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Two cases of focal atrial tachycardia probably originating from the pulmonary vein with onset later than 3 years of age are presented. Both cases had associated variable atrioventricular conduction and showed no signs of heart failure, and they converted to sinus rhythm at the time of puberty. In cases of focal atrial tachycardia originating from the pulmonary vein with onset later than 3 years of age, drug therapy may be effective. Even if drug therapy is not effective, changes in the autonomic nervous system are reflected strongly in the pulmonary veins, so that changes in autonomic nervous system regulation with growth might terminate focal atrial tachycardia. Therefore, focal atrial tachycardia originating from the pulmonary vein with onset later than 3 years of age might have a better prognosis.

Background Bone morphogenetic proteins (BMPs) induce osteogenesis in various environments. However, when BMPs are used alone in the bone marrow environment, the maintenance of new bone formation is difficult owing to vigorous bone resorption. This is because BMPs stimulate the differentiation of not only osteoblast precursor cells but also osteoclast precursor cells. The present study aimed to induce and maintain new bone formation using the topical co-administration of recombinant human BMP-2 (rh-BMP-2) and zoledronate (ZOL) on beta-tricalcium phosphate (β-TCP) composite. Methods β-TCP columns were impregnated with both rh-BMP-2 (30 µg) and ZOL (5 µg), rh-BMP-2 alone, or ZOL alone, and implanted into the left femur canal of New Zealand white rabbits (n = 56). The implanted β-TCP columns were harvested and evaluated at 3 and 6 weeks after implantation. These harvested β-TCP columns were evaluated radiologically using plane radiograph, and histologically using haematoxylin/eosin (H&E) and Masson’s trichrome (MT) staining. In addition, micro-computed tomography (CT) was performed for qualitative analysis of bone formation in each group (n = 7). Results Tissue sections stained with H&E and MT dyes revealed that new bone formation inside the β-TCP composite was significantly greater in those impregnated with both rh-BMP-2 and ZOL than in those from the other experimental groups at 3 and 6 weeks after implantations (p p Conclusions The topical co-administration of both rh-BMP-2 and ZOL on β-TCP composite promoted and maintained newly formed bone structure in the bone marrow environment.

The development of high-throughput genotyping technologies and large biobank collections, complemented with rapid methodological advances in statistical genetics, has enabled hypothesis-free genome-wide association studies (GWAS), which have identified hundreds of genetic variants across many loci associated with musculoskeletal conditions. Similarly, basic scientists have valuable molecular cellular and animal data based on musculoskeletal disease that would be enhanced by being able to determine the human translation of their findings. By integrating these large-scale human genomic musculoskeletal datasets with complementary evidence from model organisms, new and existing genetic loci can be statistically fine-mapped to plausibly causal variants, candidate genes, and biological pathways. Genes and pathways identified using this approach can be further prioritized as drug targets, including side-effect profiling and the potential for new indications. To bring together these big data, and to realize the vision of creating a knowledge portal, the International Federation of Musculoskeletal Research Societies (IFMRS) established a working group to collaborate with scientists from the Broad Institute to create the Musculoskeletal Knowledge Portal (MSK-KP)(http://mskkp.org/). The MSK consolidates omics datasets from humans, cellular experiments, and model organisms into a central repository that can be accessed by researchers. The vision of the MSK-KP is to enable better understanding of the biological mechanisms underlying musculoskeletal disease and apply this knowledge to identify and develop new disease interventions. © 2020 American Society for Bone and Mineral Research (ASBMR).

The prognosis of patients with progressive prostate cancers that are hormone refractory and/or have bone metastasis is poor. Multiple therapeutic targets to improve prostate cancer patient survival have been investigated, including orphan GPCRs. In our study, we identified G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5A) as a candidate therapeutic molecule using integrative gene expression analyses of registered data sets for prostate cancer cell lines. Kaplan-Meier analysis of TCGA data sets revealed that patients who have high GPRC5A expression had significantly shorter overall survival. PC3 prostate cancer cells with CRISPR/Cas9-mediated GPRC5A knockout exhibited significantly reduced cell proliferation both in vitro and in vivo. RNA-seq revealed that GPRC5A KO PC3 cells had dysregulated expression of cell cycle-related genes, leading to cell cycle arrest at the G2/M phase. Furthermore, the registered gene expression profile data set showed that the expression level of GPRC5A in original lesions of prostate cancer patients with bone metastasis was higher than that without bone metastasis. In fact, GPRC5A KO PC3 cells failed to establish bone metastasis in xenograft mice models. In addition, our clinical study revealed that GPRC5A expression levels in prostate cancer patient samples were significantly correlated with bone metastasis as well as the patient's Gleason score (GS). Combined assessment with the immunoreactivity of GPRC5A and GS displayed higher specificity for predicting the occurrence of bone metastasis. Together, our findings indicate that GPRC5A can be a possible therapeutic target and prognostic marker molecule for progressive prostate cancer.

C–C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Epidemiological findings suggest that the functional loss of CCR5 is correlated with a lower incidence of bone-destructive diseases as well as of HIV transmission. However, it is not clear whether CCR5 is involved in regulation of the function of bone cells, in addition to that of immune cells. Here we show that blockade of CCR5 using specific antibodies impairs human osteoclast function in vitro. Ccr5-deficient (Ccr5 −/−) mice presented with dysfunctional osteoclasts and were resistant to osteoporosis induced by receptor activator of nuclear factor kappa-B ligand (RANKL), which triggers osteoporosis independently of inflammatory and immunomodulatory pathways. Furthermore, Ccr5 deficiency impairs the cellular locomotion and bone-resorption activity of osteoclasts, which is associated with the disarrangement of podosomes and adhesion complex molecules including Pyk2. Overall, the data provides evidence that CCR5 has an essential role in bone-destructive conditions through the functional regulation of osteoclasts., CCR5 is a co-receptor for HIV, and loss of function is associated with lower incidence of HIV but also with bone-destructive diseases. Here the authors show that ablation of CCR5 impairs osteoclast function and improves resistance to osteoporosis in mouse models.

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth–promoting NF-κB pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free–based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48–linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLD(P904L) variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

Idiopathic osteonecrosis of the femoral head (IONFH) is an ischemic disorder that causes bone necrosis of the femoral head, resulting in hip joint dysfunction. IONFH is a polygenic disease and steroid and alcohol have already known to increase its risk; however, the mechanism of IONFH remains to be elucidated. We performed a genome-wide association study using ~60,000 subjects and found two novel loci on chromosome 20q12 and 12q24. Big data analyses identified LINC01370 as a candidate susceptibility gene in the 20q12 locus. Stratified analysis by IONFH risk factors suggested that the 12q24 locus was associated with IONFH through drinking capacity. Our findings would shed new light on pathophysiology of IONFH.

Osteoporosis, which patients are estimated as more than 13 million in Japan, is mainly caused by postmenopausal osteoporosis. Estrogen deficiency induced by menopause can disturb endocrine feedback and homeostasis, followed by bone loss by increased bone resorption with high bone turnover. Recent studies using systemic or conditional Estrogen Receptor α(ERα)gene knockout mice have unveiled molecular mechanisms underlying bone metabolism. In this review, it will be discussed that direct and indirect effects of estrogen and its metabolites for bone metabolism.

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.

The longitudinal growth of long bone, regulated by an epiphyseal cartilaginous component known as the “growth plate”, is generated by epiphyseal chondrocytes. The growth plate provides a continuous supply of chondrocytes for endochondral ossification, a sequential bone replacement of cartilaginous tissue, and any failure in this process causes a wide range of skeletal disorders. Therefore, the cellular and molecular characteristics of the growth plate are of interest to many researchers. Hedgehog (Hh), well known as a mitogen and morphogen during development, is one of the best known regulatory signals in the developmental regulation of the growth plate. Numerous animal studies have revealed that signaling through the Hh pathway plays multiple roles in regulating the proliferation, differentiation, and maintenance of growth plate chondrocytes throughout the skeletal growth period. Furthermore, over the past few years, a growing body of evidence has emerged demonstrating that a limited number of growth plate chondrocytes transdifferentiate directly into the full osteogenic and multiple mesenchymal lineages during postnatal bone development and reside in the bone marrow until late adulthood. Current studies with the genetic fate mapping approach have shown that the commitment of growth plate chondrocytes into the skeletal lineage occurs under the influence of epiphyseal chondrocyte-derived Hh signals during endochondral bone formation. Here, we discuss the valuable observations on the role of the Hh signaling pathway in the growth plate based on mouse genetic studies, with some emphasis on recent advances.

PHF2 is a JmjC family histone demethylase that removes the methyl group from H3K9me2 and works as a coactivator for several metabolism-related transcription factors. In this study, we examined the in vivo role of PHF2 in mice. We generated Phf2 floxed mice, systemic Phf2 null mice by crossing Phf2 floxed mice with CMV-Cre transgenic mice, and tamoxifen-inducible Phf2 knockout mice by crossing Phf2 floxed mice with Cre-ERT2 transgenic mice. Systemic Phf2 null mice had partial neonatal death and growth retardation and exhibited less adipose tissue and reduced adipocyte numbers compared with control littermates. Tamoxifen-induced conditional knockout of PHF2 resulted in impaired adipogenesis in stromal vascular cells from the adipose tissue of tamoxifen-inducible Phf2 knockout mice as well as of Phf2 knocked-down 3T3-L1 cells. PHF2 interacts with CEBPA and demethylates H3K9me2 in the promoters of CEBPA-regulated adipogenic genes. These findings suggest that PHF2 histone demethylase potentiates adipogenesis through interaction with CEBPA in vivo. Taken together, PHF2 may be a novel therapeutic target in the treatment of obesity and the metabolic syndrome.

Epigenetic regulation underlying AR(Androgen receptor)mediated transcription is important component to understand pathophysiology of osteoporosis in men. In this commentary, it is reported recent findings related to epigenetic landscape governed by AR and its cofactors including lysine-specific demethylase 1 (LSD1), and possible implication for bone metabolism.

Next generation sequencer has strongly progress big data analyses in life science. Among various kinds of sequencing data sets, epigenetic platform has just been important key to clarify the questions on broad and detail phenomenon in various forms of life. In this report, it is introduced that the research on identification of novel transcription factors in osteoclastogenesis using DNase-seq. Big data on musculoskeletal research will be organized by IFMRS and is getting more crucial.

Osteoclastogenesis is a highly regulated process governed by diverse classes of regulators. Among them, nuclear factor of activated T-cells calcineurin-dependent 1 (NFATc1) is the primary osteoclastogenic transcription factor, and its expression is transcriptionally induced during early osteoclastogenesis by receptor activation of nuclear factor κB ligand (RANKL), an osteoclastogenic cytokine. Here, we report the novel enzymatic function of JMJD5, which regulates NFATc1 protein stability. Among the tested Jumonji C (JmjC) domain-containing proteins, decreased mRNA expression levels during osteoclastogenesis were found for JMJD5 in RAW264 cells stimulated by RANKL. To examine the functional role of JMJD5 in osteoclast differentiation, we established stable JMJD5 knockdown cells, and osteoclast formation was assessed. Down-regulated expression of JMJD5 led to accelerated osteoclast formation together with induction of several osteoclast-specific genes such as Ctsk and DC-STAMP, suggesting that JMJD5 is a negative regulator in osteoclast differentiation. Although JMJD5 was recently reported as a histone demethylase for histone H3K36me2, no histone demethylase activity was detected in JMJD5 in vitro or in living cells, even for other methylated histone residues. Instead, JMJD5 co-repressed transcriptional activity by destabilizing NFATc1 protein. Protein hydroxylase activity mediated by the JmjC domain in JMJD5 was required for the observed functions of JMJD5. JMJD5 induced the association of hydroxylated NFATc1 with the E3 ubiquitin ligase Von Hippel-Lindau tumor suppressor (VHL), thereby presumably facilitating proteasomal degradation of NFATc1 via ubiquitination. Taken together, the present study demonstrated that JMJD5 is a post-translational co-repressor for NFATc1 that attenuates osteoclastogenesis.

Summary Endocrine therapies for breast cancer that target the estrogen receptor (ER) are ineffective in the 25%–30% of cases that are ER negative (ER–). Androgen receptor (AR) is expressed in 60%–70% of breast tumors, independent of ER status. How androgens and AR regulate breast cancer growth remains largely unknown. We find that AR is enriched in ER– breast tumors that overexpress HER2. Through analysis of the AR cistrome and androgen-regulated gene expression in ER–/HER2+ breast cancers we find that AR mediates ligand-dependent activation of Wnt and HER2 signaling pathways through direct transcriptional induction of WNT7B and HER3. Specific targeting of AR, Wnt or HER2 signaling impairs androgen-stimulated tumor cell growth suggesting potential therapeutic approaches for ER–/HER2+ breast cancers.

What makes treatment choice for developmental dysplasia of the hips diagnosed after walking age difficult is the poor understanding of prereduction conditions that obstruct the reduction in spatial terms. To evaluate these problems, we employed subtraction three-dimensional imaging to search for the factors involved in intraarticular obstruction. On the basis of the findings of preoperative subtraction threedimensional imaging from computed tomography, we developed a new method, a minimum invasive arthroscopic reduction with limboplasty, for reduction of developmental dysplasia of the hips after walking age. The purposes of this report were to: (1) describe the technique of the arthroscopic procedure, and (2) evaluate our new method using radiographic parameters.Ten patients with ten hips with developmental dysplasia after walking age treated by arthroscopic reduction with limboplasty were included in this study. The mean age of the patients at reduction was 22.6 months (range, 18.6-29.7 months); mean age at follow up was 7.2 years (range, 3.9-10.9 years); and mean follow up was 5.4 years (range, 1.7-9.0 years). These ten hips were evaluated using radiographic measurements.Moderate or severe avascular necrosis of the femoral head was not observed. Two hips that had a spherical-shaped head with minimal residual height loss or coxa magna were classified as Kalamchi and MacEwen grade 1. Additional surgery had been performed for two hips classified as Severin group 4 during the course of follow up. These two hips were classified as Severin group 1 at final examination. One more hip was classified as Severin group 4 at final examination, and additional surgery was recommended. The remaining seven hips (70%) therefore obtained good evaluations by arthroscopic reduction with limboplasty alone.We developed a new reduction method by using an arthroscopic procedure for the reduction of developmental dysplasia of the hips after walking age when this dysplasia failed to be reduced with nonoperative methods. The result of our new method is acceptable because good evaluations were obtained in 70% of hips 5.4 years after reduction by our new method alone.

One problem associated with clinical application of CHO-derived recombinant human bone morphogenetic protein (C-BMP-2) is its high cost due to the need for use of high doses. To solve this problem, Escherichia coli-derived BMP-2 (E-BMP-2) has been examined using the technique of molecular unfolding and refolding. However, it is unclear whether the characteristics of E-BMP-2 are appropriate for clinical application. In this study, we examined the biological activity of E-BMP-2 and its heat tolerance in in vitro and in vivo systems. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the high purity of E-BMP-2. E-BMP-2-induced alkaline phosphatase expression in osteoprogenitor cells (C2C12, ST2, and primary murine calvarial osteoblast cells) was dose-dependent, and consistently elicited ectopic new ossicles of significant size in mice, also in dose-dependent fashion. In addition, E-BMP-2 induced phosphorylation of Smad1/5/8 and mRNA expression of osteoblastic differentiation markers to the same extent as C-BMP-2. On the other hand, when E-BMP-2 was exposed to increasing heat over time, its bone-inducing capacity was maintained until reaching 70 degrees C for 2 h or 90 degrees C for 15 min. Thus, E-BMP-2 will exhibit a decrease in activity with the sterilization procedures required prior to use in surgery. These findings indicate that the biological capacity and heat stability of E-BMP-2 are almost equivalent to those of currently available C-BMP-2, and suggest that E-BMP-2 might, thus, solve current problems of cost impeding routine clinical use of rhBMP-2.

The multifarious functions of sex steroid receptors include a role as regulatory factors for bone and mineral metabolism in vivo. These functions are more complex than originally assumed. The finding of nuclear receptors in osseous tissue alludes to the existence of novel indirect and direct functions of bone tissue beyond skeletal support, hematopoiesis, and calcium homeostasis. Cell-specific gene targeting approaches are an extremely important technology for future studies that will need to be conducted to fully understand the molecular mechanisms underlying bone formation and metabolism.

There are large sex differences in the prevalence of locomotive diseases such as osteoporosis and osteoarthritis. Given that the onset of diseases is the failure of homeostasis, there might be a sex difference in the maintenance of homeostasis of locomotive organs/tissues. The factors of sex differences consist of sex chromosomes and sex hormones. Because the majority of locomotive diseases were occurred in the elderly people, the deficiency of sex hormones might play a role in pathogenesis of locomotive diseases. Sex hormones exert their functions by binding with their own specific nuclear receptors, which regulate transcription of the target genes in the target tissues. In this chapter, the transcriptional regulation by sex hormone receptors in bone and muscle will be discussed based on the reports about the mice lacking sex hormone receptor (s).

In the developed countries including Japan, population ageing has remarkably proceeded. Among such societies, fractures in the elder have raised as the issue, which should be resolved. To facilitate fracture healing in the clinical situations, it is essential to understand the molecular mechanisms underlying fracture repair. In this review, the molecular mechanisms of fracture repair, which have been recently reported, will be introduced. These clarifications on molecular mechanisms may open the new window for the treatment to accelerate fracture healing.

This study was designed to investigate effects of heat on the bone-inducing activity of recombinant human bone morphogenetic protein (rhBMP)-2. rhBMP-2 samples were heated at 50, 70, 90, or 100 degrees C for 15 min, or 1, 2, 4, or 8 h, or autoclaved at 120 degrees C for 15 min. The bone-inducing activity of the rhBMP-2 before and after heating was assayed in in vivo and in vitro systems. For the in vivo assay, 5 microg rhBMP-2 samples were impregnated into porous collagen disks (6 mm in diameter, 1 mm thickness), freeze dried, and implanted into the back muscles of ddY mice. Three weeks later, the implant was harvested from the host and examined for ectopic new bone tissue by radiography. The new bone mass was quantified by single-energy X-ray absorptiometry. The in vitro activity of the rhBMP-2 was assayed by adding the BMP sample at a concentration of 100 ng/ml to cultures of MC3T3-E1 cells. After 48 h, the alkaline phosphatase activity was measured. After heating at 50 degrees or 70 degrees C, no significant reduction in bone-inducing activity was noted in either in vivo or in vitro assay systems unless the protein was exposed to sustained heat at 70 degrees C for 8 h, based on in vitro assay data. However, heating above 90 degrees C and for longer periods led to a decrease in the biological activity of the rhBMP-2 in a time- and temperature-dependent manner. rhBMP-2 was rendered inactive when exposed to temperatures at or in excess of 120 degrees C.

Pathophysiology of postmenopausal osteoporosis has been considered due to deficiency of estrogen. However, it has been reported that the rate of bone mass loss during perimenopause is greater than that in postmenopause, probably due to increased FSH. From the recent knowledge of basic research on FSH, FSH can directly stimulate osteoclast formation and accelerate bone resorption. In contrast, FSH transgenic mice exhibit increased bone mass dependent on ovarian function. In this review, the controversies on the function of FSH in bone mass regulation will be discussed.

Postmenopausal osteoporosis is caused by estrogen deficiency. The precise mechanism was unclear how estrogen maintains bone mass and how estrogen deficiency alters bone metabolism. However, the progress of generation and analyses of estrogen receptorα (ERα) conditional knockout mice have revealed the pathophysiology of postmenopausal osteoporosis. Here we introduce recent reports related to the estrogen/ERα mediated direct and indirect regulations in bone metabolism.

During the last decade, our view on the skeleton as a mere solid physical support structure has been transformed, as bone emerged as a dynamic, constantly remodeling tissue with systemic regulatory functions including those of an endocrine organ. Reflecting this remarkable functional complexity, distinct classes of humoral and intracellular regulatory factors have been shown to control vital processes in the bone. Among these regulators, nuclear receptors (NRs) play fundamental roles in bone development, growth, and maintenance. NRs are DNA-binding transcription factors that act as intracellular transducers of the respective ligand signaling pathways through modulation of expression of specific sets of cognate target genes. Aberrant NR signaling caused by receptor or ligand deficiency may profoundly affect bone health and compromise skeletal functions. Ligand dependency of NR action underlies a major strategy of therapeutic intervention to correct aberrant NR signaling, and significant efforts have been made to design novel synthetic NR ligands with enhanced beneficial properties and reduced potential negative side effects. As an example, estrogen deficiency causes bone loss and leads to development of osteoporosis, the most prevalent skeletal disorder in postmenopausal women. Since administration of natural estrogens for the treatment of osteoporosis often associates with undesirable side effects, several synthetic estrogen receptor ligands have been developed with higher therapeutic efficacy and specificity. This review presents current progress in our understanding of the roles of various nuclear receptor-mediated signaling pathways in bone physiology and disease, and in development of advanced NR ligands for treatment of common skeletal disorders.

The pathology of rheumatoid arthritis (RA) is ameliorated during pregnancy and deteriorated after delivery. Thus, female sex hormones could be involved in the pathogenesis of RA. However, the effects of estrogen and progesterone on the development and progression of RA have been unclear. In this study, we analyzed the effects of female hormones on the pathogenesis of RA by performing ovariectomy (OVX) and hormone implantation in the SKG mouse model of human RA. OVX mice showed severe arthritis and cartilage destruction with increased serum levels of TNF-α and IL-6, when compared with sham-operated mice. In contrast, estrogen-treated mice exhibited remarkable suppression of arthritis, with no bone erosion, little synovial hyperplasia and little infiltration of immune cells. Moreover, serum levels of TNF-α and IL-6 were decreased. In progesterone-treated mice, mild synovial hyperplasia and no immune cell infiltration were observed, with decreased serum levels of IL-6. These results suggest that female hormones, estrogen and progesterone, can play roles in the remission of RA.

Based on the progress of researches in bone biology field, it has been clarified that bone metabolism can be regulated by complex network between various tissues and organs. Among them, sex steroid hormones, including estrogen and androgen, are known as one of the players in bone metabolism. Sex steroid hormones play crucial roles for maintenance of bone tissue because bone mass can be decreased by deficiencies of sex steroid hormones. Also, sex steroid hormones replacement therapies, which have various adverse effects, can rescue bone loss and prevent fractures. This review introduces the direct and indirect regulatory mechanism of sex steroid hormones for bone metabolism.

This is the brief report on basic research at ASBMR2012 held in Minneapolis, MN, USA. There were several keywords on basic research in this year, such as Osteoprogenitors, PTH, Sclerostin, Wnt signaling, BMP signaling and Networks between bone and other organs. In this brief report, I would like to introduce a part of current hot topics on basic research presented at ASBMR2012.

Estrogen is well-known to play essential roles for maintenance of bone mass. Although its osteoprotective actions are mediated mainly through suppressing bone resorption, several reports has suggested that ERα/estrogen signal in osteocytes has some osteoprotective effects. For example, ERαis reported to be involved in adaptive response to loading which is thought to be sensed by osteocytes. Estrogen is also thought to be a viable factor for osteocytes. In addition, our group recently found that osteocytic ERαexhibited decreased bone mass, suggesting that osteocytic ERαexerts osteoprotective function. Also, functions of osteocyte itself have been characterized recently. These findings may lead to an understanding of estrogen actions on osteocytes and to a comprehensive understanding of estrogen actions on bone.

Androgen and estrogen are sex steroid hormones, which bind to it own nuclear receptors, such as androgen receptor (AR) and estrogen (ER) , and exert its action through regulation of target gene expressions, and contribute to the maintenance of various homeostasis. However, the mode of binding of AR and ER binding site on the genome DNA and their intracellular behavior are diverse in various cells. From genome-wide analysis studies with the advent and remarkable development of next-generation sequencer in recent years, it has been clarified that new features and mechanism of action of sex hormone receptors in breast cancer and prostate cancer. In this paper, we focus on the AR, together with recent findings on genome-wide analysis, and we also introduce the current application for bone metabolism.

Reversible histone methylation and demethylation are highly regulated processes that are crucial for chromatin reorganization and regulation of gene transcription in response to extracellular conditions. However, the mechanisms that regulate histone-modifying enzymes are largely unknown. Here, we characterized a protein kinase A (PKA)-dependent histone lysine demethylase complex, PHF2-ARID5B. PHF2, a jmjC demethylase, is enzymatically inactive by itself, but becomes an active H3K9Me2 demethylase through PKA-mediated phosphorylation. We found that phosphorylated PHF2 then associates with ARID5B, a DNA-binding protein, and induce demethylation of methylated ARID5B. This modification leads to targeting of the PHF2-ARID5B complex to its target promoters, where it removes the repressive H3K9Me2 mark. These findings suggest that the PHF2-ARID5B complex is a signal-sensing modulator of histone methylation and gene transcription, in which phosphorylation of PHF2 enables subsequent formation of a competent and specific histone demethylase complex.

The treatment of osteoporosis has been a critical issue in today's medical situation. Various therapeutic agents and strategies have been investigated and applied, and have proven successful in the treatment of osteoporosis. However, some concerns still remain, such as the adverse effects of such treatments. From this point of view, a search for novel therapeutic targets, such as Fas signaling, remains important.

Small interfering RNA (siRNA) is useful tool for specific and efficient knockdown of disease-related genes. However, in vivo applications of siRNA are limited due to difficulty in its efficient delivery to target cells. In this study, we investigated the efficacy of a biodegradable hydrogel, poly-d,l-lactic acid-p-dioxanone-polyethylene glycol block co-polymer (PLA-DX-PEG), as a siRNA carrier. PLA-DX-PEG pellets with or without fluorescein-labeled dsRNA were implanted into mouse dosal muscle pouches. The cellular uptake of dsRNA surround the polymer was confirmed by fluorescent microscopy. The fluorescence intensity was dose-dependent of the dsRNA, and exhibited a time-dependent decrease. To investigate its biological efficiency, noggin (antagonoist to BMPs) gene-silencing with siRNA (siRNA/Noggin) was examined by the amount of suppression of BMP-2-induced noggin expression and the level of performance of BMP, indicated by ectopic bone formation. Noggin gene expression induced by BMP-2 was suppressed by addition of siRNA/Noggin to the implant, and the ectopic bone formation induced by implants with both BMP-2 and siRNA/Noggin was significantly greater than those induced by implants with BMP-2 alone. These results indicate the efficacy of local delivery of siRNAs by PLA-DX-PEG polymer, which intensified bone-inducing effects of BMP and promoted new bone formation by suppressing gene expression of Noggin.

Prostate cancer development is associated with hyperactive androgen signaling. However, the molecular link between androgen receptor (AR) function and humoral factors remains elusive. A prostate cancer mouse model was generated by selectively mutating the AR threonine 877 into alanine in prostatic epithelial cells through Cre-ER T2 –mediated targeted somatic mutagenesis. Such AR point mutant mice ( AR pe-T877A/Y ) developed hypertrophic prostates with responses to both an androgen antagonist and estrogen, although no prostatic tumor was seen. In prostate cancer model transgenic mice, the onset of prostatic tumorigenesis as well as tumor growth was significantly potentiated by introduction of the AR T877A mutation into the prostate. Genetic screening of mice identified Wnt-5a as an activator. Enhanced Wnt-5a expression was detected in the malignant prostate tumors of patients, whereas in benign prostatic hyperplasia such aberrant up-regulation was not obvious. These findings suggest that a noncanonical Wnt signal stimulates development of prostatic tumors with AR hyperfunction.

The bone mass, which is controlled by the balances between bone formation and bone resorption can be reduced by estrogen deficiency in post-menopausal osteoporosis. Reduced bone mass can be recovered by hormone replacement therapy (HRT) , however, HRT has various side effects. Although SERMs can rescue the bone mass with less side effect compared to HRT, the precise mechanisms of this effect is still elusive. From the results of the analyses for osteoclast specific estrogen receptor (ER) alphaknockout mice and the genome wide approach of ERalphabinding site, estrogen and SERMs can, at least in part, protect the bone mass by inducing the expression of Fas ligand and controling the life span of osteoclasts. More precise molecular mechanisms of the effect of SERM, especially in tissue/cell type specificity, may help to investigate new SERM, which is more specific and effective to treat post-menopausal osteoporosis.

Androgen is well established to exert anabolic action for skeletal tissues beyond sex difference. However, the target cells/genes remain to be identified. Recently, we have generated a mouse line selectively ablated androgen receptor gene in osteoclasts, and found osteoporotic phenotype in male mutants, indicating that anabolic androgen action is attributed to its repressive action for bone resorption.

The osteoprotective action of estrogen in women has drawn considerable attention because estrogen deficiency-induced osteoporosis became one of the most widely spread diseases in developed countries. In men, the significance of estrogen action for bone health maintenance is also apparent from the osteoporotic phenotype seen in male patients with genetically impaired estrogen signaling. Severe bone loss and high bone turnover, including typical osteofeatures seen in postmenopausal women, can also be recapitulated in rodents after ovariectomy. However, the expected osteoporotic phenotype is not observed in female mice deficient in estrogen receptor (ER)-α or -β or both, even though the degenerative defects are clearly seen in other estrogen target tissues together with up-regulated levels of circulating testosterone. It has also been reported that estrogens may attenuate bone remodeling by cell autonomous suppressive effects on osteoblastogenesis and osteoclastogenesis. Hence, the effects of estrogens in bone appear to be complex, and the molecular role of bone estrogen receptors in osteoprotective estrogen action remains unclear. Instead, it has been proposed that estrogens indirectly control bone remodeling. For example, the enhanced production of cytokines under estrogen deficiency induces bone resorption through stimulation of osteoclastogenesis. However, the osteoporotic phenotype without systemic defects has been recapitulated in female (but not in male) mice by osteoclast-specific ablation of the ERα, proving that bone cells represent direct targets for estrogen action. An aberrant accumulation of mature osteoclasts in these female mutants indicates that in females, the inhibitory action of estrogens on bone resorption is mediated by the osteoclastic ERα through the shortened lifespan of osteoclasts.

Estrogens play a key role in regulation of bone mass and strength by controlling activity of bone-forming osteoblasts and bone-resorbing osteoclasts. Cellular effects of estrogens are mediated predominantly by the action of estrogen receptor alpha (ERalpha). In earlier studies, ablation of the ERalpha gene in mice did not result in osteoporotic phenotypes due to systemic endocrine disturbance and compensatory effects of elevated levels of testosterone. Despite the relatively well-established effects in osteoblasts, little is known about the direct action of estrogen in osteoclasts. Development in the last decade of more sophisticated genetic manipulation approaches opened new possibilities to explore cell-specific roles of nuclear receptors in bone tissue. Recently, we have generated osteoclast-specific ERalpha gene knockout mice and shown that in vivo estrogens directly regulate the life span of mature osteoclasts by inducing the expression of pro-apoptotic Fas ligand (FasL). Inhibitory effects of estrogens on osteoclast function were further studied in vitro. We observed sufficiently detectable ERalpha expression in osteoclasts differentiating from primary bone marrow cells or RAW264 cells, although levels of ERalpha were decreasing during progression of the differentiation into mature osteoclasts. Treatment with estrogens led to reduction in expression of osteoclast-specific genes controlling bone resorption activity. However, estrogens did not affect the size of multinucleated osteoclasts or number of nuclei in a mature osteoclast. In conclusion, in osteoclasts, estrogens function to inhibit bone resorption activity and vitality rather than differentiation.

Bone morphogenetic proteins (BMPs) were originally identified as osteoinductive proteins. With cloning of BMP genes, studies of BMPs and their clinical application have advanced. However, with increasing clinical applications, drug delivery systems and production costs have become more important issues. To address these issues, we asked whether E. coli-derived rhBMP-2 (E-BMP-2)-adsorbed porous beta-TCP granules could achieve posterolateral lumbar fusion in a rabbit model similar to autogenous bone grafts. Lumbar spinal fusion masses were evaluated by 3-D computed tomography, mechanical testing, and histological analyses 8 weeks after surgery. By these measures E-BMP-2-adsorbed beta-TCP granules achieved lumbar spinal fusion in dose-dependent fashion in a rabbit model as well as autogenous bone graft. Our preliminary findings suggest E-BMP-2-adsorbed porous beta-TCP could be a novel, effective alternative to autogenous bone grafting for generating new bone and promoting regenerative repair of bone, and potentially utilizable in the clinical setting for treating spinal disorders.

When bone tissue, which plays a central role in locomotive organs, is broken by trauma and decreased in its own function of support, it is lead to be remarkable decline in the activity of daily life (ADL). Recently, in a clinical situation, various treatments have been tried for prophylaxis of fractures, and these treatments have been rewarded with good results. However, fractures would be occurred in a certain amount and there would be serious problems in ADL if the treatment fails to repair fractures. Furthermore, fractures could spontaneously heal in several weeks, it can be achieved in early rehabilitation and improvement of ADL, if we can accelerate fracture healing. Bone morphogenetic proteins, which were advocated by Dr. Urist, would be expected to be a key to establish much better society especially for the old.

Effective therapies for the regeneration of large osteochondral defects are still lacking; however, various approaches have been used. We evaluated the efficacy of Escherichia coli-derived dimeric recombinant human BMP-2 (E-rhBMP-2) for the repair of large osteochondral defects in a rabbit model. Osteochondral defects made in the femoral patellar groove of the knee were treated by transplanting gelatin sponges onto which no or various doses of E-rhBMP-2 were loaded. The outcomes were compared with those of an untreated control group four, 12 and 24 weeks after transplantation. At early time points, the cartilage tissue was repaired in a dose-dependent manner, and bone repair was accelerated in the defects treated with high doses of E-rhBMP-2. At 24 weeks, the repair of cartilage tissue was better with E-rhBMP-2 treatment, even at low doses, than without E-rhBMP-2 treatment. Our findings suggest that the use of E-rhBMP-2 improves and accelerates the repair of osteochondral defects in a rabbit model.

Treatments for locomotive organs, such as bone and joint, have been investigated and preceded in recent decades. Especially, in orthopaedic field, therapeutic alternatives for total joint arthroplasty for osteoarthritis and spinal fusion have remarkably advanced. However, bone regeneration required in massive bone defect or broad spinal fusion have been treated with bone graft mainly using auto grafted bone here in Japan. These methods have several issues such as limited bone regeneration or pain in harvest sites. We would like to describe perspectives and biology in bone regeneration focusing on bone morphogenetic protein (BMP).

Bone mass and strength is mediated by appropriate weight bearing, and it is clear that bone tissue senses and adopts mechanical stresses. However, the precise molecular mechanisms in mechanotransduction remain elusive. Recently, several studies clarified that estrogen receptor alpha (ERalpha) plays important role in mechanotransduction. For example, the lifespan of osteocytes is prolonged by mechanical loading through ERalpha action, which is produced by the plentiful expression of ERalpha in osteocytes. Although these recent findings clearly show the important role of ERalpha in mechanotransduction, the intracellular function of ERalpha is still largely unknown. In this point of view, it will be a great interest to elucidate how ERs functionally associate with regulators involved with mechanotransductions in bone cells.

Noggin is a major extracellular antagonist to bone morphogenetic proteins (BMPs) which binds to BMPs and blocks binding of them to BMP-specific receptors and negatively regulates BMP-induced osteoblastic differentiation. In this study, we investigated the effect of noggin silencing by transfection of small interfering RNA (siRNA) on BMP-induced osteoblastic differentiation in vitro and ectopic bone formation in vivo induced by recombinant human BMP-2 (rhBMP-2). Noggin mRNA expression was up-regulated in response to rhBMP-2 in C2C12 cells, a myoblastic cell line, in dose- and time-dependent fashion as determined by real-time RT-PCR assay. Silencing of noggin expression by transfection of noggin siRNA suppressed BMP-stimulated noggin expression, resulting in acceleration of BMP-induced osteoblastic differentiation. For in vivo noggin silencing, siRNA was injected locally into back muscles and transfected into local cells by electroporation, where rhBMP-2-retaining (5 microg) collagen disks had been surgically placed. The implants were harvested at 2 weeks after surgery from experimental and control group mice and analyzed by radiological and histological methods. As a result, bone mineral content of ossicles ectopically induced by rhBMP-2 was significantly increased by silencing of noggin. Our findings suggest that silencing of noggin enhances the osteoblastic differentiation of BMP-responding cells in vitro and new bone formation induced by rhBMP-2 in vivo by eliminating negative regulation of the effects of BMP. RNA interference might be useful for intensifying the effects of BMP in promoting new bone (callus) formation in repair of damaged bone.

Bone tissue protects and supports soft organs and maintains calcium homeostasis. Steroid sex hormones and fat-soluble vitamins play a pivotal role in regulation of bone homeostasis, turnover and remodeling. These molecules act as ligands of nuclear receptors, through which they control gene expression in bone cells, namely bone-forming osteoblasts, bone-resorptive osteoclasts and osteocytes. Significant advances in our understanding of nuclear receptor physiology have been achieved due to development of novel genetic manipulation approaches and generation of experimental animal models in which nuclear receptor genes were mutated in specific cell types. In this review, we summarized some aspects of recent progress in studies on molecular mechanisms of cell-specific action of nuclear hormone receptors in bone tissue.

Bone morphogenetic proteins (BMPs) were originally isolated based on their ability to induce ectopic cartilage and bone formation. The agents to promote the local bone formation with BMP would be beneficial to promote bone repair and to shorten the treatment period. For this purpose, we have examined ONO-4819, which is a prostaglandin (PG) E2 EP4 receptor selective agonist (EP4A), as a positive modulators for the efficacy of BMPs. In our previous study, the systemic and local (with biodegradable synthetic polymers) administration of EP4A led to a significant augmentation of ossicle mass. But the mechanisms how EP4A accelerates the BMP-mediated bone formation are still unknown. In this study, we have examined how EP4A facilitates the BMP signaling using in vitro system with pluripotent stromal cell line, ST2. The mRNA expressions of Osterix and ALP (a marker enzyme of osteoblastic differentiation) and enzymatic activity of ALP in the ST2 cells were elevated significantly by BMP treatment. This elevation was further elevated by addition of the EP4A. The accelerated BMP action by the EP4A was abolished by pre-treatment with PKA inhibitor. This study suggests that ONO-4819 accelerates BMP-induced osteoblastic differentiation of ST2 cells by stimulating the commitment for osteoblastic lineage. Thus PKA signaling pathway would be the main intracellular signaling pathway of the EP4 for the anabolic effect of bone and mineral metabolisms.

Isolated fracture of the calcaneal apophysis is a rare injury in children and adolescents. In this study, we report on a case of a displaced calcaneal apophyseal avulsion fracture in a child treated with open reduction and internal fixation, as well as a review of the literature. A 9-year-old female child presented to the senior surgeon complaining of acute heel pain after a gymnastic injury. She was diagnosed with a displaced, isolated fracture of the proximal calcaneal apophysis for which she underwent open reduction and internal fixation. On the magnetic resonance imaging (MRI) examination, we could diagnose that her injury was not chronic but acute because there was no change of intensity in the metaphyseal area. A combination of bioabsorbable suture tacks and pins was used to anatomically fix the fragment using the tension band wiring technique. At 2 years and 6 months follow-up, she had full range of motion, complete return of strength. We report here on the successful surgical treatment and the first case evaluated by MRI of an avulsion fracture of the calcaneal apophysis in a child.

Recombinant human bone morphogenetic protein (rhBMP)-2 in a block copolymer composed of poly-D,L-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) as a carrier and porous beta-tricalcium phosphate (beta-TCP) blocks were used to generate a new fully absorbable osteogenic biomaterial. The bone regenerability of the rhBMP-2/PLA-DX-PEG/beta-TCP composite was studied in a critical-sized rabbit bone defect model. In an initial study, a composite of PLA-DX-PEG (250 mg) and beta-TCP (300 mg) loaded with or without rhBMP2 (50 microg) was implanted into a 1.5 cm intercalated bone defect created in a rabbit femur. Defects were assessed by biweekly radiography until 8 weeks postoperatively. The bony union of the defect was recognized only in the BMP-loaded group. To obtain further data on biomechanical and remodeling properties, another BMP-loaded composites group was made and observed up to 24 weeks. All defects were completely repaired without residual traces of implants. Anatomical and mechanical properties of the repaired bone examined by histology, 3-dimensional CT (3D-CT) and mechanical testing were essentially equivalent to the nonoperated-on femur at 24 weeks. These experimental results indicate that fully absorbable rhBMP-2/PLA-DX-PEG/beta-TCP is a promising composite having osteogenicity efficient enough for repairing large bone defects.

The anabolic effects of prostaglandin E2 on bone are effected through the activation of EP4, a G protein-coupled receptor. In the present study, we examined the effects of a prostanoid receptor-selective agonist (ONO-4819) in an experimental system of ectopic bone formation using recombinant human bone morphogenetic protein-2 (rhBMP-2). Collagen pellets containing rhBMP-2 were implanted onto the back muscles of mice and then treated with ONO-4819 administered every 8 h by subcutaneous injection. The ossicles elicited ectopically by rhBMP-2 in mice treated with 30 μg/kg ONO-4819 were significantly larger in size and had a higher bone mineral density and bone mineral content when compared to the controls. We also noted that the anabolic effect of ONO-4819 was seen only in the early phase of the rhBMP-2-induced bone-forming process. These experimental results indicate that the EP4 receptor agonist enhances the rhBMP-2-induced bone formation through a selective effect on early stage mesenchymal cells, which in turn may result in increased responsiveness of the host animals to rhBMP-2.