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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.

Osteocytes are master regulators of skeletal homeostasis. However, little is known about the molecular mechanism of their differentiation. Epigenetic regulations, especially H3K27me3 modification, play critical roles in cell differentiation. Here, we found that H3K27me3 in the loci of osteocyte-expressing genes decreased during osteocyte differentiation and that H3K27me3 demethylase, Utx, was bound to the loci of those genes. To investigate the physiological functions of Utx in vivo, we generated late osteoblast-to-osteocyte specific Utx knockout mice using Dmp1-cre mice (Utx

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.

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.

Background: Vitamin D and calcium are essential nutrients for bone health. In addition, vitamin D suppresses inflammatory cytokines and increases bone resorption. Therefore, improvements in bone health by calcium and vitamin D supplementation have the potential to not only improve calcium metabolism but also suppress inflammation associated with exercise training. The purpose of this study was to determine whether ongoing vitamin D supplementation and low-fat milk intake by female high-school endurance runners would improve bone metabolism by suppressing inflammatory cytokines and the parathyroid hormone (PTH). Methods: Twenty female high-school runners were assigned to a vitamin D supplement and low-fat milk intake group (MKD) or a control group (CON). Participants in the MKD group consumed a vitamin D supplement (1,000 IU/day) and low-fat milk (Ca 315 mg/day) for 6 months. Bone mineral density measurements, blood samples, and questionnaires (regarding menses and diet) were carried out. The UMIN Clinical Trials Registry number is UMIN000027854. Results: The 25-hydroxyvitamin D (25(OH)D) concentration in MKD was sustained and PTH concentration was decreased regardless of the state of menses. The correlation coefficients of 25(OH)D or PTH concentrations and bone metabolism markers were analyzed by partial correlation coefficient via adjusting the model for frequency of menses. CTX and 25(OH)D concentration were significantly and inversely correlated at baseline (r = -0.61, P < 0.01), 3 months (r = -0.54, P = 0.02), and 6 months (r = -0.53, P = 0.02). CTX and PTH were significantly and positively correlated at 3 months (r = 0.63, P < 0.01) and 6 months (r = 0.52, P = 0.02). The bone alkaline phosphatase (BAP)/CTX ratio and 25(OH)D concentration were significantly and positively correlated at 3 months (r = 0.59, P = 0.01) and 6 months (r = 0.56, P = 0.01). The BAP/CTX ratio and PTH were significantly and inversely correlated at 3 months (r = -0.59, P = 0.01) and 6 months (r = -0.58, P < 0.01). Conclusions: This study suggested that vitamin D and low-fat milk supplementation improves bone metabolism by sustaining the 25(OH)D concentration and decreasing the PTH concentration in female high-school endurance runners regardless of the state of menses. J Clin Med Res. 2018;10(1):13-21 doi: https://doi.org/10.14740/jocmr3209w

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.

We aimed to compare site-specific bone mineral densities (BMDs) between adolescent endurance runners and sprinters and examine the relationship of fat-free mass (FFM) and nutrient intake on BMD. In this cross-sectional study, 37 adolescent female endurance runners and sprinters (16.1 ± 0.8 years) were recruited. BMD and FFM were assessed by dual-energy X-ray absorptiometry. Nutrient intake and menstrual state were evaluated by questionnaires. After adjusting for covariates, spine and total bone less head (TBLH) BMDs were significantly higher in sprinters than endurance runners (TBLH, 1.02 ± 0.05 vs. 0.98 ± 0.06 g/cm2; spine, 0.99 ± 0.06 vs. 0.94 ± 0.06 g/cm2; p < 0.05). There was no significant difference between groups in other sites. The rate of menstrual abnormality was higher in endurance runners compared with sprinters (56.3% vs. 23.8%; p < 0.05). FFM was a significant covariate for BMD on all sites except the spine (p < 0.05). Dietary intake of vitamin D was identified as a significant covariate only for pelvic BMD (p < 0.05). The BMDs of different sites among endurance runners and sprinters were strongly related to FFM. However, the association of FFM with spine BMD cannot be explained by FFM alone. Other factors, including nutrition and/or mechanical loading, may affect the spine BMD.

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.