Osteoclasts are multinucleated cells that resorb bones, and are derived from hematopoietic cells of the monocyte/macrophage lineage. The receptor activator of NF-kappaB ligand (RANKL, also called ODF/TRANCE/OPGL) stimulates both osteoclast differentiation from osteoclast progenitors and activation of mature osteoclasts. To identify genes responsible for osteoclast differentiation, we used a molecular indexing technique. Here, we report a clone of one of these genes whose transcription is induced by soluble RANKL (sRANKL) in both the RAW264.7 cells of the mouse macrophage cell line and the mouse primary bone marrow cells. The predicted protein was found to be a mouse homologue of Jun dimerization protein 2 (JDP2), a member of the AP-1 family of transcription factors, containing a basic region-leucine zipper motif. Transient transfection experiments revealed that overexpression of JDP2 leads to activation of both tartrate-resistant acid phosphatase (TRAP) and cathepsin K gene promoters in RAW264.7 cells. Infection of mouse primary bone marrow cells with retroviruses expressing JDP2-facilitated sRANKL-mediated formation of TRAP-positive multinuclear osteoclasts. Importantly, antisense oligonucleotide to JDP2 strongly suppressed sRANKL-induced osteoclast formation of RAW264.7 cells. Our findings suggest that JDP2 may play an important role in the RANK-mediated signal transduction system, especially in osteoclast differentiation.

Myelin-associated glycoprotein (MAG) has been detected in Schwann cells prior to the onset of myelination, suggesting its functions in the initiation of myelination. However, transcriptional regulatory mechanisms of MAG remain to be elucidated. Here, we analyzed the promoter of the MAG gene by using luciferase reporter systems in the primary rat Schwann cells. We identified a novel cis-acting element located 160 bp upstream from the MAG transcription initiation site. Using the identified cis-element as a bait, we performed yeast one-hybrid screening and isolated a cDNA encoding a RNF10 as a putative trans-acting protein. When overexpressed in Schwann cells, RNF10 enhanced the activity of the MAG promoter. When RNF10 expression in Schwann cells was knocked down by siRNA, endogenous MAG mRNA and protein expression decreased. Furthermore, we evaluated myelin synthesis using Schwann cell-DRG neuron cocultures. When Schwann cells were infected with retrovirus expressing RNF10 siRNA, myelin formation was inhibited. These data suggest that RNF10 regulates MAG expression and is required for myelin formation.

Rheumatoid arthritis (RA) is an autoimmune destructive arthritis associated with CD4(+) T cell-mediated immunity. Although expanded CD4(+) T cell clones (ECs) has already been confirmed, the detailed characteristics of ECs have not been elucidated in RA. Using combination of a single-cell analysis and next-generation sequencing (NGS) in TCR repertoire analysis, we here revealed the detailed nature of ECs by examining peripheral blood (PB) from 5 RA patients and synovium from 1 RA patient. When we intensively investigated the single-cell transcriptome of the most expanded clones in memory CD4(+) T cells (memory-mECs) in RA-PB, senescence-related transcripts were up-regulated, indicating circulating ECs were constantly stimulated. Tracking of the transcriptome shift within the same memory-mECs between PB and the synovium revealed the augmentations in senescence-related gene expression and the up-regulation of synovium-homing chemokine receptors in the synovium. Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs. Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes. Our approach may provide new insights into the pathophysiology of RA.

This study aimed to evaluate the validity and reliability of the Japanese version of the Neuropathic Pain Symptom Inventory (NPSI-J).

The purpose of this study was to evaluate the relations between the degree of encroachment, measured as the cross-sectional area of the dural sac, and low back pain in a large population.

Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.

Osteocyte survival is key to bone homeostasis and is perturbed in menopause and aging. However, it remains unknown how osteocyte-mediated maintenance of the skeleton is regulated by the osteoprotective factor semaphorin 3A (Sema3A), a secreted protein that is known to reduce bone resorption and enhance bone formation. Here, we show that estrogen induces osteocyte expression of Sema3A, which acts on its receptor on osteocytes to promote their survival and maintain bone homeostasis. Postnatal global and conditional deletion of Sema3a in osteoblastic cells resulted in a severe osteoporotic phenotype marked by fewer osteocytes. This phenotype was recapitulated by osteocyte-specific deficiency of either Sema3A or its receptor component neuropilin-1 (Nrp1). A stimulator of soluble guanylate cyclase-cGMP signaling mimicked Sema3A action and ameliorated bone loss after ovariectomy. We further show that serum levels of SEMA3A decreased with age or after menopause in humans. Thus, we provide a mechanistic insight into the estrogen action and a promising therapeutic approach to protect against bone-related aging.

The patient-rated Core Outcome Measures Index (COMI) assesses the multidimensional impact of back problems on the sufferer. The brevity and comprehensibility of the tool make it practical for use in clinical and research settings. Although the COMI has been cross-culturally adapted in various languages worldwide, there is currently no Japanese version. The aim of this study was to develop a Japanese version of the COMI by: (1) performing a cross-cultural adaptation of the English version and (2) evaluating the psychometric properties of the Japanese version of the COMI in Japanese volunteers with chronic back problems.

Trimethylation of histone H3 lysine 4 and lysine 27 (H3K4me3 and H3K27me3) at gene promoter regions critically regulates gene expression. Key developmental genes tend to exhibit changes in histone modification patterns from the H3K4me3/H3K27me3 bivalent pattern to the H3K4me3 monovalent pattern. Using comprehensive chromatin immunoprecipitation followed by sequencing in bone marrow-derived macrophages (BMMs) and mature osteoclasts, we found that cell surface adhesion molecule 1 (Cadm1) is a direct target of nuclear factor of activated T cells 1 (NFATc1) and exhibits a bivalent histone pattern in BMMs and a monovalent pattern in osteoclasts. Cadm1 expression was upregulated in BMMs by receptor activator of nuclear factor kappa B ligand (RANKL), and blocked by a calcineurin/NFATc1 inhibitor, FK506. Cadm1-deficient mice exhibited significantly reduced bone mass compared with wild-type mice, which was due to the increased osteoclast differentiation, survival and bone-resorbing activity in Cadm1-deficient osteoclasts. These results suggest that Cadm1 is a direct target of NFATc1, which is induced by RANKL through epigenetic modification, and regulates osteoclastic bone resorption in a negative feedback manner.

Hypertrophic peroneal tubercle (HPT) is an overgrowth of the peroneal tubercle located on the lateral aspect of the hindfoot, which could cause tenosynovitis of the peroneus longus tendon. Os peroneum (OP) is an accessory ossicle that exists in the peroneus longus tendon at the lateral aspect of the calcaneocuboid joint. Both HPT and OP can cause lateral foot pain and occasionally require surgical treatment. We encountered a case of lateral foot pain of HPT coexisting with OP. Careful preoperative magnetic resonance imaging, dynamic ultrasonographic image, and block injection suggested an impingement of HPT and OP as a cause of lateral foot pain. Surgical resection of HPT, while retaining OP, successfully achieved pain relief in the patient. To the best of our knowledge, this is the first report presenting a case of HPT coexisting with OP successfully treated without OP resection.

Numerous nontruncating missense variants of the BRCA2 gene have been identified, but there is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance and they thus remain unactionable. To assess the pathogenicity of variants of unknown significance (VUSs) within BRCA2, here we develop a method, the MANO-B method, for high-throughput functional evaluation utilizing BRCA2-deficient cells and poly (ADP-ribose) polymerase (PARP) inhibitors. The estimated sensitivity and specificity of this assay compared to those of the International Agency for Research on Cancer classification system is 95% and 95% (95% confidence intervals: 77-100% and 82-99%), respectively. We classify the functional impact of 186 BRCA2 VUSs with our computational pipeline, resulting in the classification of 126 variants as normal/likely normal, 23 as intermediate, and 37 as abnormal/likely abnormal. We further describe a simplified, on-demand annotation system that could be used as a companion diagnostic for PARP inhibitors in patients with unknown BRCA2 VUSs.

HIF-1α, an essential transcription factor under hypoxic condition, is indispensable for chondrocytes during skeletal development but its expression and roles in articular chondrocytes are yet to be revealed. We examined HIF-1α protein expression and the hypoxic condition during mouse osteoarthritis (OA) development using state of the art hypoxic probes and found that its expression decreased as OA progressed, coinciding with the change in hypoxic conditions in articular cartilage. Gain- and loss-of-function of HIF-1α in cell culture experiments showed that HIF-1α suppressed catabolic genes such as Mmp13 and Hif2a. We confirmed these anticatabolic effects by measuring glycosaminoglycan release from wild type and conditional knock-out mice femoral heads cultured ex vivo. We went on to surgically induce OA in mice with chondrocyte-specific deletion of Hif1a and found that the development of OA was exacerbated. Increased expression of catabolic factors and activation of NF-κB signalling was clearly evident in the knock-out mice. By microarray analysis, C1qtnf3 was identified as a downstream molecule of HIF-1α, and experiments showed it exerted anti-catabolic effects through suppression of NF-κB. We conclude that HIF-1α has an anti-catabolic function in the maintenance of articular cartilage through suppression of NF-κB signalling.

Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

Studies have suggested a relationship between sagittal spinal malalignment and low back pain (LBP). The current study investigated the relationship of spinal alignment with LBP and physical performance in 1491 individuals who attended the second follow-up visit of the Wakayama Spine Study. The sagittal vertical axis at C7 (C7 SVA) was measured by a spine surgeon. The occurrence of LBP within one month, pain intensity, Oswestry Disability Index (ODI), and physical performance (grip strength, 6-m walking time, chair stand test, one-leg standing test) were also evaluated. LBP in the previous month was determined using ODI, and indicators of physical performance were measured. The mean C7 SVA was 11.0 ± 42.7 mm and was significantly greater in older participants (p < 0.001). LBP was more prevalent in participants with a greater C7 SVA (< 40 mm, 35.7%; 40-95 mm, 47.3%; ≥ 95 mm, 59.4%; p < 0.001) and those with a higher ODI score (10.0%, 17.5%, and 29.4%, respectively; p < 0.001). Physical performance significantly decreased in participants with a greater C7 SVA (p < 0.001). Multiple linear regression analysis revealed that LBP and physical performance were significantly associated with C7 SVA (p < 0.001). Thus, sagittal spinal malalignment may lead to LBP and decreased physical performance.

The clinical sequencing of tumors is usually performed on formalin-fixed, paraffin-embedded samples and results in many sequencing errors. We identified that most of these errors are detected in chimeric reads caused by single-strand DNA molecules with microhomology. During the end-repair step of library preparation, mutations are introduced by the mis-annealing of two single-strand DNA molecules comprising homologous sequences. The mutated bases are distributed unevenly near the ends in the individual reads. Our filtering pipeline, MicroSEC, focuses on the uneven distribution of mutations in each read and removes the sequencing errors in formalin-fixed, paraffin-embedded samples without over-eliminating the mutations detected also in fresh frozen samples. Amplicon-based sequencing using 97 mutations confirmed that the sensitivity and specificity of MicroSEC were 97% (95% confidence interval: 82-100%) and 96% (95% confidence interval: 88-99%), respectively. Our pipeline will increase the reliability of the clinical sequencing and advance the cancer research using formalin-fixed, paraffin-embedded samples.

Cell therapy using adipose-derived mesenchymal stem cells (ASCs) is a promising avenue of regenerative medicine for the treatment of various diseases. It has been considered that ASCs exert their therapeutic effects through the secretion of multiple factors that are critical for tissue remodeling or the suppression of inflammation. Recently, conditioned medium (CM) from ASCs that contains a complex of secreted factors has received attention as a cost-effective alternative to cell therapy.

The impact of body mass index (BMI) on outcomes after lumbar spine surgery is currently unknown. Previous studies have reported conflicting evidence for patients with high BMI, while little research has been conducted on outcomes for underweight patients. This study aims to examine the impact of BMI on outcomes after lumbar spine surgery. This prospective cohort study enrolled 5622 patients; of which, 194, 5027, and 401 were in the low (< 18.5 kg/m2), normal (18.5-30), and high (≥ 30) BMI groups, respectively. Pain was assessed via the numerical pain rating scale (NPRS) for the lower back, buttock, leg, and plantar area. Quality of life was assessed via the EuroQol 5 Dimension (EQ-5D) and Oswestry Disability Index (ODI). Inverse probability weighting with propensity scores was used to adjust patient demographics and clinical characteristics between the groups. After adjustment, the 1-year postoperative scores differed significantly between groups in terms of leg pain. The proportion of patients who achieved a 50% decrease in postoperative NPRS score for leg pain was also significantly different. Obese patients reported less improvement in leg pain after lumbar spine surgery. The outcomes of patients with low BMI were not inferior to those of patients with normal BMI.

Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5'RACE analysis, and identified the 46-bp core enhancer region (CES6). We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.

TGF-ß is a multifunctional cytokine that is involved in cell proliferation, differentiation and function. We previously reported an essential role of the TGF-ß -Smad2/3 pathways in RANKL-induced osteoclastogenesis. Using chromatin immunoprecipitation followed by sequencing, we comprehensively identified Smad2/3 target genes in bone marrow macrophages. These genes were enriched in the gene population upregulated by TGF-ß and downregulated by RANKL. Recent studies have revealed that histone modifications, such as trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), critically regulate key developmental steps. We identified Nedd9 as a Smad2/3 target gene whose histone modification pattern was converted from H3K4me3(+)/H3K4me27(+) to H3K4me3(+)/H3K4me27(-) by TGF-ß. Nedd9 expression was increased by TGF-ß and suppressed by RANKL. Overexpression of Nedd9 partially rescued an inhibitory effect of a TGF-ß inhibitor, while gene silencing of Nedd9 suppressed RANKL-induced osteoclastogenesis. RANKL-induced osteoclastogenesis were reduced and stimulatory effects of TGF-ß on RANKL-induced osteoclastogenesis were partially abrogated in cells from Nedd9-deficient mice although knockout mice did not show abnormal skeletal phenotypes. These results suggest that Nedd9 is a Smad2/3 target gene implicated in RANKL-induced osteoclastogenesis.

Osteoclast differentiation is a multi-step process that involves cell proliferation, commitment, and fusion. Some adhesion molecules, including integrin alphavbeta3, have been shown to have roles in osteoclast fusion. In the course of studying with pharmacologic agents known to inhibit protein tyrosine kinases of the Src family, we found that radicicol increased cell fusion during receptor activator of nuclear factor kappaB ligand (RANKL)-driven differentiation of osteoclasts at concentrations far below the ones shown to inhibit its targets in previous studies. Treatments of low doses of radicicol to RAW 264.7 cells that undergo osteoclastic differentiation in the presence of RANKL enhanced the RANKL-induced gene expression of integrin beta3 without any effect on the expression of integrin alphav, which was constitutively high. The cell surface level of integrin alphavbeta3 complexes was consequently augmented by radicicol. In addition, sustained ERK and MEK activation was observed in cells treated with both radicicol and RANKL. More importantly, modulation of ERK activity by the MEK inhibitor U0126 or the gene transduction of a constitutively active form of MEK resulted in a suppression and increment, respectively, of integrin beta3 induction by RANKL. Our data indicate that sustained ERK activity is associated with integrin beta3 induction and subsequent cell surface expression of the alphavbeta3 integrin complex, which may contribute to cell fusion during RANKL-directed osteoclastogenesis.