Yasushi Oshima

Negative Regulation of Osteoclastogenesis by Ectodomain Shedding of Receptor Activator of NF-κB Ligand

Receptor activator of NF-κB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.

Akt1 in osteoblasts and osteoclasts controls bone remodeling.

Bone mass and turnover are maintained by the coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts, under regulation of many systemic and local factors. Phosphoinositide-dependent serine-threonine protein kinase Akt is one of the key players in the signaling of potent bone anabolic factors. This study initially showed that the disruption of Akt1, a major Akt in osteoblasts and osteoclasts, in mice led to low-turnover osteopenia through dysfunctions of both cells. Ex vivo cell culture analyses revealed that the osteoblast dysfunction was traced to the increased susceptibility to the mitochondria-dependent apoptosis and the decreased transcriptional activity of runt-related transcription factor 2 (Runx2), a master regulator of osteoblast differentiation. Notably, our findings revealed a novel role of Akt1/forkhead box class O (FoxO) 3a/Bim axis in the apoptosis of osteoblasts: Akt1 phosphorylates the transcription factor FoxO3a to prevent its nuclear localization, leading to impaired transactivation of its target gene Bim which was also shown to be a potent proapoptotic molecule in osteoblasts. The osteoclast dysfunction was attributed to the cell autonomous defects of differentiation and survival in osteoclasts and the decreased expression of receptor activator of nuclear factor-κB ligand (RANKL), a major determinant of osteoclastogenesis, in osteoblasts. Akt1 was established as a crucial regulator of osteoblasts and osteoclasts by promoting their differentiation and survival to maintain bone mass and turnover. The molecular network found in this study will provide a basis for rational therapeutic targets for bone disorders.

Regulation of RANKL-induced osteoclastogenesis by TGF-β through molecular interaction between Smad3 and Traf6.

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling.

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.

Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type x collagen gene

OBJECTIVE Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells. METHODS Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation. RESULTS RUNX-2 and type X collagen were co-localized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor beta in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between -89 and -60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells. CONCLUSION Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10A1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA.

Anti-apoptotic Molecule Bcl-2 Regulates the Differentiation, Activation, and Survival of Both Osteoblasts and Osteoclasts

The anti-apoptotic molecule Bcl-2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl-2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Bcl-2−/− mice are growth-retarded and exhibit increased bone volume of the primary spongiosa, mainly due to the decreased number and dysfunction of osteoclasts. Osteoblast function is also impaired in Bcl-2−/− mice. Ex vivo studies on osteoblasts and osteoclasts showed that Bcl-2 promoted the differentiation, activation, and survival of both cell types. Because Bcl-2−/− mice die before 6 weeks of age due to renal failure and cannot be compared with adult wild type mice, we generated Bcl-2−/−Bim+/− mice, in which a single Bim allele was inactivated, and compared them with their Bcl-2+/−Bim+/− littermates. Loss of a single Bim allele restored normal osteoclast function in Bcl-2−/− mice but did not restore the impaired function of osteoblasts, and the mice exhibited osteopenia. These data demonstrate that Bcl-2 promotes the differentiation, activity, and survival of both osteoblasts and osteoclasts. The balance between Bcl-2 and Bim regulates osteoclast apoptosis and function, whereas other pro-apoptotic members are important for osteoblasts.

Validity and Reliability of the Japanese Version of the painDETECT Questionnaire: A Multicenter Observational Study

Objectives The aim of this study was to evaluate the validity and reliability of the Japanese version of the painDETECT questionnaire (PDQ-J). Materials and Methods The translation of the original PDQ into Japanese was achieved according to the published guidelines. Subsequently, a multicenter observational study was performed to evaluate the validity and reliability of PDQ-J, including 113 Japanese patients suffering from pain. Results Factor analysis revealed that the main component of PDQ-J comprises two determinative factors, which account for 62% of the variance observed. Moreover, PDQ-J revealed statistically significant correlation with the intensity of pain (Numerical Rating Scale), Physical Component Score, and Mental Component Score of the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). The Cronbach alpha for the total score was 0.78 and for the main component was 0.80. In the analysis of test–retest method, the intraclass correlation coefficient between the two scores was 0.94. Conclusions We demonstrated the validity and reliability of PDQ-J. We encourage researchers and clinicians to use this tool for the assessment of patients who suffer suspected neuropathic pain.

Comparison of the Japanese Orthopaedic Association (JOA) Score and Modified JOA (mJOA) Score for the Assessment of Cervical Myelopathy: A Multicenter Observational Study

Objectives The Japanese Orthopaedic Association (JOA) score is widely used to assess the severity of clinical symptoms in patients with cervical compressive myelopathy, particularly in East Asian countries. In contrast, modified versions of the JOA score are currently accepted as the standard tool for assessment in Western countries. The objective of the present study is to compare these scales and clarify their differences and interchangeability and verify their validity by comparing them to other outcome measures. Materials and Methods Five institutions participated in this prospective multicenter observational study. The JOA and modified JOA (mJOA) proposed by Benzel were recorded preoperatively and at three months postoperatively in patients with cervical compressive myelopathy who underwent decompression surgery. Patient reported outcome (PRO) measures, including Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ), the Short Form-12 (SF-12) and the Neck Disability Index (NDI), were also recorded. The preoperative JOA score and mJOA score were compared to each other and the PRO values. A Bland-Altman analysis was performed to investigate their limits of agreement. Results A total of ninety-two patients were included. The correlation coefficient (Spearman’s rho) between the JOA and mJOA was 0.87. In contrast, the correlations between JOA/mJOA and the other PRO values were moderate (|rho| = 0.03 – 0.51). The correlation coefficient of the recovery rate between the JOA and mJOA was 0.75. The Bland-Altman analyses showed that limits of agreement were 3.6 to -1.2 for the total score, and 55.1% to -68.8% for the recovery rates. Conclusions In the present study, the JOA score and the mJOA score showed good correlation with each other in terms of their total scores and recovery rates. Previous studies using the JOA can be interpreted based on the mJOA; however it is not ideal to use them interchangeably. The validity of both scores was demonstrated by comparing these values to the PRO values.

Negative feedback loop in the Bim-caspase-3 axis regulating apoptosis and activity of osteoclasts.

Proapoptotic Bcl‐2 family member Bim plays an essential role in the osteoclast apoptosis and is degraded through ubiquitin/proteasome pathways in a caspase‐3–dependent manner. This negative feedback loop in the Bim–caspase‐3 axis is important for regulating the survival and activity of osteoclasts.

Pivotal Role of Bcl-2 Family Proteins in the Regulation of Chondrocyte Apoptosis

During endochondral ossification, chondrocytes undergo hypertrophic differentiation and die by apoptosis. The level of inorganic phosphate (Pi) elevates at the site of cartilage mineralization, and when chondrocytes were treated with Pi, they underwent rapid apoptosis. Gene silencing of the proapoptotic Bcl-2 homology 3-only molecule bnip3 significantly suppressed Pi-induced apoptosis. Conversely, overexpression of Bcl-xL suppressed, and its knockdown promoted, the apoptosis of chondrocytes. Bnip3 was associated with Bcl-xL in chondrocytes stimulated with Pi. Bcl-xL was expressed uniformly in the growth plate chondrocytes, whereas Bnip3 expression was exclusively localized in the hypertrophic chondrocytes. Finally, we generated chondrocyte-specific bcl-x knock-out mice using the Cre-loxP recombination system, and we provided evidence that the hypertrophic chondrocyte layer was shortened in those mice because of an increased apoptosis of prehypertrophic and hypertrophic chondrocytes, with the mice afflicted with dwarfism as a result. These results suggest the pivotal role of Bcl-2 family members in the regulation of chondrocyte apoptosis.