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Scoliosis, usually diagnosed in childhood and early adolescence, is an abnormal lateral curvature of the spine. L-type amino acid transporter 1 (LAT1), encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types. We previously demonstrated the pivotal role of LAT1 on bone homeostasis in mice, and the expression of LAT1/SLC7A5 in vertebral cartilage of pediatric scoliosis patients; however, its role in chondrocytes on spinal homeostasis and implications regarding the underlying mechanisms during the onset and progression of scoliosis, remain unknown. Here, we identified LAT1 in mouse chondrocytes as an important regulator of postnatal spinal homeostasis. Conditional inactivation of LAT1 in chondrocytes resulted in a postnatal-onset severe thoracic scoliosis at the early adolescent stage with normal embryonic spinal development. Histological analyses revealed that Slc7a5 deletion in chondrocytes led to general disorganization of chondrocytes in the vertebral growth plate, along with an increase in apoptosis and a decrease in cell proliferation. Furthermore, loss of Slc7a5 in chondrocytes activated the general amino acid control (GAAC) pathway but inactivated the mechanistic target of rapamycin complex 1 (mTORC1) pathway in the vertebrae. The spinal deformity in Slc7a5-deficient mice was corrected by genetic inactivation of the GAAC pathway, but not by genetic activation of the mTORC1 pathway. These findings suggest that the LAT1-GAAC pathway in chondrocytes plays a critical role in the maintenance of proper spinal homeostasis by modulating cell proliferation and survivability.

Extracellular signal-regulated kinase 5 (Erk5) belongs to the mitogen-activated protein kinase (MAPK) family. Previously, we demonstrated that Erk5 directly phosphorylates Smad-specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2Thr249) to activate its E3 ubiquitin ligase activity. Although we have clarified the importance of Erk5 in embryonic mesenchymal stem cells (MSCs) on skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. Leptin receptor-positive (LepR+) BM-MSCs represent a major source of bone in adult bone marrow and are critical regulators of postnatal bone homeostasis. Here, we identified Erk5 in BM-MSCs as an important regulator of bone homeostasis in adulthood. Bone marrow tissue was progressively osteosclerotic in mice lacking Erk5 in LepR+ BM-MSCs with age, accompanied by increased bone formation and normal bone resorption in vivo. Erk5 deficiency increased the osteogenic differentiation of BM-MSCs along with a higher expression of Runx2 and Osterix, essential transcription factors for osteogenic differentiation, without affecting their stemness in vitro. Erk5 deficiency decreased Smurf2Thr249 phosphorylation and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. The genetic introduction of the Smurf2T249E mutant (a phosphomimetic mutant) suppressed the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5-Smurf2Thr249 axis in BM-MSCs plays a critical role in the maintenance of proper bone homeostasis by preventing excessive osteogenesis in adult bone marrow.

PURPOSE To assess the performance of artificial intelligence in the automated classification of images taken with a tablet device of patients with blepharoptosis and subjects with normal eyelid. METHODS This is a prospective and observational study. A total of 1276 eyelid images (624 images from 347 blepharoptosis cases and 652 images from 367 normal controls) from 606 participants were analyzed. In order to obtain a sufficient number of images for analysis, 1 to 4 eyelid images were obtained from each participant. We developed a model by fully retraining the pre-trained MobileNetV2 convolutional neural network. Subsequently, we verified whether the automatic diagnosis of blepharoptosis was possible using the images. In addition, we visualized how the model captured the features of the test data with Score-CAM. k-fold cross-validation (k = 5) was adopted for splitting the training and validation. Sensitivity, specificity, and the area under the curve (AUC) of the receiver operating characteristic curve for detecting blepharoptosis were examined. RESULTS We found the model had a sensitivity of 83.0% (95% confidence interval [CI], 79.8-85.9) and a specificity of 82.5% (95% CI, 79.4-85.4). The accuracy of the validation data was 82.8%, and the AUC was 0.900 (95% CI, 0.882-0.917). CONCLUSION Artificial intelligence was able to classify with high accuracy images of blepharoptosis and normal eyelids taken using a tablet device. Thus, the diagnosis of blepharoptosis with a tablet device is possible at a high level of accuracy. TRIAL REGISTRATION Date of registration: 2021-06-25. TRIAL REGISTRATION NUMBER UMIN000044660. Registration site: https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000051004.

The mechanistic regulation of bone mass in aged animals is poorly understood. In this study, we examined the role of SIRT6, a longevity-associated factor, in osteocytes, using mice lacking Sirt6 in Dmp-1-expressing cells (cKO mice) and the MLO-Y4 osteocyte-like cell line. cKO mice exhibited increased osteocytic expression of Sost, Fgf23 and senescence inducing gene Pai-1 and the senescence markers p16 and Il-6, decreased serum phosphate levels, and low-turnover osteopenia. The cKO phenotype was reversed in mice that were a cross of PAI-1-null mice with cKO mice. Furthermore, senescence induction in MLO-Y4 cells increased the Fgf23 and Sost mRNA expression. Sirt6 knockout and senescence induction increased HIF-1α binding to the Fgf23 enhancer sequence. Bone mass and serum phosphate levels were higher in PAI-1-null aged mice than in wild-type mice. Therefore, SIRT6 agonists or PAI-1 inhibitors may be promising therapeutic options for aging-related bone metabolism disruptions.

Ess2, also known as Dgcr14, is a transcriptional co-regulator of CD4

Differentiation of mesenchymal stem cells (MSCs) into osteoblasts is a critical process for proper skeletal development and acquisition/maintenance of bone mass. However, since this regulatory mechanism has not yet been fully elucidated, the treatment of severe osteoporosis and fractures is a challenge. Here, through a comprehensive analysis of gene expression during the differentiation of MSCs into osteoblasts, we show that the forkhead transcription factor Foxf2 is a crucial regulator of this process. Foxf2 expression transiently increased during MSC osteoblastic differentiation. Overexpression of Foxf2 in MSCs inhibited osteoblastic differentiation, and conversely, knockdown of Foxf2 expression promoted this process. Osteoprogenitor-specific Foxf2 knockout mice developed a high bone mass phenotype due to increased bone formation. RNA-seq analysis and molecular experiments revealed that Foxf2 regulation of bone formation is mediated by Wnt2b. Knockdown of Foxf2 in mouse femurs enhanced bone regeneration in vivo. FOXF2 expression was correlated with hip bone mineral density in postmenopausal women with low bone mass. Finally, inhibition of FOXF2 promoted osteoblastic differentiation of human MSCs. This study uncovers a critical role of Foxf2 in the differentiation of MSCs into osteoblasts and provides insight into the pathogenesis associated with bone-related diseases such as osteoporosis and nonunion after fracture.

Satellite cell proliferation is an essential step in proper skeletal muscle development and muscle regeneration. However, the mechanisms regulating satellite cell proliferation are relatively unknown compared to the knowledge associated with the differentiation of satellite cells. Moreover, it is still unclear whether overload muscle fiber hypertrophy is dependent on satellite cell proliferation. In general, cell proliferation is regulated by the activity of cell cycle regulators, such as cyclins and cyclin-dependent kinases (CDKs). Despite recent reports on the function of CDKs and CDK inhibitors in satellite cells, the physiological role of Cdk1 in satellite cell proliferation remains unknown. Herein, we demonstrate that Cdk1 regulates satellite cell proliferation, muscle regeneration, and muscle fiber hypertrophy. Cdk1 is highly expressed in myoblasts and is downregulated upon myoblast differentiation. Inhibition of CDK1 activity inhibits myoblast proliferation. Deletion of Cdk1 in satellite cells leads to inhibition of muscle recovery after muscle injury due to reduced satellite cell proliferation in vivo. Finally, we provide direct evidence that Cdk1 expression in satellite cells is essential for overload muscle fiber hypertrophy in vivo. Collectively, our results demonstrate that Cdk1 is essential for myoblast proliferation, muscle regeneration, and muscle fiber hypertrophy. These findings could help to develop treatments for refractory muscle injuries and muscle atrophy, such as sarcopenia.

Sarcopenia is among the most common medical problems of the aging population worldwide and a major social concern. Here, we explored the therapeutic potential of TM5484, a novel orally available PAI-1 inhibitor, to prevent sarcopenia. The sarcopenic phenotypes of the calf muscle of 12- and 6-month-old middle-aged mice were compared. Although significant decline of isometric gastrocnemius muscle force was detected in the older untreated mice, those administered TM5484 had significantly greater calf muscle force, as determined using isometric measurements by electrical stimulation. Histological analysis indicated that cross-sectional gastrocnemius muscle fibers in untreated older mice were thinner than those in younger mice; however, TM5484-treated group showed thicker fibers than younger mice. Treatment with TM5484 for 6 months enhanced Igf1, Atrogin-1, Mt-Co1, and Chrna1 mRNA expression in the mice gastrocnemius muscle, with increased serum IGF-1 concentration. TM5484 induced dose-dependent Igf1, Atrogin-1, and Chrna1 expression in C2C12 myoblastic cells, confirming cell autonomous effect. Further, the presence of plasmin for 72 h caused significantly increased Igf1 expression in C2C12 cells. These findings suggest that oral PAI-1 inhibitors represent a promising therapeutic candidate for preventing sarcopenia progression in humans.

Nervous cell functions are known to be physiologically regulated by mechanical factors in the brain. However, it remains unclear whether mechanical interventions can modulate the pathophysiological processes underlying brain-related disorders and modify their consequences. Here we show that passive head motion of hypertensive rats, which reproduces mechanical accelerations generated at their heads during treadmill running at a moderate velocity, decreases the expression of angiotensin II type 1 receptor (AT1R) in astrocytes in their rostral ventrolateral medulla (RVLM). This decrease results in lowering their blood pressure. Passive head motion generates interstitial fluid movement that is estimated to exert shear stress with average magnitude of a few Pa on cells in rats’ brainstem. Fluid shear stress of a relevant magnitude decreases AT1R expression in cultured astrocytes, but not in neuronal cells. Furthermore, in hypertensive rats, inhibition of movement of interstitial fluid by its gelation with reactive polyethylene glycol injected into the RVLM eliminates the ability of passive head motion to decrease their blood pressure and AT1R expression in RVLM astrocytes. Consistent with these results from animal experiments, vertically oscillating chair riding of hypertensive adult humans, which reproduces mechanical accelerations generated at their heads during light jogging or fast walking, lowers their blood pressure. Our findings indicate that moderate mechanical impact on the head has an antihypertensive effect by modulating the function of RVLM astrocytes through interstitial fluid shear stress. We anticipate mechanical regulation to underlie a variety of positive effects of physical exercise on human health, particularly those related to brain functions.

The aim of this study was to examine the effect of long-term locking plate fixation on the cortical bone of the canine radius. Locking compression plates were fixed to the left and right radius in dogs (n = 3). The left radius was fixed with a locking head screw (Locking Plate group, LP). The locking compression plate was compressed periosteally in the right radius using a cortex screw (Compression Plate group, CP). Radial bones from dogs that were euthanized for other purposes were collected as an untreated control group (Control group). After euthanasia at 36 weeks following plate fixation, radial bones were evaluated for bone mineral density and underwent histological analysis. Bone metabolic markers were analyzed by quantitative polymerase chain reaction (qPCR). Statistical analyses were performed for comparisons between groups. The LP group showed no significant difference in bone mineral density after plate fixation, whereas the CP group showed significantly lower bone mineral density. Histological analysis indicated that the number of osteoclasts and rate of empty lacunae increased significantly in the CP group relative to the Control and LP groups. qPCR analysis indicated increased expression of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-6, and tumor necrosis factor ligand superfamily member 11 in the CP group, whereas Runt-related transcription factor 2, an osteoblast marker, was similar in all groups. The expression of hypoxia-inducible factor-1α in the CP group was also increased relative to that in the Control and LP groups. Thus, locking plate fixation is a biologically superior fixation method that does not cause implant-induced osteoporosis in the bone in the long term.

Leptin regulates bone remodeling through the central nervous system, in particular, the β-adrenergic system. β2-adrenergic receptor signaling in osteoblasts inhibits osteoblast differentiation and regulates RANKL expression and then increases bone resorption. Furthermore, β-blockers increase bone mass in animal models and humans. In some human epidemiologic studies, selective β1-blockers have been reported to have a more beneficial effect in protecting bone reduction in postmenopausal women than nonselective β-blockers. In contrast, the parasympathetic nervous system positively regulates bone remodeling via regulation of the sympathetic nervous system. The results from these studies have provided us with important information for the treatment of osteoporosis by targeting the adrenergic system with agonists and antagonists.

Summary: Mechanical stimuli including loading after birth promote bone growth. However, little is known about how mechanical force triggers biochemical signals to regulate bone growth. Here, we identified a periosteal-osteoblast-derived secretory peptide, Osteocrin (OSTN), as a mechanotransducer involved in load-induced long bone growth. OSTN produced by periosteal osteoblasts regulates growth plate growth by enhancing C-type natriuretic peptide (CNP)-dependent proliferation and maturation of chondrocytes, leading to elongation of long bones. Additionally, OSTN cooperates with CNP to regulate bone formation. CNP stimulates osteogenic differentiation of periosteal osteoprogenitors to induce bone formation. OSTN binds to natriuretic peptide receptor 3 (NPR3) in periosteal osteoprogenitors, thereby preventing NPR3-mediated clearance of CNP and consequently facilitating CNP-signal-mediated bone growth. Importantly, physiological loading induces Ostn expression in periosteal osteoblasts by suppressing Forkhead box protein O1 (FoxO1) transcription factor. Thus, this study reveals a crucial role of OSTN as a mechanotransducer converting mechanical loading to CNP-dependent bone formation.

Osteoporosis is a progressive bone disease caused by an imbalance between bone resorption and formation. Recently, plasminogen activator inhibitor-1 (PAI-1) was shown to play an important role in bone metabolism using PAI-1-deficient mice. In this study, we evaluated the therapeutic benefits of novel, orally available small-molecule PAI-1 inhibitor (iPAI-1) in an estrogen deficiency-induced osteoporosis model. Eight-week-old C57BL/6J female mice were divided into three groups: a sham + vehicle (Sham), ovariectomy + vehicle (OVX + v), and OVX + iPAI-1 (OVX + i) group. iPAI-1 was administered orally each day for 6 weeks starting the day after the operation. Six weeks of iPAI-1 treatment prevented OVX-induced trabecular bone loss in both the femoral bone and lumbar spine. Bone formation activity was significantly higher in the OVX + i group than in the OVX + v and Sham groups. Unexpectedly, OVX-induced osteoclastogenesis was partially, but significantly reduced. Fluorescence-activated cell sorting analyses indicated that the number of bone marrow stromal cells was higher in the OVX + i group than that in the OVX + v group. A colony-forming unit-osteoblast assay indicated enhanced mineralized nodule formation activity in bone marrow cells isolated from iPAI-1-treated animals. Bone marrow ablation analysis indicated that the remodeled trabecular bone volume was significantly higher in the iPAI-1-treated group than that in the control group. In conclusion, our results suggest PAI-1 blockade via a small-molecule inhibitor is a new therapeutic approach for the anabolic treatment of postmenopausal osteoporosis.

We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]-induced receptor activator of nuclear factor κB ligand (Rankl) expression was more strongly enhanced in both Bmal1-deficient bone and cultured osteoblasts, whereas overexpression of Bmal1/Clock conversely inhibited it in osteoblasts. These results suggest that bone resorption and bone mass are regulated at a sophisticated level by osteoblastic Clock system through a mechanism relevant to the modulation of 1,25(OH)2 D3 -induced Rankl expression in osteoblasts. © 2017 American Society for Bone and Mineral Research.

L-type amino acid transporter 1 (LAT1), which is encoded by solute carrier transporter 7a5 (Slc7a5), plays a crucial role in amino acid sensing and signaling in specific cell types, contributing to the pathogenesis of cancer and neurological disorders. Amino acid substrates of LAT1 have a beneficial effect on bone health directly and indirectly, suggesting a potential role for LAT1 in bone homeostasis. Here, we identified LAT1 in osteoclasts as important for bone homeostasis. Slc7a5 expression was substantially reduced in osteoclasts in a mouse model of ovariectomy-induced osteoporosis. The osteoclast-specific deletion of Slc7a5 in mice led to osteoclast activation and bone loss in vivo, and Slc7a5 deficiency increased osteoclastogenesis in vitro. Loss of Slc7a5 impaired activation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway in osteoclasts, whereas genetic activation of mTORC1 corrected the enhanced osteoclastogenesis and bone loss in Slc7a5-deficient mice. Last, Slc7a5 deficiency increased the expression of nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1) and the nuclear accumulation of NFATc1, a master regulator of osteoclast function, possibly through the canonical nuclear factor κB pathway and the Akt-glycogen synthase kinase 3β signaling axis, respectively. These findings suggest that the LAT1-mTORC1 axis plays a pivotal role in bone resorption and bone homeostasis by modulating NFATc1 in osteoclasts, thereby providing a molecular connection between amino acid intake and skeletal integrity.

During development of long bones, two mechanistically distinct processes contribute to long- and short-axis growth. Endochondral ossification in the growth plate leads to the long-axis growth, while intramembranous ossification including apposition in the periosteum regulates the short axis growth. Here, we show that periosteal osteoblast-derived secretory peptide, Osteocrin (OSTN), promotes both types of long bone growth through potentiation of signaling by C-type natriuretic peptide (CNP), because OSTN inhibits the clearance of CNP by binding to natriuretic peptide receptor 3 (NPR3). The mice lacking OSTN showed less bone mass in trabecular and cortical regions than the control mice, suggesting the dual functions of OSTN in long bone growth. We found that OSTN regulated trabecular bone formation by inducing proliferation and maturation of chondrocytes possibly through enhancing CNP-dependent signaling. Besides the contribution of OSTN to long axis growth, we demonstrated that OSTN together with CNP induced osteoblast differentiation of periosteum-derived multipotent progenitor cells expressing NPR3. These data suggest that OSTN induces long bone growth through endochondral ossification and osteoblast specification of multipotent progenitor cells in the periosteum.

Bone is one of common metastasis sites for many types of cancer. In bone metastatic microenvironment, tumor-bone interactions play a significant role in the regulation of osteolytic or osteoblastic bone metastasis. In order to investigate the direct interaction between tumor cells and bone tissue, it is essential to generate appropriate animal models that mimic the behavior of tumor cells in bone metastatic lesions. Calvarial implantation model (bone invasion model) is a newly-established animal model that accurately recapitulates the behavior of tumor cells in the tumor-bone microenvironment. The surgical technique for tumor cell implantation is simpler than intracardiac, intra-arterial, or intraosseous injection techniques. This model can be useful for the identification of key factors driving tumor-induced osteolytic or osteoblastic changes.

In the past decade, a growing importance has been placed on understanding the significance of long noncoding RNAs (lncRNAs) in regulating development, metabolism, and homeostasis. Osteoblast proliferation and differentiation are essential elements in skeletal development, bone metabolism, and homeostasis. However, the underlying mechanisms of lncRNAs in the process of osteoblast proliferation and differentiation remain largely unknown. Through comprehensive analysis of lncRNAs during bone formation, we show that colorectal neoplasia differentially expressed (Crnde), previously viewed as a cancer-related lncRNA, is an important regulator of osteoblast proliferation and differentiation. Crnde was found to be expressed in osteoblasts, and its expression was induced by parathyroid hormone. Furthermore, Crnde knockout mice developed a low bone mass phenotype due to impaired osteoblast proliferation and differentiation. Overexpression of Crnde in osteoblasts promoted their proliferation, and conversely, reduced Crnde expression inhibited osteoblast proliferation. Although ablation of Crnde inhibited osteoblast differentiation, overexpression of Crnde restored it. Finally, we provided evidence that Crnde modulates bone formation through Wnt/β-catenin signaling. Therefore, our data suggest that Crnde is a novel regulator of bone metabolism.

Bone mass is maintained by a balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. Although recent genetic studies have uncovered various mechanisms that regulate osteoblast differentiation, the molecular basis of osteoblast proliferation remains unclear. Here, using an osteoblast-specific loss-of-function mouse model, we demonstrate that cyclin-dependent kinase 1 (Cdk1) regulates osteoblast proliferation and differentiation. Quantitative RT-PCR analyses revealed that Cdk1 is highly expressed in bone and is down-regulated upon osteoblast differentiation. We also noted that Cdk1 is dispensable for the bone-anabolic effects of parathyroid hormone (PTH). Cdk1 deletion in osteoblasts led to osteoporosis in adult mice due to low bone formation, but did not affect osteoclast formation in vivo. Cdk1 overexpression in osteoblasts promoted proliferation, and conversely, Cdk1 knockdown inhibited osteoblast proliferation and promoted differentiation. Of note, we provide direct evidence that PTH's bone-anabolic effects occur without enhancing osteoblast proliferation in vivo. Furthermore, we found that Cdk1 expression in osteoblasts is essential for bone fracture repair. These findings may help reduce the risk of nonunion after bone fracture and identify patients at higher risk for nonresponse to PTH treatment. Collectively, our results indicate that Cdk1 is essential for osteoblast proliferation and that it functions as a molecular switch that shifts osteoblast proliferation to maturation. We therefore conclude that Cdk1 plays an important role in bone formation.

Significance Prostate cancer is one of most common cancers in men worldwide, and osteoblastic bone metastasis is frequently observed in prostate cancer patients. However, the mechanisms responsible for the predominantly osteoblastic phenotype have not been fully elucidated. Cancer-secreted microRNAs (miRNAs) were recently shown to be significant in the modification of the tumor microenvironment. Here, hsa-miR-940, which was highly secreted by prostate cancer cells, promoted osteogenic differentiation of human mesenchymal stem cells in vitro, and induced extensive osteoblastic lesions in the bone metastatic microenvironment in vivo. Our study provides a demonstration that osteoblastic bone metastasis can be induced by miRNAs secreted by cancer cells in the bone microenvironment., Bone metastatic lesions are classified as osteoblastic or osteolytic lesions. Prostate and breast cancer patients frequently exhibit osteoblastic-type and osteolytic-type bone metastasis, respectively. In metastatic lesions, tumor cells interact with many different cell types, including osteoblasts, osteoclasts, and mesenchymal stem cells, resulting in an osteoblastic or osteolytic phenotype. However, the mechanisms responsible for the modification of bone remodeling have not been fully elucidated. MicroRNAs (miRNAs) are transferred between cells via exosomes and serve as intercellular communication tools, and numerous studies have demonstrated that cancer-secreted miRNAs are capable of modifying the tumor microenvironment. Thus, cancer-secreted miRNAs can induce an osteoblastic or osteolytic phenotype in the bone metastatic microenvironment. In this study, we performed a comprehensive expression analysis of exosomal miRNAs secreted by several human cancer cell lines and identified eight types of human miRNAs that were highly expressed in exosomes from osteoblastic phenotype-inducing prostate cancer cell lines. One of these miRNAs, hsa-miR-940, significantly promoted the osteogenic differentiation of human mesenchymal stem cells in vitro by targeting ARHGAP1 and FAM134A. Interestingly, although MDA-MB-231 breast cancer cells are commonly known as an osteolytic phenotype-inducing cancer cell line, the implantation of miR-940–overexpressing MDA-MB-231 cells induced extensive osteoblastic lesions in the resulting tumors by facilitating the osteogenic differentiation of host mesenchymal cells. Our results suggest that the phenotypes of bone metastases can be induced by miRNAs secreted by cancer cells in the bone microenvironment.

Objective Bone mass is maintained through a balance of bone formation and resorption. This homeostatic balance is regulated by various systems involving humoral and local factors. The discovery that the anorexigenic hormone leptin regulates bone mass via neuronal pathways revealed that neurons and neuropeptides are intimately involved in bone homeostasis. Ghrelin is a stomach-derived orexigenic hormone that counteracts leptin's action. However, the physiological role of ghrelin in bone homeostasis remains unknown. In this study, through the global knockout of ghrelin receptor (Ghsr) followed by tissue-specific re-expression, we addressed the molecular basis of the action of ghrelin in bone remodeling in vivo . Methods We performed molecular, genetic and cell biological analyses of Ghsr-null mice and Ghsr-null mice with tissue specific Ghsr restoration. Furthermore, we evaluated the molecular mechanism of ghrelin by molecular and cell-based assays. Results Ghsr-null mice showed a low bone mass phenotype with poor bone formation. Restoring the expression of Ghsr specifically in osteoblasts, and not in osteoclasts or the central nervous system, ameliorated bone abnormalities in Ghsr-null mice. Cell-based assays revealed ghrelin induced the phosphorylation of CREB and the expression of Runx2, which in turn accelerated osteoblast differentiation. Conclusions Our data show that ghrelin regulates bone remodeling through Ghsr in osteoblasts by modulating the CREB and Runx2 pathways.

Vitamin E is a lipid-soluble antioxidant that inhibits lipid peroxidation by scavenging reactive oxygen species, and it is thought to protect against the aging process. Indeed, it is one of the most popular supplements in the US. However, recent studies have revealed that vitamin E has dual effects on the aging process. We discovered that α-tocopherol, the major form of vitamin E in the body, stimulates osteoclast fusion and bone resorption as well as induces an osteoporosis-like phenotype in rodents. Clinical intervention trials have also demonstrated that supplementation with vitamin E is neutral or even harmful for preventing age-related diseases in humans. Therefore, the role of vitamin E as an ‘anti-ager' has been called into question. This review outlines the present understanding of the role of vitamin E in age-related disease prevention.

Intervertebral disc degeneration (IVDD) greatly affects the quality of life. The nucleus pulposus (NP) of chondrodystrophic dog breeds (CDBs) is similar to the human NP because the cells disappear with age and are replaced by fibrochondrocyte-like cells. Because IVDD develops as early as within the first year of life, we used canines as a model to investigate the in vitro mechanisms underlying IVDD. The mechanism underlying age-related IVDD, however, is poorly understood. Several research groups have suggested that Wnt/β-catenin signaling plays an important role in IVDD. However, the role of Wnt/β-catenin signals in IVD cells is not yet well understood. Here, we demonstrate that Wnt/β-catenin signaling could enhance Runx2 expression in IVDD and lead to IVD calcification. Nucleus pulposus (NP) tissue was obtained from Beagle dogs after evaluation of the degeneration based on magnetic resonance imaging (MRI). Histological analysis showed that lack of Safranin-O staining, calcified area, and matrix metalloproteinase (MMP) 13-positive cells increased with progression of the degeneration. Furthermore, the levels of β-catenin- and Runx2-positive cells also increased. Real-time reverse-transcription polymerase chain reaction analysis showed that the MRI signal intensity and mRNA expression levels of β-catenin and Runx2 are correlated in NP tissues. Moreover, supplementation of LiCl induced β-catenin accumulation and Runx2 expression. In contrast, FH535 inhibited LiCl-induced upregulation. These results suggest that Runx2 transcript and protein expression, potentially in combination with β-catenin accumulation, are enhanced in degenerated and calcified intervertebral discs of CDBs.

In this study, we examined the antimicrobial susceptibility of the enterococci isolated from dogs and cats in Japan during 2011–2012. Fecal samples were collected from 84 dogs and 16 cats that underwent antibiotic treatment. Enterococci were detected in 70 of 84 dogs (83.3%) and 7 of 16 cats (43.8%). The most prevalent Enterococcus species was Enterococcus faecalis (64.9%); Enterococccus faecium and Enterococcus durans were also isolated from 14 of 77 (18.2%) and 5 of 77 (6.5%) of these animals, respectively. The most active resistance was observed for erythromycin (44.2%) and oxytetracycline (44.2%), and there was considerable resistance to lincomycin (41.6%), gentamicin (31.2%) and kanamycin (31.2%). Compared with the results of a similar study conducted in 2006 and 2007, enterococci susceptibility to enrofloxacin and ampicillin had significantly increased. Enterococcus gallinarum harboring vanC1 and Enterococcus casseliflavus harboring vanC2/3 were isolated from 4 of 77 enterococcal isolates. However, no enterococcal isolates were resistant to vancomycin. Multidrug resistance was found for as few as two and as many as nine antimicrobials regardless of the class. These results demonstrate that dogs and cats treated with antibiotics are commonly colonized with antimicrobial-resistant enterococci.

We have previously shown that endochondral ossification is finely regulated by the Clock system expressed in chondrocytes during postnatal skeletogenesis. Here we show a sophisticated modulation of bone resorption and bone mass by the Clock system through its expression in bone-forming osteoblasts. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and Period1 (Per1) were expressed with oscillatory rhythmicity in the bone in vivo, and circadian rhythm was also observed in cultured osteoblasts of Per1::luciferase transgenic mice. Global deletion of murine Bmal1, a core component of the Clock system, led to a low bone mass, associated with increased bone resorption. This phenotype was recapitulated by the deletion of Bmal1 in osteoblasts alone. Co-culture experiments revealed that Bmal1-deficient osteoblasts have a higher ability to support osteoclastogenesis. Moreover, 1α,25-dihydroxyvitamin D

Pericytes are mesenchymal cells that surround the endothelial cells of small vessels in various organs. These cells express several markers, such as NG2, CD146, and PDGFR&beta, and play an important role in the stabilization and maturation of blood vessels. It was also recently revealed that like mesenchymal stem cells (MSCs), pericytes possess multilineage differentiation capacity, especially myogenic, adipogenic, and fibrogenic differentiation capacities. Although some previous studies have reported that pericytes also have osteogenic potential, the osteogenesis of pericytes can still be further elucidated. In the present study, we established novel methods for isolating and culturing primary murine pericytes. An immortalized pericyte line was also established. Multilineage induction of the pericyte line induced osteogenesis, adipogenesis, and chondrogenesis of the cells in vitro. In addition, pericytes that were injected into the fracture site of a bone fracture mouse model contributed to callus formation. Furthermore, in vivo pericyte-lineage-tracing studies demonstrated that endogenous pericytes also differentiate into osteoblasts and osteocytes and contribute to bone fracture healing as a cellular source of osteogenic cells. Pericytes can be a promising therapeutic candidate for treating bone fractures with a delayed union or nonunion as well as bone diseases causing bone defects.

Global gene deletion studies in mice and humans have established the pivotal role of runt related transcription factor-2 (Runx2) in both intramembranous and endochondral ossification processes during skeletogenesis. In this study, we for the first time generated mice carrying a conditional Runx2 allele with exon 4, which encodes the Runt domain, flanked by loxP sites. These mice were crossed with α1(I)-collagen-Cre or α1(II)-collagen-Cre transgenic mice to obtain osteoblast-specific or chondrocyte-specific Runx2 deficient mice, respectively. As seen in Runx2(-/-) mice, perinatal lethality was observed in α1(II)-Cre;Runx2(flox/flox) mice, but this was not the case in animals in which α1(I)-collagen-Cre was used to delete Runx2. When using double-staining with Alizarin red for mineralized matrix and Alcian blue for cartilaginous matrix, we observed previously that mineralization was totally absent at embryonic day 15.5 (E15.5) throughout the body in Runx2(-/-) mice, but was found in areas undergoing intramembranous ossification such as skull and clavicles in α1(II)-Cre;Runx2(flox/flox) mice. In newborn α1(II)-Cre;Runx2(flox/flox) mice, mineralization impairment was restricted to skeletal areas undergoing endochondral ossification including long bones and vertebrae. In contrast, no apparent skeletal abnormalities were seen in mutant embryo, newborn, and 3-week-old to 6-week old-mice in which Runx2 had been deleted with the α1(I)-collagen-Cre driver. These results suggest that Runx2 is absolutely required for endochondral ossification during embryonic and postnatal skeletogenesis, but that disrupting its expression in already committed osteoblasts as achieved here with the α1(I)-collagen-Cre driver does not affect overtly intramembranous and endochondral ossification. The Runx2 floxed allele established here is undoubtedly useful for investigating the role of Runx2 in particular cells.