Search

Background Muscle wasting is a common condition concomitant with aging. Androgens significantly increase skeletal muscle mass, but the role of the androgen receptor (AR) in skeletal muscle is not well established. TEI‐SARM2, a novel selective androgen receptor modulator (SARM), was developed as a pharmaceutical candidate for the treatment of muscle wasting diseases. Methods The efficacy and specificity of TEI‐SARM2 were analysed in vitro assays, and efficacy was also evaluated in vivo using orchiectomized male rats, rat tail‐suspension, rat and mice cancer‐induced muscle atrophy models, and female cynomolgus monkeys. Male myofibre‐specific AR‐knockout (mARKO) mice were orchiectomized to investigate the role of AR in muscle using TEI‐SARM2. RNA‐seq analysis of bulbocavernosus muscle was performed. The effects of spermidine were evaluated in C2C12 myoblasts. Results Selective, potent anabolic effects of TEI‐SARM2 in muscle were detected in rats and monkeys. TEI‐SARM2 inhibited muscle loss and promoted muscle recovery in a model of muscle disuse and prevented muscle wasting and improved survival rate in cancer models. In vivo and RNA‐seq analyses revealed that AR regulates skeletal muscle mass in myofibres and extra‐myofibres, both directly and indirectly. Among them, polyamine synthesis‐related genes were focused, and spermidine treatment could induce C2C12 cell proliferation. Conclusions TEI‐SARM2 exhibited potent and selective anabolic effects in muscles, as well as tissue specificity in various animal models. The results support that TEI‐SARM2 is a promising drug candidate for muscle wasting diseases. TEI‐SARM2 induced anabolic effects on skeletal muscle via AR in myofibres as well as other androgen target cells.

Epigenetic transcriptional regulation is essential for the differentiation of various types of cells, including skeletal muscle cells. DNA methyltransferase 1 (Dnmt1) is responsible for maintenance of DNA methylation patterns via cell division. Here, we investigated the relationship between Dnmt1 and skeletal muscle regeneration. We found that Dnmt1 is upregulated in muscles during regeneration. To assess the role of Dnmt1 in satellite cells during regeneration, we performed conditional knockout (cKO) of Dnmt1 specifically in skeletal muscle satellite cells using Pax7CreERT2 mice and Dnmt1 flox mice. Muscle weight and the cross-sectional area after injury were significantly lower in Dnmt1 cKO mice than in control mice. RNA sequencing analysis revealed upregulation of genes involved in cell adhesion and apoptosis in satellite cells from cKO mice. Moreover, satellite cells cultured from cKO mice exhibited a reduced number of cells. These results suggest that Dnmt1 is an essential factor for muscle regeneration and is involved in positive regulation of satellite cell number.

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.

Summary Estrogens are female sex hormones that are important for comprehensively maintaining muscle function, and an insufficiency affects muscle strength and regeneration in females. However, it is still unclear whether estrogen signaling is mediated through receptors. To investigate the specific role of estrogen receptor β (ERβ) in skeletal muscle and satellite cells (muscle stem cells), we generated muscle-specific ERβ-knockout (mKO) and satellite cell-specific ERβ-knockout (scKO) mice, respectively. Young female mKO mice displayed a decrease in fast-type dominant muscle mass. Female, but not male, scKO mice exhibited impaired muscle regeneration following acute muscle injury, probably due to reduced proliferation and increased apoptosis of satellite cells. RNA-sequencing analysis revealed that loss of ERβ in satellite cells altered gene expression of extracellular matrix components, including laminin and collagen. The results indicate that the estrogen-ERβ pathway is a sex-specific regulatory mechanism that controls muscle growth and regeneration in female mice., Graphical Abstract, Highlights • ERβ controls muscle growth in young female mice • ERβ is essential for muscle regeneration in female mice • Inactivation of ERβ causes an increase in apoptosis • ERβ is required for satellite cell population expansion, In this article, Seko et al. demonstrate that ERβ is a female-specific regulator of muscle growth and regeneration. Muscle-specific ERβ-deleted young female mice exhibited a decrease in muscle mass, while satellite cell-specific ERβ inactivation resulted in impaired muscle regeneration following acute muscle injury in female mice due to reduced proliferation and increased apoptosis of satellite cells.

Macrophages are progenitors of osteoclasts as well as regulators of bone metabolism. Macrophages mediate not only bone formation by osteoblasts under physiological conditions, but also bone regeneration after fracture. The mechanisms of macrophages regulation of bone formation and regeneration remain unclear, however. Here, we demonstrate that the liposome-encapsulated Clodronate (Clod-lip) injected mouse model with cortical bone defect induced by drill-hole injury and targeted depletion of phagocytic macrophages exhibits impaired angiogenesis of type H vessels that couple angiogenesis and osteogenesis. Moreover, we identify Tgfbi (encoding TGFBI), Plau (encoding uPA) and Tgfb1 (encoding TGF-β1), through RNA-seq analysis, as genes of macrophage-secreted factors mediating angiogenesis and wound healing. The relevant mRNA was highly expressed in bone marrow-derived macrophages among bone cells, as determined through qRT-PCR. Finally, we disclose that treatment with uPA inhibitor or TGF-β receptor I, receptor II inhibitor impairs bone regeneration after injury, confirming the importance of uPA and TGF-β1 during bone regeneration. Our findings reveal a novel mechanism of bone regeneration mediated by macrophages.

Case-control study of an animal model.To investigate the factors that are upregulated and potentially related to degenerative changes in the ligamentum flavum (LF) upon mechanical stress concentration.LF hypertrophy is reported to be associated with mechanical stress. However, few studies, using exhaustive analysis with control subjects, on the molecular mechanisms of LF hypertrophy have been published.Fourteen rabbits were used for this study. The first group underwent L2-3 and L4-5 posterolateral fusion with instrumentation and resection of the L3-4 supraspinal muscle to concentrate the mechanical stress on L3-4, whereas the other group underwent a sham operation. The deep layer of the LF from L2-3 to L4-5 in both groups was harvested after 16 weeks. Gene expression was evaluated exhaustively using DNA microarray and real-time polymerase chain reaction (RT-PCR). Fibroblast growth factor 9 (FGF9) protein expression was subsequently examined by immunohistological staining.A total of 680 genes were found to be upregulated upon mechanical stress concentration and downregulated upon mechanical shielding compared with those in the sham group. Functional annotation analysis revealed that these genes not only included those related to the extracellular matrix but also those related to certain FGF families. On RT-PCR validation and immunohistological analysis, we identified that the FGF9 protein increases in the LF upon mechanical stress, especially in the area wherein degenerative changes were frequently identified in the previous literature.FGF9 and its pathway are suggested to contribute to the degenerative changes in the LF following mechanical stress. This finding will be helpful in further understanding the molecular mechanism of human LF degeneration.N/A.

We investigated the effects of ibandronate, a bisphosphonate; eldecalcitol, an active vitamin D3 analogue; and combination treatment with both agents on secondary osteoporosis and arthritis using rats with adjuvant-induced arthritis. Arthritis was induced in 8-week-old male Lewis rats. Rats were randomized into four treatment groups and an untreated normal control group: ibandronate, eldecalcitol, ibandronate + eldecalcitol, vehicle, and control. Paw thickness was measured to evaluate arthritis. Joint destruction was evaluated histomorphometrically by the ankle joint stained with Fast Green and safranin O. The femur and lumbar spine were scanned using dual-energy X-ray absorptiometry, and the distal femur was scanned using micro-computed tomography for bone mineral density (BMD) and trabecular microstructural evaluations. Ibandronate and/or eldecalcitol increased BMD in both the lumbar vertebrae and femur and improved several microstructural parameters (bone volume/total volume, structure model index, trabecular number, and trabecular separation of the distal femur). In addition, there was an additive effect of combination treatment compared with single treatments for most trabecular parameters, including BMD and bone volume. However, ibandronate and/or eldecalcitol did not inhibit arthritis and joint destruction. Combination treatment with ibandronate and eldecalcitol may be effective for secondary osteoporosis associated with arthritis.

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.

Estrogen deficiency impairs fracture healing and homeostasis of bone tissue. OVX-induced estrogen deficiency in mice attenuates fracture healing and changes the expression ratio of estrogen receptor (ER) α and ERβ in callus during the process of fracture healing. Therefore, ERs may be involved in the regulation of fracture healing. However, the roles of ERs in fracture healing are largely unknown. The purpose of this study was to clarify the significance of ERs during fracture healing using osteoblast-specific ER knockout mice in a mono-cortical drill hole bone regeneration model. The mature osteoblast-specific ER knockout mice were generated using osteocalcin (OCN)-Cre mice, and ERα and ERβ flox mice (OCN-Cre; ERαf/f, ERαΔOb/ΔOb and OCN-Cre; ERβf/f, ERβΔOb/ΔOb). Drill hole surgery was conducted on the tibiae of 8-week-old female mice. The mice were sacrificed 10 or 14 days after surgery and the bones were analyzed by DXA, μCT and bone histomorphometry. DXA analysis revealed that intact femoral BMD was significantly decreased in ERαΔOb/ΔOb mice compared with ERαf/f mice, but there was no difference in bone mass between ERβΔOb/ΔOb and ERβf/f mice. Micro CT analyses showed that the callus volume at the restricted drill hole site in tibiae was significantly less in ERαΔOb/ΔOb compared to ERαf/f mice only at day 14 but not at day 10. In addition to femoral BMD, there was no significant difference in callus volume between ERβΔOb/ΔOb and ERβf/f mice. Bone histomorphometric analyses showed that Ob.S/BS and N.Ob/B.Pm were significantly less in ERαΔOb/ΔOb mice compared with ERαf/f mice only at day 10. In addition, Oc.S/BS and N.Oc/B.Pm were significantly less in ERαΔOb/ΔOb mice compared with ERαf/f mice only at day 14. These results suggest that ERα but not ERβ in osteocalcin-positive osteoblasts may contribute to the late stage of bone regeneration.

Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation with aberrant epigenetic alterations, eventually leading to joint destruction. However, epigenetic regulatory mechanisms underlying RA pathogenesis remain largely unknown. Here, we showed that Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) , a key molecule involved in maintenance of DNA methylation during cell division, is a central epigenetic regulator that orchestrates the suppression of expression of multiple factors that exacerbate RA. We found that murine arthritis tissue and human RA tissue, particularly synovial fibroblasts (SF), exhibit remarkable up-regulation of expression of Uhrf1. SF-specific Uhrf1 conditional knockout mice showed more severe arthritic phenotypes and apoptosis-resistant SF. Integrative analysis of the transcriptome and methylome showed that expression of several cytokines including Ccl20 was up-regulated in Uhrf1-deficient SF. In RA patients, disease activity scores, CCL20 expression, Th17 accumulation and apoptosis resistance were negatively correlated with UHRF1 expression in synovium. Finally, stabilization of UHRF1 by Ryuvidine administration diminished disease pathogenesis in arthritis model mice. Our results demonstrated that UHRF1 expressed in SF can contribute to suppression of multiple pathogenic events associated with RA such as Th17 recruitment, SF apoptosis and bone destruction, suggesting that targeting UHRF1 could represent a novel therapeutic strategy for RA.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on Hh functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0&ndash, 48 h (early stage of osteoclast differentiation) or 48&ndash, 96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.

The anabolic effects of androgen on skeletal muscles are thought to be mediated by androgen receptor (AR). Although multiple studies concerning the effects of AR in males have been performed, the molecular mechanisms of AR in skeletal muscles remain unclear. Here we first confirmed that satellite cells from mouse hindlimb muscles express AR. We then generated satellite cell-specific AR knockout mice using Pax7CreERT2 and ARL2/Y mice to test whether AR in satellite cells is necessary for muscle regeneration. Surprisingly, we found that muscle regeneration was compromised in both Pax7CreERT2(Fan)/+ control mice and Pax7CreERT2(Fan)/+;ARL2/Y mice compared to ARL2/Y mice. However, Pax7CreERT2(Gaka)/+;ARL2/Y;R26tdTomato/+ mice showed no significant differences between control and mutant muscle regeneration. These findings indicate that AR in satellite cells is not essential for muscle regeneration. We propose that Pax7CreERT2(Fan)/+ control mice should be included in all experiments, because these mice negatively affect the muscle regeneration and show the mild regeneration phenotype.

Breast cancer subtypes have been classified based on the gene expression profiles, and the specifically effective treatments for each subtype have been developed and applied in clinical situations. However, there is no effective treatment for metaplastic breast carcinomas, which are pathological tissue types with poor prognosis. Metaplastic breast carcinomas are almost resistant to existing chemotherapy because it has not been identified the specific therapeutic target molecules. Recently, Lehmann et al. reported that breast cancers with chondroid metaplasia are categorized in Mesenchymal (M) type, which is one subtype out of six triple negative breast cancer (TNBC) subtypes based on drug susceptibility. From the knowledge on this report, we hypothesized that integrative analyses using the gene expression profile data sets obtained from various types of breast cancers with or without cartilage formation may enable us to identify the therapeutic target genes for metaplastic breast carcinomas, especially for breast cancers with chondroid metaplasia. Bioinformatics analyses were performed using the gene expression profile data sets of TNBC, which are registered in Gene Expression Omnibus (GEO). When compared with metaplastic breast carcinomas (28 data sets), 200 genes were specifically fluctuated over 2-fold in breast cancer with chondroid metaplasia (8 data sets). On the other hand, M type (11 data sets) specific 578 genes were identified over 56 TNBC data sets. In addition, 57 genes were overlapped between chondoroid metaplastic 200 genes and M type 578 genes. Therefore, these 57 overlapped genes might be considered as possible poor prognostic factors in breast cancer with chondroid metaplasia. As a result of Gene Ontology analysis on these 57 genes, skeletal system development and extracellular matrix structural constituent were significantly enriched in Biological process and Molecular function, respectively. Also, SCRG1, SOX8 and SOX10, which are genes related to cartilage differentiation, were included. Next, we focused 14 genes because these genes could serve as therapeutic targets. Then, we validated the expression levels of these 14 genes using tissues obtained from surgically resected human breast cancer with chondroid metaplasia dividing into three parts, the invasive ductal carcinoma, the chondroid metaplasia and normal mammary gland. As a result, SCRG1, CD86, HCLS1, EPSTI1, LYZ, and SLA were significantly increased in chondroid metaplasia part, suggesting that these validated genes were identified as possible therapeutic targets for breast cancer with chondroid metaplasia. Further study will help us to investigate a novel and effective therapeutic strategy for metaplastic breast carcinomas. Citation Format: Yamashita M, Kamei Y, Kusakabe E, Yamasawa H, Aoki R, Taguchi K, Nishiyama K, Murakami A, Sawada Y, Takada Y, Imai Y. Exploring novel therapeutic target molecules for metaplastic breast carcinoma using comprehensive genome-wide gene expression analyses [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-07.

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.

Estrogen deficiency caused by bilateral ovariectomy (OVX) has been reported to lead to morphological changes in otoconia. Thus, we examined the morphological changes in the otoconial layer after OVX. We also investigated whether micro-computed tomography (µCT) is useful for the detection of morphological changes in the otoconial layer.The otic capsules of C57BL/6 J mice were removed and evaluated using histological techniques and µCT at 2, 4, and 8 weeks after OVX or sham surgery. The volume of the utricle otoconial layer was measured and compared between the OVX and sham groups. The µCT scan and histological study results were also compared.The volume of the utricle otoconial layer was significantly increased 4 weeks after OVX compared to the sham group in both histological and µCT studies (p 0.05). The volume of the otoconial layer measured using µCT was significantly correlated with the histological study results (p 0.05).The volume of the utricle otoconial layer increased after OVX. These morphological changes could be detected by µCT.

Background Postoperative pericardial adhesions are considered a risk factor for redo cardiac surgery. Several large- and medium-size animal models of pericardial adhesions have been reported, but small animal models for investigating the development of anti-adhesion materials and molecular mechanisms of this condition are lacking. In this study, we aimed to establish a simple mouse model of pericardial adhesions to address this gap. Methods We administered blood, minocycline, picibanil, and talc into the murine pericardial cavity via one-shot injection. Micro-computed tomography analyses of contrast agent-injected mice were carried out for methodological evaluation. We investigated various dosages and treatment durations for molecules identified to be inducers of pericardial adhesion. The adhesive grade was quantified by scoring the strength and volume of adhesion tissues at sacrificed time points. Histological staining with hematoxylin and eosin and Masson’s trichrome, and immunostaining for F4/80 or αSMA was performed to investigate the structural features of pericardial adhesions, and pathological features of the pericardial adhesion tissue were compared with human clinical specimens. Results Administration of talc resulted in the most extensive pericardial adhesions. Micro-computed tomography imaging data confirmed that accurate injection into the pericardial cavity was achieved. We found the optimal condition for the formation of strong pericardial adhesions to be injection of 2.5 mg/g talc for 2 weeks. Furthermore, histological analysis showed that talc administration led to an invasion of myofibroblasts and macrophages in the pericardial cavity and epicardium, consistent with pathological findings in patients with left ventricular assistive devices. Conclusions We successfully established a simple mouse model of talc-induced pericardial adhesions, which mimics human pathology and could contribute to solving the clinical issues related to pericardial adhesions.

Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor that is involved in osteoclast differentiation in our previous report. However, the biological functions of Zscan10 are not fully understood except its roles in the maintenance of genome stability and pluripotency of embryonic stem cells. Therefore, the purpose of this study was to clarify the function of Zscan10 in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established by genome editing using CRISPR/Cas9 and single cell sorting. Then, control (Ctrl) and KO cells were differentiated into osteoclasts by RANKL stimulation. We observed that TRAP activity and the expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells compared to Ctrl cells. These results suggest that Zscan10 might regulate transcription of the genes that negatively control osteoclastogenesis. To understand gene expression profiles controlled by Zscan10, RNA-seq was performed and stringent analyses identified the haptoglobin gene (Hp) as a possible target of Zscan10. In addition, ChIP against Zscan10 revealed that Zscan10 could interact with its binding motif located near the Hp gene locus as well as the transcription start site of Hp, suggesting that Zscan10 can directly regulate transcription of Hp. Finally, to examine the effects of Hp on osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). rHp treatment suppressed TRAP activity of KO cells without affecting cell viability. Furthermore, it has been reported that Hp KO mice exhibit decreased bone mass and increased osteoclast number. Importantly, hemolytic disease patients exhibited decreased serum level of Hp as well as low bone mineral density. Taken together, this study suggests that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

Summary Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of Myosin light-chain kinase 4 (Mylk4) as a target of AR in skeletal muscles. Mylk4 knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis., Graphical abstract, Highlights • DHT increases muscle strength through myofiber AR • Myofiber AR increases a fast-type muscle-specific novel splicing variant of Mylk4 • MYLK4 regulates muscle strength and muscle stiffness • MYLK4 induces phosphorylation of MYOM1, Animal Physiology; Molecular Physiology; Molecular Biology; Endocrinology

Bone Morphogenetic Proteins (BMPs) strongly induce the recruitment and differentiation of mesenchymal progenitor cells into mature osteoblasts, but also directly and indirectly stimulate differentiation of osteoclast progenitor cells and acceleration of mature osteoclasts function leading excessive bone resorption. Bisphosphonates, such as zoledronate (ZOL), inhibit osteoclasts function and osteoclasts mediated bone resorption. The short or middle term effect of BMPs and bisphosphonates on bone formation were previously reported, but there was no study that argue about the long term effect of bisphosphonates on BMP-induced bone anabolism. The present study demonstrated that the local administration of ZOL with recombinant human BMP-2 (rh-BMP-2) using beta tricalcium phosphate (β-TCP) as a carrier had superior efficacy not only to augment the BMP-induced new ectopic bone formation but to maintain the trabecular bone structure inside the new bone for long period. Histological analysis showed that rh-BMP-2/β-TCP composite induced trabecular bone resorption especially inside the new bone nodules over time, whereas no trabecular bone resorption was seen in rh-BMP-2/ZOL/β-TCP composite reducing the number of TRAP-positive cells. Thus, inhibition of bone resorption by bisphosphonate, such as ZOL, would be one of the advantageous ways to augment the new bone formation induced by rh-BMP-2, and moreover local co-application of ZOL using β-TCP as a carrier can be a useful material for long term suppression of osteoclastic resorption and thereby maintain the structure of new bone formation induced by rh-BMP-2.

DNA methylation is closely involved in the regulation of cellular differentiation, including chondrogenic differentiation of mesenchymal stem cells. Recent studies showed that Ten–eleven translocation (TET) family proteins converted 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, 5-formylcytosine and 5carboxylcytosine by oxidation. These reactions constitute potential mechanisms for active demethylation of methylated DNA. However, the relationship between the DNA methylation patterns and the effects of TET family proteins in chondrocyte differentiation is still unclear. In this study, we showed that DNA hydroxylation of 5mC was increased during chondrocytic differentiation of C3H10T1/2 cells and that the expression of Tet1 was particularly enhanced. Moreover, knockdown experiments revealed that the downregulation of Tet1 expression caused decreases in chondrogenesis markers such as type 2 and type 10 collagens. Furthermore, we found that TET proteins had a site preference for hydroxylation of 5mC on the Insulin-like growth factor 1 (Igf1) promoter in chondrocytes. Taken together, we showed that the expression of Tet1 was specifically facilitated in chondrocyte differentiation and Tet1 can regulate chondrocyte marker gene expression presumably through its hydroxylation activity for DNA., Highlights • The level of 5hmc increases concomitant with chondrocyte differentiation. • Among the TET family proteins, the expression of TET1 is specifically facilitated in chondrocyte differentiation. • Knock-down of TET1 leads to decrease in the expression of chondrocyte differentiation marker. • TET proteins have a site preference for hydroxylation of 5mC on the Igf1 promoter.

Bone mineral density (BMD) is a commonly used diagnostic indicator for bone fracture risk in osteoporosis. Along with low BMD, bone fragility accounts for reduced bone quality in addition to low BMD, but there is no diagnostic method to directly assess the bone quality. In this study, we investigated changes in bone quality using the Raman spectroscopic technique. Sciatic neurectomy (NX) was performed in male C57/BL6J mice (NX group) as a model of disuse osteoporosis, and sham surgery was used as an experimental control (Sham group). Eight months after surgery, we acquired Raman spectral data from the anterior cortical surface of the proximal tibia. We also performed a BMD measurement and micro-CT measurement to investigate the pathogenesis of osteoporosis. Quantitative analysis based on the Raman peak intensities showed that the carbonate/phosphate ratio and the mineral/matrix ratio were significantly higher in the NX group than in the Sham group. There was direct evidence of alterations in the mineral content associated with mechanical properties of bone. To fully understand the spectral changes, we performed principal component analysis of the spectral dataset, focusing on the matrix content. In conclusion, Raman spectroscopy provides reliable information on chemical changes in both mineral and matrix contents, and it also identifies possible mechanisms of disuse osteoporosis.

To detect the bone quality loss in osteoporosis, we performed Raman spectroscopic analysis of sciatic nerve resection (NX) mice. Eight months after surgery, lower limbs were collected from the mice and fixed with 70% ethanol. Raman spectra of anterior cortical surface of the proximal tibia at 5 points in each bone were measured by RENISHAW inVia Raman Microscope. Excitation wave length was 785 nm. We also performed DXA and micro CT measurement to confirm the bone mineral density and bone microstructure in the osteoporotic model induced by sciatic nerve resection. In the result of Raman spectroscopy, we detected changes of Raman peak intensity ratio in carbonate/phosphate, mineral/combined proline and hydroxyproline and mineral/phenylalanine. In addition, in the result of micro CT, we found significant changes in VOX BV/TV, Trabecular number, thickness, cancellous bone mineral density, cortical thickness and cortical bone mineral density. The results suggest that not only the bone mineral density but also bone quality reduced in the NX mice. We conclude that Raman spectroscopy is a useful for bone quality assessment as a complementary technique for conventional diagnostics.

Longitudinal bone growth progresses by continuous bone replacement of epiphyseal cartilaginous tissue, known as "growth plate", produced by columnar proliferated- and differentiated-epiphyseal chondrocytes. The endochondral ossification process at the growth plate is governed by paracrine signals secreted from terminally differentiated chondrocytes (hypertrophic chondrocytes), and hedgehog signaling is one of the best known regulatory signaling pathways in this process. Here, to investigate the developmental relationship between longitudinal endochondral bone formation and osteogenic progenitors under the influence of hedgehog signaling at the growth plate, genetic lineage tracing was carried out with the use of Gli1CreERT2 mice line to follow the fate of hedgehog-signal-responsive cells during endochondral bone formation. Gli1CreERT2 genetically labeled cells are detected in hypertrophic chondrocytes and osteo-progenitors at the chondro-osseous junction (COJ); these progeny then commit to the osteogenic lineage in periosteum, trabecular and cortical bone along the developing longitudinal axis. Furthermore, in ageing bone, where longitudinal bone growth ceases, hedgehog-signal responsiveness and its implication in osteogenic lineage commitment is significantly weakened. These results show, for the first time, evidence of the developmental contribution of endochondral progenitors under the influence of epiphyseal chondrocyte-derived secretory signals in longitudinally growing bone. This study provides a precise outline for assessing the skeletal lineage commitment of osteo-progenitors in response to growth-plate-derived regulatory signals during endochondral bone formation.

Nuclear factor-κB (NF-κB) proteins are transcription factors that play key roles in regulating most immune responses and cell death. Constitutively active NF-κB has been shown to exhibit chemoresistance by inducing anti-apoptosis in tumor cells. Multiple myeloma is known as a constitutive NF-κB activating disease, and the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma. We demonstrate here that DANFIN (N,N'-bis-(2,4-dimethyl-phenyl)-ethane-1,2-diamine) functions as an inhibitor of the p65 family proteins and induces chemosensitization to bortezomib in multiple myeloma. DANFIN was found to be an inhibitor of interactions between p65 and IκBα without the inhibition of the DNA binding activity of the p65 protein. In addition, DANFIN affected the IκBα binding region in Rel Homology Domain (RHD) and suppressed the nuclear translocalization of the p65 protein in cells. Furthermore, in multiple myeloma cells, DANFIN suppressed the expression level of NF-κB target genes and induced apoptosis. The combination therapy of DANFIN with bortezomib dramatically enhanced the apoptosis of multiple myeloma cells and indicated a remarkable anti-tumor effect in a multiple-myeloma xenograft mouse model.

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.

The acquisition of T cell functions seems to be closely linked to the reprogramming of the metabolic pathway. However, the impact of metabolic changes on the differentiation of helper T cell subsets remains unclear. We found that TCR-mediated activation of glutamine metabolism regulates Th2 cell differentiation via the supplementation of α-ketogulutalate (α-KG) and histone H3K27 demethylation. Deprivation of glutamine or pharmacological inhibition of glutamine metabolism blocks the induction of Th2 cell differentiation without affecting Stat6 phosphorylation. The methylation status of H3K27 at the Th2 cytokine gene locus was significantly increased in Th2 cells cultured under glutamine-deprived conditions. The inhibitory effect of glutamine-deprivation was antagonized by α-KG, and the α-KG-dependent induction of Th2 cell differentiation was reduced in utx- and jmjd3-deficient naïve CD4 T cells. These findings show that the glutamine-a-ketoglutarate axis is crucial to regulating the epigenetic status at the Th2 cytokine gene locus and subsequent Th2 cell differentiation.

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice (Uhrf1ΔLimb/ΔLimb) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1ΔLimb/ΔLimb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1, which is reported to be an IL-1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation such as Mmp13. These results indicate that Uhrf1 governs cell-type specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.

To evaluate the bone quality in the osteoporosis, we generated sciatic nerve resection (NX) mice as an osteoporosis model and analyzed by Raman spectroscopy. Raman spectra were measured in anterior cortical surface of the proximal tibia at 5 points in each bone. After that, the samples were fixed with 70% ethanol. We then performed DXA and μCT measurement. Raman peak intensity ratios were significantly different between NX and Control. Those changes in the Raman peak intensity ratios may reflect loss of bone quality in the osteoporosis model. Raman spectroscopy is a promising technique for measuring the bone quality and bone strength.

Clarification of the mechanisms underlying osteoclast differentiation enables us to understand the physiology of bone metabolism as well as the pathophysiology of bone diseases such as osteoporosis. Recently, it has been reported that epigenetics can determine cell fate and regulate cell type-specific gene expression. However, little is known about epigenetics during osteoclastogenesis. To reveal a part of epigenetics, especially focused on chromatin dynamics, during early osteoclastogenesis and to identify novel transcription factors involved in osteoclastogenesis, we performed a genome-wide analysis of open chromatin during receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis using DNase I hypersensitive sites sequencing (DNase-seq). DNase-seq was performed using the extracted nuclei from RAW264 cells treated with or without RANKL for 24 hours, followed by several bioinformatic analyses. DNase I hypersensitive sites (DHSs) were dynamically changed during RANKL-induced osteoclastogenesis and they accumulated in promoter regions. The distributions of DHSs among cis-regulatory DNA regions were identical regardless of RANKL stimulation. Motif discovery analysis successfully identified well-known osteoclastogenic transcription factors including Jun, CREB1, FOS, ATF2, and ATF4, but also novel transcription factors for osteoclastogenesis such as Zscan10, Atf1, Nrf1, and Srebf2. siRNA knockdown of these identified novel transcription factors impaired osteoclastogenesis. Taken together, DNase-seq is a useful tool for comprehension of epigenetics, especially chromatin dynamics during osteoclastogenesis and for identification of novel transcription factors involved in osteoclastogenesis. This study may reveal underlying mechanisms that determine cell type-specific differentiation of bone cells and may lead to investigation of novel therapeutic targets for osteoporosis.

Bone mass is regulated by various molecules including endogenous factors as well as exogenous factors, such as nutrients and pollutants. Aryl hydrocarbon receptor (AhR) is known as a dioxin receptor and is responsible for various pathological and physiological processes. However, the role of AhR in bone homeostasis remains elusive because the cell type specific direct function of AhR has never been explored in vivo. Here, we show the cell type specific function of AhR in vivo in bone homeostasis. Systemic AhR knockout (AhRKO) mice exhibit increased bone mass with decreased resorption and decreased formation. Meanwhile, osteoclast specific AhRKO (AhR(ΔOc/ΔOc)) mice have increased bone mass with reduced bone resorption, although the mice lacking AhR in osteoblasts have a normal bone phenotype. Even under pathological conditions, AhR(ΔOc/ΔOc) mice are resistant to sex hormone deficiency-induced bone loss resulting from increased bone resorption. Furthermore, 3-methylcholanthrene, an AhR agonist, induces low bone mass with increased bone resorption in control mice, but not in AhR(ΔOc/ΔOc) mice. Taken together, cell type specific in vivo evidence for AhR functions indicates that osteoclastic AhR plays a significant role in maintenance of bone homeostasis, suggesting that inhibition of AhR in osteoclasts can be beneficial in the treatment of osteoporosis.

The bulbocavernosus (BC) is a sexually dimorphic muscle observed only in males. Androgen receptor knockout mouse studies show the loss of BC formation. This suggests that androgen signaling plays a vital role in its development. Androgen has been known to induce muscle hypertrophy through satellite cell activation and myonuclei accretion during muscle regeneration and growth. Whether the same mechanism is present during embryonic development is not yet elucidated. To identify the mechanism of sexual dimorphism during BC development, the timing of morphological differences was first established. It was revealed that the BC was morphologically different between male and female mice at embryonic day (E) 16.5. Differences in the myogenic process were detected at E15.5. The male BC possesses a higher number of proliferating undifferentiated myoblasts. To identify the role of androgen signaling in this process, muscle-specific androgen receptor (AR) mutation was introduced, which resulted in no observable phenotypes. Hence, the expression of AR in the BC was examined and found that the AR did not colocalize with any muscle markers such as Myogenic differentiation 1, Myogenin, and paired box transcription factor 7. It was revealed that the mesenchyme surrounding the BC expressed AR and the BC started to express AR at E15.5. AR mutation on the nonmyocytic cells using spalt-like transcription factor 1 (Sall1) Cre driver mouse was performed, which resulted in defective BC formation. It was revealed that the number of proliferating undifferentiated myoblasts was reduced in the Sall1 Cre:ARL−/Y mutant embryos, and the adult mutants were devoid of BC. The transition of myoblasts from proliferation to differentiation is mediated by cyclin-dependent kinase inhibitors. An increased expression of p21 was observed in the BC myoblast of the Sall1 Cre:ARL−/Y mutant and wild-type female. Altogether this study suggests that the nonmyocytic AR may paracrinely regulate the proliferation of myoblast possibly through inhibiting p21 expression in myoblasts of the BC.

Estrogens are well known steroid hormones necessary to maintain bone health. In addition, mechanical loading, in which estrogen signaling may intersect with the Wnt/β-catenin pathway, is essential for bone maintenance. As osteocytes are known as the major mechanosensory cells embedded in mineralized bone matrix, osteocyte ERα deletion mice (ERα(ΔOcy/ΔOcy)) were generated by mating ERα floxed mice with Dmp1-Cre mice to determine the role of ERα in osteocytes. Trabecular bone mineral density of female, but not male ERα(ΔOcy/ΔOcy) mice was significantly decreased. Bone formation parameters in ERα(ΔOcy/ΔOcy) were significantly decreased while osteoclast parameters were unchanged. This suggests that ERα in osteocytes exerts osteoprotective function by positively controlling bone formation. To identify potential targets of ERα, gene array analysis of Dmp1-GFP osteocytes sorted by FACS from ERα(ΔOcy/ΔOcy) and control mice was performed. Gene expression microarray followed by gene ontology analyses revealed that osteocytes from ERα(ΔOcy/ΔOcy) highly expressed genes categorized in 'Secreted' when compared to control osteocytes. Among them, expression of Mdk and Sostdc1, both of which are Wnt inhibitors, was significantly increased without alteration of expression of the mature osteocyte markers such as Sost and β-catenin. Moreover, hindlimb suspension experiments showed that trabecular bone loss due to unloading was greater in ERα(ΔOcy/ΔOcy) mice without cortical bone loss. These data suggest that ERα in osteocytes has osteoprotective functions in trabecular bone formation through regulating expression of Wnt antagonists, but conversely plays a negative role in cortical bone loss due to unloading.

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.

Resistance training (RT) has been known to be effective in maintaining and improving bone strength, which is based on bone mineral density (BMD) and bone quality. However, it is not clear whether RT is effective in improving bone strength in patients with type‐2 diabetes mellitus (T2DM), who have a high risk of fracture. Therefore, we tested the effects of a 6‐week RT regimen using percutaneous electrical stimulation in T2DM model rats, male Otsuka Long‐Evans Tokushima Fatty (OLETF), and its control, Long‐Evans Tokushima Otsuka (LETO). After 6 weeks of RT, tibial BMD in RT legs was significantly higher than that in control (CON) legs in both groups. In diaphyseal cortical bone, bone area/tissue area, and cortical thickness was significantly increased in RT legs compared with CON legs in both groups. Cortical porosity was highly observed in OLETF compared with LETO, but RT improved cortical porosity in both groups. Interestingly, trabecular number, trabecular thickness and trabecular space as well as BMD and bone volume/tissue volume in proximal tibial metaphyseal trabecular bone were significantly improved in RT legs compared with CON legs in both groups. In contrast, connectivity density and structural model index were not affected by RT. These results indicate that the 6‐week RT regimen effectively increased BMD and improved bone quality in T2DM model rats as well as control rats. Therefore, RT may have the potential to improve bone strength and reduce fracture risk, even in patients with T2DM.

Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire−/− mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2. Taken together, Aire might play a role as an active regulator of chondrocyte differentiation, which leads to new insights into the regulatory mechanisms of chondrocyte differentiation.

sFRP4 is an extracellular Wnt antagonist that fine-tunes its signal activity by direct binding to Wnts. Bone fragility under oxidative stress by diabetes and aging is partly related to the suppression of the Wnt signal through upregulated sFRP4. Here, to explore the functions of sFRP4 as a balancer molecule in bone development and remodeling, we analyzed the sFRP4 knock-in mouse strain. X-gal and immunohistochemically stained signals in sFRP4-LacZ heterozygous mice were detectable in restricted areas, mostly in osteoblasts and osteoclasts, of the femoral diaphysis after neonatal and postnatal stages. Histological and μCT analyses showed increased trabecular bone mass with alteration of the Wnt signal and osteogenic activity in sFRP4 mutants; this augmented the effect of the buildup of trabecular bone during the ageing period. Our results indicate that sFRP4 plays a critical role in bone development and remodeling by regulating osteoblasts and osteoclasts and that its functional loss prevents age-related bone loss in the trabecular bone area. These findings imply that sFRP4 functions as a key potential endogenous balancer of the Wnt signaling pathway by efficiently having direct influence on both bone formation and bone absorption during skeletal bone development and maintenance through remodeling.

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.

Multinucleation is indispensable for the bone-resorbing activity of mature osteoclasts. Although multinucleation is evident in mature osteoclasts and certain other cell types, putative regulatory networks among nuclei remain poorly characterized. To address this issue, transcriptional activity of each nucleus in a multinucleated osteoclast was assessed by detecting the distributions of nuclear proteins by immunocytochemistry and primary transcripts by RNA FISH. Patterns of epigenetic histone markers governing transcription as well as localization of tested nuclear receptor proteins appeared indistinguishable among nuclei in differentiated Raw264 cells and mouse mature osteoclasts. However, RNAPII-Ser5P/2P and NFATc1 proteins were selectively distributed in certain nuclei in the same cell. Similarly, the distributions of primary transcripts for osteoclast-specific genes (Nfatc1, Ctsk and Acp5) as well as a housekeeping gene (beta-tubulin) were limited in certain nuclei within individual cells. By fusing two Raw264 cell lines that stably expressed ZsGreen-NLS and DsRed-NLS proteins, transmission of nuclear proteins across all of the nuclei in a cell could be observed, presumably through the shared cytoplasm. Taken together, we conclude that although nuclear proteins are diffusible among nuclei, only certain nuclei within a multinucleated osteoclast are transcriptionally active.