2 patients. ADI was less than 3 mm in 5 patients. Frequent association of OALL (6 patients) and marked decrease in C2 to C7 range of motion (mean, 17.6°; range, 3°–36°) were noted. Ankylosis of O-C1 was observed in 4 patients and C2 to C3 in 6. Severe degenerative change of C2 to C3 zygapophysial joint was observed in 4 patients. The patients underwent occipito-cervical fusion (9 patients) or direct removal of the pseudotumor (1 patient). Postoperative magnetic resonance imaging invariably demonstrated the mass regression. Conclusion. Retro-odontoid pseudotumors were not always associated with radiographic atlantoaxial instability. Our data indicate that extensive OALL and ankylosis of the adjacent segments are risk factors for the formation of the pseudotumor. Retro-odontoid pseudotumors may develop as an “adjacent segment disease” after altered biomechanics of the cervical spine, especially those in the adjacent segments. Posterior fusion was effective even in cases without radiographic atlantoaxial instability.

Mortality and morbidity after high-dose methylprednisolone treatment in patients with acute cervical spinal cord injury: a propensity-matched analysis using a nationwide administrative database

Objective To examine the magnitude of the adverse impact of high-dose methylprednisolone treatment in patients with acute cervical spinal cord injury (SCI). Methods We examined the abstracted data from the Japanese Diagnosis Procedure Combination database, and included patients with ICD-10 code S141 who were admitted on an emergency basis between 1 July and 31 December in 2007–2009. The investigation evaluated the patients’ sex, age, comorbidities, Japan Coma Scale, hospital volume and the amount of methylprednisolone administered. One-to-one propensity-score matching between high-dose methylprednisolone group (>5000 mg) and control group was performed to compare the rates of in-hospital death and major complications (sepsis; pneumonia; urinary tract infection; gastrointestinal ulcer/bleeding; and pulmonary embolism). Results We identified 3508 cervical SCI patients (2652 men and 856 women; mean age, 60.8±18.7 years) including 824 (23.5%) patients who received high-dose methylprednisolone. A propensity-matched analysis with 824 pairs of patients showed a significant increase in the occurrence of gastrointestinal ulcer/bleeding (68/812 vs 31/812; p<0.001) in the high-dose methylprednisolone group. Overall, the high-dose methylprednisolone group demonstrated a significantly higher risk of complications (144/812 vs 96/812;OR, 1.66; 95% CI 1.23 to 2.24; p=0.001) than the control group. There was no significant difference in in-hospital mortality between the high-dose methylprednisolone group and the control group (p=0.884). Conclusions Patients receiving high-dose methylprednisolone had a significantly increased risk of major complications, in particular, gastrointestinal ulcer/bleeding. However, high-dose methylprednisolone treatment was not associated with any increase in mortality.