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Amino acids have recently emerged as important regulators of osteoblast differentiation and bone formation. Osteoblasts require a continuous supply of amino acids to sustain biomass production to fuel cell proliferation, osteoblast differentiation and bone matrix production. We recently identified proline as an essential amino acid for bone development by fulfilling unique synthetic demands that are associated with osteoblast differentiation. Osteoblasts rely on the amino acid transporter SLC38A2 to provide proline to fuel endochondral ossification. Despite this, very little is known about the function or substrates of SLC38A2 during bone homeostasis. Here we demonstrate that the neutral amino acid transporter SLC38A2 is expressed in osteoblast lineage cells and provides proline and alanine to osteoblast lineage cells. Genetic ablation of SLC38A2 using Prrx1Cre results in decreased bone mass in both male and female mice due to a reduction in osteoblast numbers and bone forming activity. Decreased osteoblast numbers are attributed to impaired proliferation and osteogenic differentiation of skeletal stem and progenitor cells. Collectively, these data highlight the necessity of SLC38A2- mediated proline and alanine uptake during postnatal bone formation and bone homeostasis. [ABSTRACT FROM AUTHOR]

Introduction: Type 2 diabetes mellitus (T2DM) is well-known to be associated with normal bone density but, concurrently, low bone turnover and increased risk for fracture. One of the proposed mechanisms is possible derangement in bone precursor cells, which could be represented by deficiencies in circulating osteogenic progenitor (COP) cells and osteoclast precursors (OCP). The objective of our study is to understand whether extent of glycemic control has an impact on these cells, and to identify other factors that may as well. Methods: This was a secondary analysis of baseline data from 51 male participants, aged 37-65 in an ongoing clinical trial at Michael E. DeBakey VA Medical Center, Houston, Texas, USA. At study entry serum Hemoglobin A1c was measured by high-performance liquid chromatography osteocalcin (OCN) and C-terminal telopeptide of type 1 collagen (CTx) were measured by ELISA, and testosterone and estradiol by liquid-chromatography/mass-spectrometry. Areal bone mineral density (BMD), trabecular bone score and body composition were measured by dual energy x-ray absorptiometry, while COP and OCP were measured by flow cytometry. Results: When adjusted for serum testosterone, parathyroid hormone, and 25-hydroxyvitamin D, those with poor long-term glycemic control had significantly higher percentage of COP (p = 0.04). COP correlated positively with visceral adipose tissue (VAT) volume (r = 0.37, p = 0.01) and negatively with free testosterone (r = -0.28, p = 0.05) and OCN (r = -0.28, p = 0.07), although only borderline for the latter. OCP correlated positively with age, FSH, lumbar spine BMD, and COP levels, and negatively with glucose, triglycerides, and free estradiol. Multivariable regression analyses revealed that, in addition to being predictors for each other, another independent predictor for COP was VAT volume while age, glucose, and vitamin D for OCP. Conclusion: Our results suggest that high COP could be a marker of poor metabolic control. However, given the complex nature and the multitude of factors influencing osteoblastogenesis/adipogenesis, it is possible that the increase in COP is a physiologic response of the bone marrow to increased osteoblast apoptosis from poor glycemic control. Alternatively, it is also likely that a metabolically unhealthy profile may retard the development of osteogenic precursors to fully mature osteoblastic cells. [ABSTRACT FROM AUTHOR]

Multiple myeloma is an incurable haematological cancer. The increase in targeted therapies has improved the number of myeloma patients achieving a complete response and improved progression-free survival following therapy. However, a low level of disease or minimal residual disease (MRD) still persists which contributes to the inevitable relapse in myeloma patients. MRD has been attributed to the presence of dormant myeloma cells and their subsequent reactivation, which is controlled by the microenvironment and specialised niches within the bone marrow. This contributes to the evasion of the immune system and chemotherapy, eventually leading to relapse. The growth of myeloma tumours are heavily dependent on environmental stimuli from the bone marrow microenvironment, and this plays a key role in myeloma progression. The bone microenvironment also plays a critical role in myeloma bone disease and the development of skeletal-related events. This review focuses on the bone marrow microenvironment in relation to myeloma pathogenesis and cancer dormancy. Moreover, it reviews the current therapies targeting the bone microenvironment to treat myeloma and myeloma bone disease. Lastly, it identifies novel therapeutic targets for myeloma treatment and the associated bone disease. [ABSTRACT FROM AUTHOR]

Osteoporosis is a bone metabolic disorder characterized by decreased bone density and deteriorated microstructure, which increases the risk of fractures. The imbalance between bone formation and bone resorption results in the occurrence and progression of osteoporosis. Osteoblast-mediated bone formation, osteoclast-mediated bone resorption and macrophage-regulated inflammatory response play a central role in the process of bone remodeling, which together maintain the balance of the osteoblast-osteoclast-macrophage (OB-OC-MΦ) axis under physiological conditions. Bone formation and bone resorption disorders caused by the imbalance of OB-OC-MΦ axis contribute to osteoporosis. Many microRNAs are involved in the regulation of OB-OC-MΦ axis homeostasis, with microRNA-23a (miR-23a) being particularly crucial. MiR-23a is highly expressed in the pathological process of osteoporosis, which eventually leads to the occurrence and further progression of osteoporosis by inhibiting osteogenesis, promoting bone resorption and inflammatory polarization of macrophages. This review focuses on the role and mechanism of miR-23a in regulating the OB-OC-MΦ axis to provide new clinical strategies for the prevention and treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

IntroductionType 2 diabetes mellitus (T2DM) is well-known to be associated with normal bone density but, concurrently, low bone turnover and increased risk for fracture. One of the proposed mechanisms is possible derangement in bone precursor cells, which could be represented by deficiencies in circulating osteogenic progenitor (COP) cells and osteoclast precursors (OCP). The objective of our study is to understand whether extent of glycemic control has an impact on these cells, and to identify other factors that may as well.MethodsThis was a secondary analysis of baseline data from 51 male participants, aged 37-65 in an ongoing clinical trial at Michael E. DeBakey VA Medical Center, Houston, Texas, USA. At study entry serum Hemoglobin A1c was measured by high-performance liquid chromatography osteocalcin (OCN) and C-terminal telopeptide of type 1 collagen (CTx) were measured by ELISA, and testosterone and estradiol by liquid-chromatography/mass-spectrometry. Areal bone mineral density (BMD), trabecular bone score and body composition were measured by dual energy x-ray absorptiometry, while COP and OCP were measured by flow cytometry.ResultsWhen adjusted for serum testosterone, parathyroid hormone, and 25-hydroxyvitamin D, those with poor long-term glycemic control had significantly higher percentage of COP (p = 0.04). COP correlated positively with visceral adipose tissue (VAT) volume (r = 0.37, p = 0.01) and negatively with free testosterone (r = -0.28, p = 0.05) and OCN (r = -0.28, p = 0.07), although only borderline for the latter. OCP correlated positively with age, FSH, lumbar spine BMD, and COP levels, and negatively with glucose, triglycerides, and free estradiol. Multivariable regression analyses revealed that, in addition to being predictors for each other, another independent predictor for COP was VAT volume while age, glucose, and vitamin D for OCP.ConclusionOur results suggest that high COP could be a marker of poor metabolic control. However, given the complex nature and the multitude of factors influencing osteoblastogenesis/adipogenesis, it is possible that the increase in COP is a physiologic response of the bone marrow to increased osteoblast apoptosis from poor glycemic control. Alternatively, it is also likely that a metabolically unhealthy profile may retard the development of osteogenic precursors to fully mature osteoblastic cells.

Multiple myeloma is an incurable haematological cancer. The increase in targeted therapies has improved the number of myeloma patients achieving a complete response and improved progression-free survival following therapy. However, a low level of disease or minimal residual disease (MRD) still persists which contributes to the inevitable relapse in myeloma patients. MRD has been attributed to the presence of dormant myeloma cells and their subsequent reactivation, which is controlled by the microenvironment and specialised niches within the bone marrow. This contributes to the evasion of the immune system and chemotherapy, eventually leading to relapse. The growth of myeloma tumours are heavily dependent on environmental stimuli from the bone marrow microenvironment, and this plays a key role in myeloma progression. The bone microenvironment also plays a critical role in myeloma bone disease and the development of skeletal-related events. This review focuses on the bone marrow microenvironment in relation to myeloma pathogenesis and cancer dormancy. Moreover, it reviews the current therapies targeting the bone microenvironment to treat myeloma and myeloma bone disease. Lastly, it identifies novel therapeutic targets for myeloma treatment and the associated bone disease.

Regulator of G protein signaling (RGS) proteins are critical negative molecules of G protein-coupled receptor (GPCR) signaling, which mediates a variety of biological processes in bone homeostasis and diseases. The RGS proteins are divided into nine subfamilies with a conserved RGS domain which plays an important role in regulating the GTPase activity. Mutations of some RGS proteins change bone development and/or metabolism, causing osteopathy. In this review, we summarize the recent findings of RGS proteins in regulating osteoblasts, chondrocytes, and osteoclasts. We also highlight the impacts of RGS on bone development, bone remodeling, and bone-related diseases. Those studies demonstrate that RGS proteins might be potential drug targets for bone diseases. [ABSTRACT FROM AUTHOR]

The strictly regulated bone remodeling process ensures that osteoblastic bone formation is coupled to osteoclastic bone resorption. This coupling is regulated by a panel of coupling factors, including clastokines promoting the recruitment, expansion, and differentiation of osteoprogenitor cells within the eroded cavity. The osteoprogenitor cells on eroded surfaces are called reversal cells. They are intermixed with osteoclasts and become bone-forming osteoblast when reaching a critical density and maturity. Several coupling factors have been proposed in the literature, but their effects and expression pattern vary between studies depending on species and experimental setup. In this study, we investigated the mRNA levels of proposed secreted and membrane-bound coupling factors and their receptors in cortical bone remodeling events within the femur of healthy adolescent human controls using high-sensitivity RNA in situ hybridization. Of the proposed coupling factors, human osteoclasts showed mRNA-presence of LIF, PDGFB, SEMA4D, but no presence of EFNB2, and OSM. On the other hand, the osteoblastic reversal cells proximate to osteoclasts presented with LIFR, PDGFRA and PLXNB1, but not PDGFRB, which are all known receptors of the proposed coupling factors. Although EFNB2 was not present in mature osteoclasts, the mRNA of the ligand-receptor pair EFNB2:EPHB4 were abundant near the central blood vessels within intracortical pores with active remodeling. EPHB4 and SEMA4D were also abundant in mature bone-forming osteoblasts. This study highlights that especially LIF:LIFR, PDGFB:PDGFRA, SEMA4D:PLXNB1 may play a critical role in the osteoclast-osteoblast coupling in human remodeling events, as they are expressed within the critical cells.

Regulator of G protein signaling (RGS) proteins are critical negative molecules of G protein-coupled receptor (GPCR) signaling, which mediates a variety of biological processes in bone homeostasis and diseases. The RGS proteins are divided into nine subfamilies with a conserved RGS domain which plays an important role in regulating the GTPase activity. Mutations of some RGS proteins change bone development and/or metabolism, causing osteopathy. In this review, we summarize the recent findings of RGS proteins in regulating osteoblasts, chondrocytes, and osteoclasts. We also highlight the impacts of RGS on bone development, bone remodeling, and bone-related diseases. Those studies demonstrate that RGS proteins might be potential drug targets for bone diseases.

The bone is a mechanosensitive organ that is also a common metastatic site for prostate cancer. However, the mechanism by which the tumor interacts with the bone microenvironment to further promote disease progression remains to be fully understood. This is largely due to a lack of physiological yet user-friendly models that limit our ability to perform in-depth mechanistic studies. Here, we report a tunable bioreactor which facilitates the 3D culture of the osteocyte cell line, MLO-Y4, in a hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold under constant fluidic shear stress and tunable hydrostatic pressure within physiological parameters. Increasing hydrostatic pressure was sufficient to induce a change in the expression of several bone remodeling genes such as Dmp1, Rankl, and Runx2. Furthermore, increased hydrostatic pressure induced the osteocytes to promote the differentiation of the murine macrophage cell line RAW264.7 toward osteoclast-like cells. These results demonstrate that the bioreactor recapitulates the mechanotransduction response of osteocytes to pressure including the measurement of their functional ability in a 3D environment. In conclusion, the bioreactor would be useful for exploring the mechanisms of osteocytes in bone health and disease.

Several studies have shown that the G-protein coupled cannabinoid receptor CB2 and its interaction partner p62 are molecularly involved in bone remodeling processes. Pharmacological activation of the CB2 receptor enhanced bone volume in postmenopausal osteoporosis and arthritis models in rodents, whereas knockout or mutation of the p62 protein in aged mice led to Paget’s disease of bone-like conditions. Studies of pharmacological CB2 agonist effects on bone metabolism in p62 KO mice have not been performed to date. Here, we assessed the effect of the CB2-specific agonist JWH133 after a short-term (5 days in 3-month-old mice) or long-term (4 weeks in 6-month-old mice) treatment on structural, dynamic, and cellular bone morphometry obtained by μCT of the femur and histomorphometry of the vertebral bodies in p62 KO mice and their WT littermates in vivo. A genotype-independent stimulatory effect of CB2 on bone formation, trabecular number, and trabecular thickness after short-term treatment and on tissue mineral density after long-term treatment was detected, indicating a weak osteoanabolic function of this CB2 agonist. Moreover, after short-term systemic CB2 receptor activation, we found significant differences at the cellular level in the number of osteoblasts and osteoclasts only in p62 KO mice, together with a weak increase in trabecular number and a decrease in trabecular separation. Long-term treatment showed an opposite JWH133 effect on osteoclasts in WT versus p62 KO animals and decreased cortical thickness only in treated p62 KO mice. Our results provide new insights into CB2 receptor signaling in vivo and suggest that CB2 agonist activity may be regulated by the presence of its macromolecular binding partner p62.

Several studies have shown that the G-protein coupled cannabinoid receptor CB2 and its interaction partner p62 are molecularly involved in bone remodeling processes. Pharmacological activation of the CB2 receptor enhanced bone volume in postmenopausal osteoporosis and arthritis models in rodents, whereas knockout or mutation of the p62 protein in aged mice led to Paget's disease of bone-like conditions. Studies of pharmacological CB2 agonist effects on bone metabolism in p62 KO mice have not been performed to date. Here, we assessed the effect of the CB2-specific agonist JWH133 after a short-term (5 days in 3-month-old mice) or long-term (4 weeks in 6-month-old mice) treatment on structural, dynamic, and cellular bone morphometry obtained by μCT of the femur and histomorphometry of the vertebral bodies in p62 KO mice and their WT littermates in vivo. A genotype-independent stimulatory effect of CB2 on bone formation, trabecular number, and trabecular thickness after short-term treatment and on tissue mineral density after long-term treatment was detected, indicating a weak osteoanabolic function of this CB2 agonist. Moreover, after short-term systemic CB2 receptor activation, we found significant differences at the cellular level in the number of osteoblasts and osteoclasts only in p62 KO mice, together with a weak increase in trabecular number and a decrease in trabecular separation. Long-term treatment showed an opposite JWH133 effect on osteoclasts in WT versus p62 KO animals and decreased cortical thickness only in treated p62 KO mice. Our results provide new insights into CB2 receptor signaling in vivo and suggest that CB2 agonist activity may be regulated by the presence of its macromolecular binding partner p62. [ABSTRACT FROM AUTHOR]

Inflammatory osteolysis is a pathological skeletal disease associated with not only the production of inflammatory cytokines but also local oxidative status. Excessive reactive oxygen species (ROS) promote bone resorption by osteoclasts and induce the apoptosis of osteoblasts. In consideration of the lack of effective preventive or treatments options against osteolysis, the exploitation of novel pharmacological compounds/agents is critically required. In our study, we found that a novel antioxidant compound, JSH-23, plays a role in restoring bone homeostasis by scavenging intracellular ROS during both osteoclastogenesis and osteoblastogenesis. Mechanically, JSH-23 suppressed RANKL-induced osteoclastogenesis, bone resorption and the expression of specific genes (including NFATc1, c-Fos, TRAP, CTSK and DC-STAMP) via inhibition of the NF-κB signaling pathway. Meanwhile, JSH-23 suppressed RANKL-induced ROS generation via the TRAF6/Rac1/NOX1 pathway and the enhanced expression of Nrf2/HO-1. In addition, JSH-23 attenuated H2O2-induced apoptosis and mineralization reduction in osteoblasts by reducing ROS production and enhancing Nrf2/HO-1 expression. Our in vivo results further revealed that JSH-23 exerts its protective effects on bone mass through its antioxidant activity. In conclusion, our results show that the application of JSH-23 might be a novel and plausible strategy for the treatment of osteolysis-related disease.

Bone tissue is organized at the molecular level to resist fracture with the minimum of bone material. This implies that several modifications of the extracellular matrix, including enzymatic collagen crosslinking, take place. We previously highlighted the role of several gut hormones in enhancing collagen maturity and bone strength. The present study investigated the effect of proglucagon-derived peptides on osteoblast-mediated collagen post-processing. Briefly, MC3T3-E1 murine osteoblasts were cultured in the presence of glucagon (GCG), [D-Ala²]-glucagon-like peptide-1 ([D-Ala²]-GLP-1), and [Gly²]-glucagon-like peptide-2 ([Gly²]-GLP-2). Gut hormone receptor expression at the mRNA and protein levels were investigated by qPCR and Western blot. Extent of collagen postprocessing was examined by Fourier transform infrared microspectroscopy. GCG and GLP-1 receptors were not evidenced in osteoblast cells at the mRNA and protein levels. However, it is not clear whether the known GLP-2 receptor is expressed. Nevertheless, administration of [Gly²]-GLP-2, but not GCG or [D-Ala²]-GLP-1, led to a dose-dependent increase in collagen maturity and an acceleration of collagen post-processing. This mechanism was dependent on adenylyl cyclase activation. In conclusion, the present study highlighted a direct effect of [Gly²]-GLP-2 to enhance collagen post-processing and crosslinking maturation in murine osteoblast cultures. Whether this effect is translatable to human osteoblasts remains to be elucidated.

Inflammatory osteolysis is a pathological skeletal disease associated with not only the production of inflammatory cytokines but also local oxidative status. Excessive reactive oxygen species (ROS) promote bone resorption by osteoclasts and induce the apoptosis of osteoblasts. In consideration of the lack of effective preventive or treatments options against osteolysis, the exploitation of novel pharmacological compounds/agents is critically required. In our study, we found that a novel antioxidant compound, JSH-23, plays a role in restoring bone homeostasis by scavenging intracellular ROS during both osteoclastogenesis and osteoblastogenesis. Mechanically, JSH-23 suppressed RANKL-induced osteoclastogenesis, bone resorption and the expression of specific genes (including NFATc1, c-Fos, TRAP, CTSK and DC-STAMP) via inhibition of the NF-κB signaling pathway. Meanwhile, JSH-23 suppressed RANKL-induced ROS generation via the TRAF6/Rac1/NOX1 pathway and the enhanced expression of Nrf2/HO-1. In addition, JSH-23 attenuated H2O2-induced apoptosis and mineralization reduction in osteoblasts by reducing ROS production and enhancing Nrf2/HO-1 expression. Our in vivo results further revealed that JSH-23 exerts its protective effects on bone mass through its antioxidant activity. In conclusion, our results show that the application of JSH-23 might be a novel and plausible strategy for the treatment of osteolysis-related disease. [ABSTRACT FROM AUTHOR]

Bone tissue is organized at the molecular level to resist fracture with the minimum of bone material. This implies that several modifications of the extracellular matrix, including enzymatic collagen crosslinking, take place. We previously highlighted the role of several gut hormones in enhancing collagen maturity and bone strength. The present study investigated the effect of proglucagon-derived peptides on osteoblast-mediated collagen post-processing. Briefly, MC3T3-E1 murine osteoblasts were cultured in the presence of glucagon (GCG), [D-Ala²]-glucagon-like peptide-1 ([D-Ala²]-GLP-1), and [Gly²]-glucagon-like peptide-2 ([Gly²]-GLP-2). Gut hormone receptor expression at the mRNA and protein levels were investigated by qPCR and Western blot. Extent of collagen postprocessing was examined by Fourier transform infrared microspectroscopy. GCG and GLP-1 receptors were not evidenced in osteoblast cells at the mRNA and protein levels. However, it is not clear whether the known GLP-2 receptor is expressed. Nevertheless, administration of [Gly²]-GLP-2, but not GCG or [D-Ala²]-GLP-1, led to a dose-dependent increase in collagen maturity and an acceleration of collagen post-processing. This mechanism was dependent on adenylyl cyclase activation. In conclusion, the present study highlighted a direct effect of [Gly²]-GLP-2 to enhance collagen post-processing and crosslinking maturation in murine osteoblast cultures. Whether this effect is translatable to human osteoblasts remains to be elucidated. [ABSTRACT FROM AUTHOR]