Selenium Protects Bone Marrow Stromal Cells Against Hydrogen Peroxide-Induced Inhibition of Osteoblastic Differentiation by Suppressing Oxidative Stress and ERK Signaling Pathway

Osteoporosis is a bone disease that leads to an increased risk of fracture. Oxidative stress may play a major role in the development of osteoporosis in part by inhibiting osteoblastic differentiation of bone marrow stromal cells (MSCs). Some evidence suggested that antioxidant selenium could prevent osteoporosis, but the underlying mechanism remains unclear. In this work, the effect of sodium selenite on H₂O₂-induced inhibition of osteoblastic differentiation of primary rat bone MSCs and the related mechanisms were examined. Pretreatment with selenite inhibited the adverse effect of H₂O₂ on osteoblastic differentiation of MSCs, based on alkaline phosphatase activity, gene expression of type I collagen and osteocalcin, and matrix mineralization. In addition, selenite pretreatment also suppressed the activation of extracellular signal-regulated kinase (ERK) induced by H₂O₂. The above effects were mediated by the antioxidant effect of selenite. Selenite enhanced the gene expression and activity of glutathione peroxidase, reversed the decreased total antioxidant capacity and reduced glutathione, and suppressed reactive oxygen species production and lipid peroxidation level in H₂O₂-treated MSCs. These results showed that selenite protected MSCs against H₂O₂-induced inhibition of osteoblastic differentiation through inhibiting oxidative stress and ERK activation, which provided, for the first time, the mechanistic explanation for the negative association of selenium status and risk of osteoporosis in terms of bone formation.     

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.     

Resveratrol improves oxidative stress and prevents the progression of periodontitis via the activation of the Sirt1/AMPK and the Nrf2/antioxidant defense pathways in a rat periodontitis model

Oxidative stress is a key factor regulating the systemic pathophysiological effects associated with periodontitis. Resveratrol is a phytochemical with antioxidant and anti-inflammatory properties that can reduce oxidative stress and inflammation. We hypothesized that resveratrol may prevent the progression of periodontitis and reduce systemic oxidative stress through the activation of the sirtuin 1 (Sirt1)/AMP-activated protein kinase (AMPK) and the nuclear factor E2-related factor 2 (Nrf2)/antioxidant defense pathways. Three groups of male Wistar rats (periodontitis treated with melinjo resveratrol, periodontitis without resveratrol, and control rats with no periodontitis or resveratrol treatment) were examined. A ligature was placed around the maxillary molars for 3 weeks to induce periodontitis, and the rats were then given drinking water with or without melinjo resveratrol. In rats with periodontitis, ligature placement induced alveolar bone resorption, quantified using three-dimensional images taken by micro-CT, and increased proinflammatory cytokine levels in gingival tissue. Melinjo resveratrol intake relieved alveolar bone resorption and activated the Sirt1/AMPK and the Nrf2/antioxidant defense pathways in inflamed gingival tissues. Further, melinjo resveratrol improved the systemic levels of 8-hydroxydeoxyguanosine, dityrosine, nitric oxide metabolism, nitrotyrosine, and proinflammatory cytokines. We conclude that oral administration of melinjo resveratrol may prevent the progression of ligature-induced periodontitis and improve systemic oxidative and nitrosative stress.     

Hesperetin suppresses RANKL-induced osteoclastogenesis and ameliorates lipopolysaccharide-induced bone loss

Destructive bone diseases caused by osteolysis are increasing in incidence. They are characterized by an excessive imbalance of osteoclast formation and activation. During osteolysis, the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways are triggered by receptor activator of NF-κB ligand (RANKL), inflammatory factors, and oxidative stress. Previous studies have indicated that the common flavanone glycoside compound hesperetin exhibits anti-inflammatory and antioxidant activity by inhibition of NF-κB and MAPK signaling pathways. However, the direct relationship between hesperetin and osteolysis remain unclear. In the present study, we investigated the effects of hesperetin on lipopolysaccharide (LPS)-induced osteoporosis and elucidated the related mechanisms. Hesperetin effectively suppressed RANKL-induced osteoclastogenesis, osteoclastic bone resorption, and F-actin ring formation in a dose-dependent manner. It also significantly suppressed the expression of osteoclast-specific markers including tartrate-resistant acid phosphatase, matrix metalloproteinase-9, cathepsin K, c-Fos, and nuclear factor of activated T-cells cytoplasmic 1. Furthermore, it inhibited osteoclastogenesis by inhibiting activation of NF-κB and MAPK signaling, scavenging reactive oxygen species, and activating the nuclear factor E2 p45-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway. Consistent with in vitro results, hesperetin effectively ameliorated LPS-induced bone loss, reduced osteoclast numbers, and decreased the RANKL/OPG ratio in vivo. As such, our results suggest that hesperetin may be a great candidate for developing a novel drug for destructive bone diseases such as periodontal disease, tumor bone metastasis, rheumatoid arthritis, and osteoporosis.     

Lack of bone resorption in osteosclerotic (oc/oc) mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.

In a co-culture system of mouse spleen cells and osteoblastic cells, we have demonstrated that a suitable microenvironment must be provided by osteoblastic cells in order for osteoclast-like multinucleated cell (MNC) formation. Using this co-culture system, we examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. Numerous tartrate-resistant acid phosphatase (TRAP, an osteoclast marker enzyme)-positive MNCs were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] both in co-cultures of oc/oc spleen cells and normal osteoblastic cells and in those of normal spleen cells and oc/oc osteoblastic cells. TRAP-positive MNCs derived from normal spleen cells tended to spread out on culture dishes, whereas those from oc/oc spleen cells remained as small, compact MNCs. When TRAP-positive MNCs enriched from co-cultures of normal spleen cells and oc/oc osteoblastic cells were cultured on dentine slices, they formed numerous resorption pits with ruffled borders and clear zones. In contrast, none of the TRAP-positive MNCs derived from oc/oc spleen cells formed either ruffled borders or resorption pits. These results indicate that the lack of bone resorption in oc/oc mice is due to a defect in osteoclast progenitors rather than the local microenvironment provided by osteoblastic cells.     

The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

An excess of osteoclastic bone resorption relative to osteoblastic bone formation results in progressive bone loss, characteristic of osteoporosis. Understanding the mechanisms of osteoclast differentiation is essential to develop novel therapeutic approaches to prevent and treat osteoporosis. We showed previously that Wrch1/RhoU is the only RhoGTPase whose expression is induced by RANKL during osteoclastogenesis. It associates with podosomes and the suppression of Wrch1 in osteoclast precursors leads to defective multinucleated cell formation. Here we further explore the functions of this RhoGTPase in osteoclasts, using RAW264.7 cells and bone marrow macrophages as osteoclast precursors. Suppression of Wrch1 did not prevent induction of classical osteoclastic markers such as NFATc1, Src, TRAP (Tartrate-Resistant Acid Phosphatase) or cathepsin K. ATP6v0d2 and DC-STAMP, which are essential for fusion, were also expressed normally. Similar to the effect of RANKL, we observed that Wrch1 expression increased osteoclast precursor aggregation and reduced their adhesion onto vitronectin but not onto fibronectin. We further found that Wrch1 could bind integrin ß3 cytoplasmic domain and interfered with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 also acted as an inhibitor of M-CSF-induced prefusion osteoclast migration. In mature osteoclasts, high Wrch1 activity inhibited podosome belt formation. Nevertheless, it had no effect on mineralized matrix resorption. Our observations suggest that during osteoclastogenesis, Wrch1 potentially acts through the modulation of αvß3 signaling to regulate osteoclast precursor adhesion and migration and allow fusion. As an essential actor of osteoclast differentiation, the atypical RhoGTPase Wrch1/RhoU could be an interesting target for the development of novel antiresorptive drugs.     

A Prunus mume extract stimulated the proliferation and differentiation of osteoblastic MC3T3-E1 cells

Osteoporosis is a serious disease caused by decreased bone mass. There is constant matrix remodeling in bones, by which bone formation is performed by osteoblastic cells, whereas bone resorption is accomplished by osteoclast cells. We investigated the effect of a Japanese apricot (Prunus mume SIBE. et ZUCC.) extract on the proliferation and osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. An alkaline phosphatase (ALP) activity assay, cell proliferation assay, alizarin red staining and expression analysis of osteoblastic genes were carried out to assess the proliferation and osteoblastic differentiation. The water-soluble fraction of Prunus mume (PWF) increased the ALP activity, cell proliferation and mineralization. The gene expression of osteopontin and bone morphogenetic protein-2, which are markers in the early period of osteoblastic differentiation, were significantly enhanced by the PWF treatment. PWF therefore stimulated the proliferation and osteoblastic differentiation of cells and may have potential to prevent osteoporosis.     

Bcl-2 protects against oxidative stress while inducing premature senescence

Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.     

Asperpyrone A attenuates RANKL‐induced osteoclast formation through inhibiting NFATc1, Ca2+ signalling and oxidative stress

Imbalance of osteoblast and osteoclast in adult leads to a variety of bone‐related diseases, including osteoporosis. Thus, suppressing the activity of osteoclastic bone resorption becomes the main therapeutic strategy for osteoporosis. Asperpyrone A is a natural compound isolated from Aspergillus niger with various biological activities of antitumour, antimicrobial and antioxidant. The present study was designed to investigate the effects of Asperpyrone A on osteoclastogenesis and to explore its underlining mechanism. We found that Asperpyrone A inhibited RANKL‐induced osteoclastogenesis in a dose‐dependent manner when the concentration reached 1 µm, and with no cytotoxicity until the concentration reached to 10 µm. In addition, Asperpyrone A down‐regulated the mRNA and protein expression of NFATc1, c‐fos and V‐ATPase‐d2, as well as the mRNA expression of TRAcP and Ctsk. Furthermore, Asperpyrone A strongly attenuated the RNAKL‐induced intracellular Ca²⁺ oscillations and ROS (reactive oxygen species) production in the process of osteoclastogenesis and suppressed the activation of MAPK and NF‐κB signalling pathways. Collectively, Asperpyrone A attenuates RANKL‐induced osteoclast formation via suppressing NFATc1, Ca²⁺ signalling and oxidative stress, as well as MAPK and NF‐κB signalling pathways, indicating that this compound may become a potential candidate drug for the prevention or treatment of osteoporosis.     

氧化损伤和微重力致骨质疏松症的研究进展

伴随着人口老龄化日益严重,骨质疏松症作为"悄无声息的流行病"逐渐引起人们的注意。氧化损伤和力学刺激是造成骨质疏松的两个主要原因。一方面氧化损伤可通过刺激FoxOs信号通路抑制成骨细胞分化,造成骨质疏松,另一方面机体在长期缺乏负荷力刺激时也会发生废用性骨丢失,二者之间存在着紧密的联系。Nrf2作为细胞应对氧化损伤的主要防御机制,可调控多种抗氧化蛋白酶转录,在氧化损伤所造成的骨质疏松中扮演着重要角色。本文综述了氧化损伤和微重力造成骨质疏松的机制以及Nrf2对抗氧化损伤的调节和对修复骨质发育的影响。     

Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha

The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.     

Regulation of bone mass, bone loss and osteoclast activity by cannabinoid receptors

Accelerated osteoclastic bone resorption has a central role in the pathogenesis of osteoporosis and other bone diseases. Identifying the molecular pathways that regulate osteoclast activity provides a key to understanding the causes of these diseases and to the development of new treatments. Here we show that mice with inactivation of cannabinoid type 1 (CB1) receptors have increased bone mass and are protected from ovariectomy-induced bone loss. Pharmacological antagonists of CB1 and CB2 receptors prevented ovariectomy-induced bone loss in vivo and caused osteoclast inhibition in vitro by promoting osteoclast apoptosis and inhibiting production of several osteoclast survival factors. These studies show that the CB1 receptor has a role in the regulation of bone mass and ovariectomy-induced bone loss and that CB1- and CB2-selective cannabinoid receptor antagonists are a new class of osteoclast inhibitors that may be of value in the treatment of osteoporosis and other bone diseases.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Inhibition of osteoblastic metalloproteinases in mice prevents bone loss induced by oestrogen deficiency

Matrix metalloproteinases (MMPs) are key mediators in extra-cellular matrix remodelling and implicated primarily in bone growth, and particularly in osteoclastic bone resorption. We hypothesise that MMPs have a role in the increased bone remodelling resulting from oestrogen deficiency. Transgenic (TG) mice overexpressing TIMP-1 in their osteoblastic cells and their wild-type (WT) littermates were ovariectomised. One month after surgery, bone mineral density (BMD) and bone microarchitecture were assessed. Primary cells from WT and TG mice were used to determine how TIMP-1 affects osteoclast and osteoblastic cells. The reduction of BMD induced by ovariectomy in WT mice was not observed in the transgenic mice. The transgene overexpression also dampened the post-ovariectomy increase in bone resorption in contrast to the WT mice. In vivo, osteoclastic surfaces and D-pyridinoline were not increased in TG mice, and ex vivo, the differentiation of osteoclasts from TG bone marrow precursor cells were unaffected by in vivo oestrogen deficiency or treatment. We showed also that TIMP-1 overexpression reduces and delays the osteoblastic proliferation and differentiation respectively, and reduced the generation of the active form of TGFbeta1 in the supernatant of TG osteoblasts. Our findings support the hypothesis that in vivo inhibition of osteoblastic MMPs prevented the bone loss induced by oestrogen deficiency, with a significant decrease in bone resorption. This effect was presumably resulting from (1) a direct inhibition of osteoclastic resorption activity by the TIMP-1 and (2) the modification in the local activation of extra-cellular signalling factors such as TGFbeta1 and the OPG/RANKL ratio.