Taro Funamoto

Regulation of ER molecular chaperone prevents bone loss in a murine model for osteoporosis

Endoplasmic reticulum (ER) stress response is important for protein maturation in the ER. Some murine models for bone diseases have provided significant insight into the possibility that pathogenesis of osteoporosis is related to ER stress response of osteoblasts. We examined a possible correlation between osteoporosis and ER stress response. Bone specimens from 8 osteoporosis patients and 8 disease-controls were used for immunohistochemical analysis. We found that ER molecular chaperones, such as BiP (immunoglobulin heavy-chain binding protein) and PDI (protein-disulfide isomerase) are down-regulated in osteoblasts from osteoporosis patients. Based on this result, we hypothesized that up-regulation of ER molecular chaperones in osteoblasts could restore decreased bone formation in osteoporosis. Therefore, we investigated whether treatment of murine model for osteoporosis with BIX (BiP inducer X), selective inducer BiP, could prevent bone loss. We found that oral administration of BIX effectively improves decline in bone formation through the activation of folding and secretion of bone matrix proteins. Considering these results together, BIX may be a potential therapeutic agent for the prevention of bone loss in osteoporosis patients.

Roles of the endoplasmic reticulum stress transducer OASIS in fracture healing

A new type of endoplasmic reticulum (ER) stress transducer, Old Astrocyte Specifically Induced Substance (OASIS), which is induced by bone morphogenetic protein-2 (BMP2), has been reported to activate the transcription of type I collagen and contribute to the secretion of bone matrix proteins in osteoblasts. Here, we examined the role of OASIS in fracture healing using the fracture models in wild-type (WT) and OASIS(-/-) mice. We found that the expression of OASIS mRNA was induced after fracture. Micro-computed tomography indicated that the callus density of OASIS(-/-) mice was less than that of WT mice, and the newly formed bone in OASIS(-/-) mice exhibited a decrease of the bone volume by bone morphometric analysis. Biomechanically, the callus bone strength of OASIS(-/-) mice was inferior to that of WT mice. Based on RT-PCR, in situ hybridization, immunohistochemical, and electrophoretic analyses, it was clarified that the synthesis of type I collagen was impaired in OASIS(-/-) mice. Electron microscopic analysis revealed that OASIS(-/-) osteoblasts in the fracture callus contained the abnormal expansion of the ERs, similar to OASIS(-/-) osteoblasts in the normal skeletal development. Thus, OASIS may play a role in bone formation through the expression of type I collagen and the secretion of bone matrix proteins in fracture healing.

Angiotensin II Stimulation of Cardiac Hypertrophy and Functional Decompensation in Osteoprotegerin-Deficient Mice.

Circulating and myocardial expressions of receptor activator of nuclear factor-&kgr;b ligand and osteoprotegerin are activated in heart failure; however, it remains to be determined their pathophysiological roles on left ventricular structure and function in interaction with renin–angiotensin system. We conducted experiments using 8-week-old osteoprotegerin−/− mice and receptor activator of nuclear factor-&kgr;b ligand-transgenic mice to assess whether they affect the angiotensin II–induced left ventricular remodeling. Subcutaneous infusion of angiotensin II to osteoprotegerin−/− mice progressed the eccentric hypertrophy, resulting in left ventricular systolic dysfunction for 28 days, and this was comparable with wild-type mice, showing concentric hypertrophy, irrespective of equivalent elevation of systolic blood pressure. The structural alteration was associated with reduced interstitial fibrosis, decreased procollagen &agr;1 and syndecan-1 expressions, and the increased number of apoptotic cells in the left ventricle, compared with wild-type mice. In contrast, angiotensin II infusion to the receptor activator of nuclear factor-&kgr;b ligand-transgenic mice revealed the concentric hypertrophy with preserved systolic contractile function. Intraperitoneal administration of human recombinant osteoprotegerin, but not subcutaneous injection of anti-receptor activator of nuclear factor-&kgr;b ligand antibody, to the angiotensin II–infused osteoprotegerin−/− mice for 28 days ameliorated the progression of heart failure without affecting systolic blood pressure. These results underscore the biological activity of osteoprotegerin in preserving myocardial structure and function during the angiotensin II–induced cardiac hypertrophy, independent of receptor activator of nuclear factor-&kgr;b ligand activity. In addition, the antiapoptotic and profibrotic actions of osteoprotegerin that emerged from our data might be involved in the mechanisms.

Segmental copy number loss in the region of Semaphorin 4D gene in patients with acetabular dysplasia.

Acetabular dysplasia (AD) appears to be a multi‐factorial disease, which may involve both genetic and environmental factors and whose pathogenesis remains obscure. The present study aims to identify a genetic variation that might confer risk of AD. We performed whole‐genome screening of a copy number variation (CNV) using a deCODE‐Illumina CNV beadchip with 20 female AD patients and 131 control subjects. Subsequently, Agilents region‐targeted high‐density oligonucleotide tiling microarray was used to analyze 64 female AD patients and 32 female control subjects. By sequential analyses, we found a copy number loss in 18 of 64 AD patients, but none in the 32 controls. The loss occurred within a 472 kb region on 9q22.2, which harbors the gene for Semaphorin 4D (Sema4D; 18/64 vs. 0/32, p = 4.81 × 10−4, OR = 25.86). We suggest that a copy number loss of the Sema4D gene region may play a role in the etiology of AD.

Copy number loss in the region of the ASPN gene in patients with acetabular dysplasia

Objectives We have previously investigated an association between the genome copy number variation (CNV) and acetabular dysplasia (AD). Hip osteoarthritis is associated with a genetic polymorphism in the aspartic acid repeat in the N-terminal region of the asporin (ASPN) gene; therefore, the present study aimed to investigate whether the CNV of ASPN is involved in the pathogenesis of AD. Methods Acetabular coverage of all subjects was evaluated using radiological findings (Sharp angle, centre-edge (CE) angle, acetabular roof obliquity (ARO) angle, and minimum joint space width). Genomic DNA was extracted from peripheral blood leukocytes. Agilent’s region-targeted high-density oligonucleotide tiling microarray was used to analyse 64 female AD patients and 32 female control subjects. All statistical analyses were performed using EZR software (Fisher’s exact probability test, Pearson’s correlation test, and Student’s t-test). Results CNV analysis of the ASPN gene revealed a copy number loss in significantly more AD patients (9/64) than control subjects (0/32; p = 0.0212). This loss occurred within a 60 kb region on 9q22.31, which harbours the gene for ASPN. The mean radiological parameters of these AD patients were significantly worse than those of the other subjects (Sharp angle, p = 0.0056; CE angle, p = 0.0076; ARO angle, p = 0.0065), and all nine patients required operative therapy such as total hip arthroplasty or pelvic osteotomy. Moreover, six of these nine patients had a history of operative or conservative therapy for developmental dysplasia of the hip. Conclusions Copy number loss within the region harbouring the ASPN gene on 9q22.31 is associated with severe AD. A copy number loss in the ASPN gene region may play a role in the aetiology of severe AD. Cite this article: T. Sekimoto, M. Ishii, M. Emi, S. Kurogi, T. Funamoto, Y. Yonezawa, T. Tajima, T. Sakamoto, H. Hamada, E. Chosa. Copy number loss in the region of the ASPN gene in patients with acetabular dysplasia: ASPN CNV in acetabular dysplasia. Bone Joint Res 2017;6:439–445. DOI: 10.1302/2046-3758.67.BJR-2016-0094.R1.

Possible association of single nucleotide polymorphisms in the 3' untranslated region of HOXB9 with acetabular overcoverage

Objectives Excessive acetabular coverage is the most common cause of pincer-type femoroacetabular impingement. To date, an association between acetabular over-coverage and genetic variations has not been studied. In this study we investigated the association between single nucleotide polymorphisms (SNPs) of paralogous Homeobox (HOX)9 genes and acetabular coverage in Japanese individuals to identify a possible genetic variation associated with acetabular over-coverage. Methods We investigated 19 total SNPs in the four HOX9 paralogs, then focused in detail on seven of those located in the 3’ untranslated region of HOXB9 (rs8844, rs3826541, rs3826540, rs7405887, rs2303485, rs2303486, rs79931349) using a case-control association study. The seven HOXB9 SNPs were genotyped in 316 subjects who had all undergone radiological examination. The association study was performed by both single-locus and haplotype-based analyses. Results The genotype and allele frequencies of the five HOXB9 SNPs showed significant association with acetabular over-coverage compared with controls (rs7405887 OR = 3.16, p = 5.29E-6, 95% CI 1.91 to 5.25). A significant difference was also detected when haplotypes were evaluated (OR = 2.59, p = 2.61E-5, 95% CI 1.65 to 4.08). The two HOXB9 SNPs (rs2303485, rs2303486) were associated with decreased acetabular coverage (rs2303485 OR = 0.524, p = 0.0091, 95% CI 0.322 to 0.855; rs2303486 OR = 0.519, p = 0.011, 95% CI 0.312 to 0.865). Conclusions The five HOXB9 SNPs (rs8844, rs3826541, rs3826540, rs7405887, rs79931349) were associated with acetabular over-coverage. On the other hand, the two SNPs (rs2303485 and rs2303486) were associated with the lower acetabular coverage. The association of rs2303486 would be consistent with the previous study. Therefore, the HOXB9 SNPs might be involved in the morphogenesis of acetabular coverage, and could be an independent risk factor for developing pincer-type femoroacetabular impingement. Cite this article: Bone Joint Res 2015;4:50–5.

Screening for musculoskeletal problems in Japanese schoolchildren: a cross-sectional study nested in a cohort

OBJECTIVES To clarify the frequency of musculoskeletal problems in public elementary and junior high school children and to determine the advantages and problems of musculoskeletal examinations. STUDY DESIGN School-based cross-sectional study nested in a cohort. METHODS We examined 41,376 public elementary and junior high school children (aged 6-15 years) in Miyazaki, Japan, from 2008 to 2014. Participation was voluntary. Participants received an in-school primary musculoskeletal examination (clinical examination with check items and a questionnaire) and a secondary examination at an orthopaedic outpatient clinic as indicated. Estimated prevalence rates for musculoskeletal problems were calculated from the results of both examinations. RESULTS The total estimated prevalence of musculoskeletal problems was 8.6%. Prevalence by school grade ranged from 3.2% to 13.7%. Estimated prevalence rates increased as grade increased and were higher in junior high school students than in elementary school students. The secondary examination identified musculoskeletal problems on the back (65.4%), knee (8.1%), ankle or feet (7.3%) and elbow (5.4%). Of those referred for a secondary examination, 44.4% had not reported musculoskeletal complaints on the initial questionnaire. Overall, 69.8% of problems diagnosed in the secondary examination were previously undiagnosed. CONCLUSIONS School-based musculoskeletal examination enables early detection of abnormal growth and disorders of the locomotive organs and is expected to support childrens musculoskeletal growth and development. We recommend musculoskeletal examinations as part of school check-ups in Japan. Our findings suggest musculoskeletal examinations should be conducted for students in higher elementary school grades and for all junior high school students. Evaluation should include both direct clinical examination and questionnaires.

Development of an efficient screening system to identify novel bone metabolism-related genes using the exchangeable gene trap mutagenesis mouse models.

Despite numerous genetic studies on bone metabolism, understanding of the specific mechanisms is lacking. We developed an efficient screening system to identify novel genes involved in bone metabolism using mutant mouse strains registered with the Exchangeable Gene Trap Clones (EGTC) database. From 1278 trap clones in the EGTC database, 52 candidate lines were selected in the first screening, determined based on “EST profile”, “X-gal”, “Related article”, and “Novel gene”. For the second screening, bone morphometric analysis, biomechanical strength analysis, bone X-gal staining, etc. were performed on candidate lines. Forty-two male trap lines (80.8%) showed abnormalities with either bone morphometric analysis or biomechanical strength analysis. In the screening process, X-gal staining was significantly efficient (P = 0.0057). As examples, Lbr and Nedd4 trap lines selected using the screening system showed significant bone decrease and fragility, suggesting a relationship with osteoblast differentiation. This screening system using EGTC mouse lines is extremely efficient for identifying novel genes involved in bone metabolism. The gene trap lines identified as abnormal using this screening approach are highly likely to trap important genes for bone metabolism. These selected trap mice will be valuable for use as novel bio-resources in bone research.

Results of Overhead Traction in Congenital Dislocation of the Hip

当科では先天性股関節脱臼に対しRBによる整復を試みるが，整復困難例や初診時に生後1年以上経過している例に対してはオーバーヘッドトラクション（OHT）による整復を行う．これらの保存的治療に抵抗する例に対しては手術療法を考慮する．今回当科にてOHTによる整復を行って1年以上経過した例について検討した．【対象】平成3年から平成19年までに当科外来を受診した6例7関節（全例女児）を対象とした．初診時平均月齢は1歳（5ヶ月～1歳10ヶ月），牽引開始平均年齢は1歳2ヶ月（8ヶ月～2歳）平均観察期間は8年（1年～15年）である．【結果と考察】牽引期間は平均で65日（51日～74日）であり，7関節全例に整復が得られた．しかしOHT開始が1歳以降の4例中2例に補正手術が必要であった．また，残りの2例も1例は補正手術を予定し，1例は外転装具装着中である．早期の脱臼整復が予後に関係するものと考えられた．