<Emphasis Type="SmallCaps">l</Emphasis>-Carnitine protects against apoptosis of murine MC3T3-E1 osteoblastic cells

L-carnitine (LC), an amino acid with a major role in cellular energy metabolism, has positive effects on bone metabolism. However, the effect of LC on apoptosis of osteoblast in vitro has not been reported. The aim of this study was to investigate the action of LC on apoptosis of mouse osteoblastic cell line MC3T3-E1. Cell apoptosis was measured by sandwich-enzyme-immunoassay. Release of cytochrome c from mitochondria into cytosol and Bcl-2, Bax protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-9. LC inhibited MC3T3-E1 cell apoptosis induced by serum deprivation. Our study also shows that LC decreased cytochrome c release and caspase-3 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Furthermore, LC protected against MC3T3-E1 cell apoptosis induced by the glucocorticoid (GC) dexamethasone (Dex).     

Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells

Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H₂S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H₂S-producing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H₂S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPKα1-shRNA. In summary, we show that H₂S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H₂S signaling might be further investigated as a novel target for anti-osteoporosis treatment.     

Effect of progesterone on apoptosis of murine MC3T3-E1 osteoblastic cells

Progesterone (P) has been suggested as a bone-trophic hormone. Previous studies have shown that P promoted bone formation by stimulating the proliferation and differentiation of osteoblasts. But, the effect of P on apoptosis of osteoblast in vitro has not been reported. We propose that P may promote bone formation by suppressing the apoptosis of osteoblast. The present study was performed to investigate the effect of P on apoptosis of murine MC3T3-E1 osteoblastic cells. Cell apoptosis was measured by acidine orange/ethidium bromide (AO/EB) staining and sandwich-enzyme-immunoassay. Progesterone receptor (PR), cytochrome c, caspase-9 and caspase-3 protein levels were determined by Western blot analysis. The enzyme substrate was also used to assess the activation of caspase-3 and caspase-9. Progesterone suppressed MC3T3-E1 cells apoptosis induced by serum deprivation, and this effect was blocked by a PR antagonist RU486. Furthermore, the suppressive effects of P on cytochrome c release and caspase-9 and caspase-3 activation in serum-deprived MC3T3-E1 cells were also reversed by RU486. Our study demonstrated that P protects osteoblast against apoptosis through PR and the downstream mitochondrial pathway. Thus, the data suggest that the effects of P on osteoblast apoptosis may contribute to the mechanisms by which P exerts its action on bone formation.     

Arbutin ameliorates glucocorticoid-induced osteoporosis through activating autophagy in osteoblasts

Chronic long-term glucocorticoid use causes osteoporosis partly by interrupting osteoblast homeostasis and exacerbating bone loss. Arbutin, a natural hydroquinone glycoside, has been reported to have biological activities related to the differentiation of osteoblasts and osteoclasts. However, the role and underlying mechanism of arbutin in glucocorticoid-induced osteoporosis are elusive. In this study, we demonstrated that arbutin administration ameliorated osteoporotic disorders in glucocorticoid dexamethasone (Dex)-induced mouse model, including attenuating the loss of bone mass and trabecular microstructure, promoting bone formation, suppressing bone resorption, and activating autophagy in bone tissues. Furthermore, Dex-stimulated mouse osteoblastic MC3T3-E1 cells were treated with arbutin. Arbutin treatment rescued Dex-induced repression of osteoblast differentiation and mineralization, the downregulation of osteogenic gene expression, reduced autophagic marker expression, and decreased autophagic puncta formation. The application of autophagy inhibitor 3-MA decreased autophagy, differentiation, and mineralization of MC3T3-E1 cells triggered by arbutin. Taken together, our findings suggest that arbutin treatment fends off glucocorticoid-induced osteoporosis, partly through promoting differentiation and mineralization of osteoblasts by autophagy activation.     

Recombinant tissue metalloproteinase inhibitor-3 protein induces apoptosis of murine osteoblast MC3T3-E1

Tissue inhibitor of metalloproteinases (TIMPs) plays an essential role in the regulation of bone metabolism. Here we report that recombinant tissue metalloproteinase inhibitor-3 (TIMP-3) protein induces the apoptosis of MC3T3-E1 osteoblasts. Cell apoptosis was detected by sandwich-enzyme-immunoassay. Fas and Fasl protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-8. The phosphorylation of JNK, p38 and ERK1/2 was examined by Western blot analysis. The ELISA suggested that TIMP-3 promoted MC3T3-E1 cell apoptosis. TIMP-3 treatment induced the expression of Fas and Fasl proteins, and the activation of caspase-8 and caspase-3. TIMP-3 treatment induced p38 and ERK phosphorylation. SB203580 and PD98059, the inhibitor of p38 and ERK, respectively, abolished the TIMP-3 effect on osteoblast apoptosis. In conclusion, the signal pathway through which TIMP-3 induces MC3T3-E1 cell apoptosis, mediated by Fas and involves the p38 and ERK signal transduction pathways.     

Glucocorticoid receptor and sequential P53 activation by dexamethasone mediates apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells

Glucocorticoids play a pivotal role in the proliferation of osteoblasts, but the underlying mechanism has not been successfully elucidated. In this report, we have investigated the molecular mechanism which elucidates the inhibitory effects of dexamethasone on murine osteoblastic MC3T3-E1 cells. It was found that the inhibitory effects were largely attributed to apoptosis and G1 phase arrest. Both the cell cycle arrest and apoptosis were dependent on glucocorticoid receptor (GR), as they were abolished by GR blocker RU486 pre-treatment and GR interference. G1 phase arrest and apoptosis were accompanied with a p53-dependent up-regulation of p21 and pro-apoptotic genes NOXA and PUMA. We also proved that dexamethasone can't induce apoptosis and cell cycle arrest when p53 was inhibited by p53 RNA interference. These data demonstrate that proliferation of MC3T3-E1 cell was significantly and directly inhibited by dexamethasone treatment via aberrant GR activation and subsequently P53 activation.     

Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes: MT1-MMP maintains osteocyte viability

Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from apoptosis when transdifferentiating into osteocytes. By examination of osteoblasts and osteocytes embedded in calvarial bone in the MT1-MMP deficient mice, we found that MT1-MMP deficient mice had 10-fold higher levels of apoptotic osteocytes than wild-type controls. We have previously shown that MT1-MMP activates latent Transforming Growth Factorbeta (TGF-beta). These findings strongly suggest that MT1-MMP-activated TGF-beta maintains osteoblast survival during transdifferentiation into osteocytes, and maintains mature osteocyte viability. Thus, the interrelationship of MMPs and TGF-beta may play an important role in bone formation and maintenance.     

Soybean ethanol extract increases the function of osteoblastic MC3T3-E1 cells

To investigate the bioactivities of soybean, which act on bone metabolism, we studied the effect of a soybean ethanol extract on the activity of osteoblast MC3T3-E1 cells. Soy extract (0.01-0.1 g/l) dose-dependently increased survival (P<0.05) and DNA synthesis (P<0.05) of MC3T3-E1 cells. In addition, soy extract (0.05 g/l) increased alkaline phosphatase activity (P<0.05) and collagen synthesis (P<0.05) of MC3T3-E1 cells. Moreover, the anti-estrogen tamoxifen eliminated the stimulation of MC3T3-E1 cells on the proliferation, ALP activity and collagen synthesis by soy extract, indicating that the main action of the soy extract on osteoblastic MC3T3-E1 cells is similar to that of estrogen effects. Treatment with soy extract prevented apoptosis, as assessed by a one-step sandwich immunoassay and DNA gel electrophoresis studies. This effect may be associated with the activation of the estrogen receptor, since we observed soy extract-mediated survival against apoptosis was blocked by the estrogen receptor antagonist tamoxifen in cells, further supporting a receptor-mediated mechanism of cell survival. These results suggest that osteoblast function is promoted by soy extract and that the estrogen receptor is involved in the response, thereby playing an important role in bone remodeling. In conclusion, soy extract has a direct stimulatory effect on bone formation in cultured osteoblastic cell in vitro. Presumably, dietary soy products are useful in the prevention of osteoporosis.     

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-kappaB (NF-kappaB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-kappaB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast- and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.     

Matrix metalloproteinase-dependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis

Upon termination of bone matrix synthesis, osteoblasts either undergo apoptosis or differentiate into osteocytes or bone lining cells. In this study, we investigated the role of matrix metalloproteinases (MMPs) and growth factors in the differentiation of osteoblasts into osteocytes and in osteoblast apoptosis. The mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts were either grown on two-dimensional (2-D) collagen-coated surfaces, where they morphologically resemble flattened, cuboidal bone lining cells, or embedded in three-dimensional (3-D) collagen gels, where they resemble dendritic osteocytes constituting a network of cells. When MC3T3-E1 osteoblasts were grown in a 3-D matrix in the presence of an MMP inhibitor (GM6001), the cell number was dose-dependently reduced by approximately 50%, whereas no effect was observed on a 2-D substratum. In contrast, the murine mature osteocyte cell line, MLO-Y4, was unaffected by GM6001 under all culture conditions. According to TUNEL assay, the osteoblast apoptosis was increased 2.5-fold by 10 microm GM6001. To investigate the mechanism by which MMPs mediate the survival of osteoblasts, we examined the effect of GM6001 on MC3T3-E1 osteoblasts in the presence of extracellular matrix components and growth factors, including tenascin, fibronectin, laminin, collagenase-cleaved collagen, gelatin, parathyroid hormone, basic fibroblast growth factor, vascular epidermal growth factor, insulin-like growth factor, interleukin-1, and latent and active transforming growth factor-beta (TGF-beta). Only active TGF-beta, but not latent TGF-beta or other agents tested, restored cell number and apoptosis to control levels. Furthermore, we found that the membrane type MMP, MT1-MMP, which is produced by osteoblasts, could activate latent TGF-beta and that antibodies neutralizing endogenous TGF-beta led to a similar decrease in cell number as GM6001. Whereas inhibitors of other protease families did not induce osteoblast apoptosis, an inhibitor of the p44/42 mitogen-activated protein kinase showed the same but non-synergetic effect as GM6001. These findings suggest that MMP-activated TGF-beta maintains osteoblast survival during trans-differentiation into osteocytes by a p44/42-dependent pathway.     

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.     

高浓度葡萄糖通过p38MAPK-TXNIP/TRX-ROS通路抑制MC3T3-E1细胞成骨分化

目的：研究高浓度葡萄糖抑制MC3T3-E1细胞成骨分化的机理。方法：建立MC3T3-E1细胞成骨分化诱导体系,观察不同浓度葡萄糖（5.5mM和22mM）对MC3T3-E1细胞成骨分化的影响;用不同浓度的p38 MAPK抑制剂Fr167653（0.1μM、1.0μM和10μM）进行药物干预,观察MC3T3-E1细胞在22mM葡萄糖浓度下成骨分化的变化情况。通过钙含量检测、Real time PCR检测相关分化的变化;用Western Blot方法检测MC3T3-E1细胞分化过程中p38 MAPK磷酸化状态、TXNIP表达水平的变化;使用胰岛素二硫键还原法检测细胞内TRX活性水平;使用活性氧检测试剂盒检测细胞内自由氧生成水平。结果：体外诱导条件下,高浓度（22mM）葡萄糖通过升高p38 MAPK磷酸化水平,上调TXNIP表达水平,同时降低TRX活性,使细胞内自由氧生成增加,抑制MC3T3-E1细胞的成骨分化;Fr167653通过抑制p38 MAPK磷酸化,下调TXNIP表达同时升高TRX活性,抑制细胞内自由氧生成,解除高浓度葡萄糖对细胞成骨分化的抑制作用。结论：高浓度葡萄糖通过p38 MAPK-TXNIP/TRX-ROS信号通路抑制MC3T3-E1细胞成骨分化。     

Dexamethasone interferes with osteoblasts formation during osteogenesis through altering IGF-1-mediated angiogenesis

Dexamethasone (Dex), a synthetic glucocorticoid (GC) with long-lasting treatment effects, has been proved to exert a modulatory effect on osteoblast proliferation and differentiation during embryonic osteogenesis. However, it is still controversial if Dex exposure influences endochondral ossification and the underlying mechanism. In this study, chick embryos in vivo and preosteoblast cell cultures in vitro were utilized to investigate the effects of Dex on osteoblast formation and differentiation during the skeletal development. We first demonstrated that Dex exposure could shorten the long bones of 17-day chick embryos in vivo, and also downregulated the expressions of osteogenesis-related genes. Next, we established that Dex exposure inhibited the proliferation and viability of preosteoblasts-MC3TC-E1 cells, and the addition of insulin-like growth factor 1 (IGF-1) could dramatically rescue these negative effects. On the basis of remarkable changes in the rescue experiments, we next verified the important role of angiogenesis in osteogenesis by culturing isolated embryonic phalanges in Dulbecco's modified Eagle's medium culture or on the chick chorioallantoic membrane (CAM). Then, we transplanted MC3T3-E1 cell masses onto the CAM. The data showed that Dex exposure reduced the vessel density within the developed cell mass, concomitantly with the downregulation of IGF-1 pathway. We verified that the inhibition of blood vessel formation caused by Dex could be rescued by IGF-1 treatment using the CAM angiogenesis model. Eventually, we demonstrated that the shortened length of the phalanges in the presence of Dex could be reversed by IGF-1 addition. In summary, these findings suggested that the inhibition of Igf-1 signal caused by Dex exposure exerts a detrimental impact on the formation of osteoblasts and angiogenesis, which consequently shortens long bones during osteogenesis.     

TIMP-1 suppressed by miR-138 participates in endoplasmic reticulum stress-induced osteoblast apoptosis in osteoporosis

The aim of this study was to investigate the role of miR-138 in osteoporosis and its underlying mechanism. Hydrogen peroxide (H₂O₂) was used to induce osteoporotic injury of osteoblasts. The cell viability and apoptosis of MC3T3-E1 cells was assessed using MTT assay and flow cytometry, respectively. The cell transfection was carried out to modulate the expression levels of miR-138 and TIMP-1 in MC3T3-E1 cells. Luciferase reporter gene assay was performed to determine the interaction between miR-138 and TIMP-1 3′UTR. In the present study, H₂O₂ inhibited osteoblasts growth and induced intracellular endoplasmic reticulum (ER) stress accompanied by high expression of miR-138. We also confirmed that miR-138 promoted osteoblasts apoptosis in vitro and in vivo. MiR-138 was further indicated to inhibit osteoblast survival via negative regulating TIMP-1 expression. Moreover, the downregulated TIMP-1 also mediated the ER stress-induced apoptosis of osteoblasts. We confirmed that miR-138 and ER stress were induced in osteoporosis and then promoted the apoptosis of osteoblasts, at least in part, through TIMP-1.