Apoptosis In Osteoblastic MC3T3-E1 Cells Research Articles

Depletion of Zinc Causes Osteoblast Apoptosis with Elevation of Leptin Secretion and Phosphorylation of JAK2/STAT3.

Zinc (Zn) has been reported to mediate leptin secretion, and thus leptin can be an important candidate molecule linking Zn with bone formation. The present study investigated whether zinc deficiency induces leptin secretion by activating a JAK2/STAT3 signaling pathway and leads to osteoblastic apoptosis. MC3T3-E1 cells were incubated for 24 h in normal osteogenic differentiation medium (OSM) or OSM treated with either 1 μM (Low Zn) or 15 μM (High Zn) of ZnCl2 containing 5 μM TPEN (Zn chelator). Our results demonstrated that low Zn stimulated extracellular leptin secretion and increased mRNA and protein expression of leptin in osteoblastic MC3T3-E1 cells. The OB-Rb (long isoform of leptin receptor) expressions were also elevated in osteoblasts under depletion of Zn. Leptin-signaling proteins, JAK2 and p-JAK2 in the cytosol of low Zn osteoblast conveyed leptin signaling, which ultimately induced higher p-STAT3 expression in the nucleus. Apoptotic effects of JAK2/STAT3 pathway were shown by increased caspase-3 in low Zn osteoblasts as well as apoptotic morphological features observed by TEM. Together, these data suggest that low Zn modulates leptin secretion by activating JAK2/STAT3 signaling pathway and induces apoptosis of osteoblastic MC3T3-E1 cells.

Tissue inhibitor of metalloproteinase 1 suppresses growth and differentiation of osteoblasts and differentiation of osteoclasts by targeting the AKT pathway

Tissue inhibitor of metalloproteinase 1 (TIMP1) has various biological activities including the regulation of cell growth and differentiation. However, its role in bone homeostasis and remodeling remains poorly understood. In this study, we investigate the effects of TIMP1 on osteoblast and osteoclast activity at both cellular and molecular level using siRNA-mediated knockdown technique. Our results show that knockdown of TIMP1 stimulates proliferation and survival, but decreases apoptosis in osteoblastic MC3T3-E1 cells, suggesting that TIMP1 inhibits cell growth. TIMP1 also dampens differentiation of committed osteoblasts, as well as osteoblastogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). We further show that the modulation of TIMP1 on osteoblast activity is independent of its MMP inhibition. Importantly, we uncover that TIMP1 suppresses osteoblast growth and differentiation by targeting the AKT pathway, and this is associated with TIMP1-mediated induction of PTEN via its binding to the cell surface receptor CD44. Therefore, our results highlight a novel TIMP1/CD44/PTEN/AKT signaling nexus that functions as a suppressor of osteoblast activity. Moreover, we show that TIMP1 also inhibits osteoclast differentiation in osteoclast precursor RAW 264.7 cells by targeting the AKT. In conclusion, our results demonstrate that TIMP1 can act as a suppressor of growth and differentiation of osteoblasts and differentiation of osteoclasts through the negative regulation of the AKT pathway. We propose that TIMP1 may serve as a potential target for low bone mass-related skeletal diseases, such as osteoporosis.

Silencing FOXO1 attenuates dexamethasone-induced apoptosis in osteoblastic MC3T3-E1 cells

Dexamethasone (DEX), a widely used glucocorticoid with strong anti-inflammatory and immunosuppressive activities, has been reported to induce apoptosis in osteoblasts, but the underlying mechanisms are still not comprehensively investigated. FOXO1 plays an important role in the regulation of cell proliferation and apoptosis. Our study aims to explore the role of FOXO1 in DEX-induced apoptosis of osteoblastic MC3T3-E1 cells through bioinformatics and experiments. We first employed bioinformatics to identify DEX-related genes and revealed their functions by GO enrichment analysis including FOXO1 associated biological processes. Expression level of FOXO1 was validated by GEO data. Then, experiments were performed to verify the hypothesis. CCK8 was used to detect cell viability and apoptosis was detected by flow cytometry. SiRNA was used to silence FOXO1 and western-blot was employed to detect protein expression. Results demonstrated DEX-related genes involved in cell proliferation, apoptosis and angiogenesis and FOXO1 was a regulator of apoptosis. DEX could up-regulate FOXO1 expression, inhibit cell viability, promote apoptosis. SiRNA-FOXO1 could attenuate DEX-induced apoptosis in MC3T3-E1. These findings suggested DEX could affect some vital biological processes of MC3T3-E1 and FOXO1 played an essential role in DEX-induced apoptosis in MC3T3-E1.

Effect of the three Enterococcus faecalis strains on apoptosis in MC3T3 cells.

Osteoblast apoptosis is critical for the development and repairing of bone destruction in persistent apical periodontitis (PAP). Enterococcus faecalis is considered as a frequently isolated pathogen of PAP. This study aimed to explore the effect of E.faecalis on apoptosis in osteoblastic MC3T3-E1 cells via an in vitro model. MC3T3 cells were incubated with live clinically isolated strains of E.faecalis at a multiplicity of infection (MOI) of 1,000:1 for 2hr. Flow cytometry analysis using annexin V-FITC and PI staining, JC-1 staining and TUNEL assay were conducted to detect the apoptosis in the infected cells. Western blotting and quantitative real-time PCR were used to determine the expression of caspase-3, Bcl-2 and Bax. The proliferation of the infected cells was inhibited. Decreased mitochondrial membrane potential (ΔΨm) and enhanced DNA fragmentation of the infected cells were observed. The relative expression of Bax and cleavage caspase-3 was upregulated, and the expression of Bcl-2 and Bcl-2/Bax was downregulated in the infected cells. Together, the clinically isolated strains of E.faecalis can induce apoptosis in MC3T3 osteoblasts, which may be attributed to the regulation of interaction between members of the Bcl-2 family.

Liraglutide Inhibits the Apoptosis of MC3T3-E1 Cells Induced by Serum Deprivation through cAMP/PKA/β-Catenin and PI3K/AKT/GSK3β Signaling Pathways.

In recent years, the interest towards the relationship between incretins and bone has been increasing. Previous studies have suggested that glucagon-like peptide-1 (GLP-1) and its receptor agonists exert beneficial anabolic influence on skeletal metabolism, such as promoting proliferation and differentiation of osteoblasts via entero-osseous-axis. However, little is known regarding the effects of GLP-1 on osteoblast apoptosis and the underlying mechanisms involved. Thus, in the present study, we investigated the effects of liraglutide, a glucagon-like peptide-1 receptor agonist, on apoptosis of murine MC3T3-E1 osteoblastic cells. We confirmed the presence of GLP-1 receptor (GLP-1R) in MC3T3-E1 cells. Our data demonstrated that liraglutide inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as detected by Annexin V/PI and Hoechst 33258 staining and ELISA assays. Moreover, liraglutide upregulated Bcl-2 expression and downregulated Bax expression and caspase-3 activity at intermediate concentration (100 nM) for maximum effect. Further study suggested that liraglutide stimulated the phosphorylation of AKT and enhanced cAMP level, along with decreased phosphorylation of GSK3β, increased β-catenin phosphorylation at Ser675 site and upregulated nuclear β-catenin content and transcriptional activity. Pretreatment of cells with the PI3K inhibitor LY294002, PKA inhibitor H89, and siRNAs GLP-1R, β-catenin abrogated the liraglutide-induced activation of cAMP, AKT, β-catenin, respectively. In conclusion, these findings illustrate that activation of GLP-1 receptor by liraglutide inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation through cAMP/PKA/β-catenin and PI3K/Akt/GSK3β signaling pathways.

P21Waf1/Cip1 depletion promotes dexamethasone-induced apoptosis in osteoblastic MC3T3-E1 cells by inhibiting the Nrf2/HO-1 pathway.

Dexamethasone (Dex), a glucocorticoid with strong anti-inflammatory and immunosuppressive activities, has been shown to exhibit marked cytotoxicity and apoptosis in osteoblasts, but the underlying mechanisms have not yet been comprehensively investigated. P21Waf1/Cip1 (p21) plays a critical role in the regulation of cell cycle progression and apoptosis. The present study aims to investigate the role of p21 in Dex-induced apoptosis in osteoblastic MC3T3-E1 cells, and to explore its mechanisms. Results demonstrated that Dex-induced apoptosis decreased the phosphorylation of Akt in a concentration-dependent manner. Moreover, LY294002, an inhibitor of the PI3K/Akt pathway enhanced the Dex-induced apoptosis of osteoblasts. On the contrary, insulin-like growth factor-1 (IGF-1), an activator of PI3K/Akt, attenuated the apoptosis of Dex in MC3T3-E1 cells. The protein level of p21 was downregulated by shortening its half-life, which was associated with inhibition of the PI3K/Akt pathway by Dex. Furthermore, depletion of p21 by siRNA enhanced Dex-induced caspase-3 activation and ROS generation, and promoted apoptosis of MC3T3-E1 cells. In addition, suppression of p21 led to a reduction of Dex-induced upregulation of nuclear Nrf2 and heme oxygenase-1 (HO-1) protein levels. These findings demonstrate that p21 depletion promotes Dex-induced apoptosis of MC3T3-E1 cells by inhibiting the antioxidant Nrf2/HO-1 pathway, which highlights the anti-apoptotic effect of p21 in MC3T3-E1 cells.

Oxidative stress-induced apoptosis of osteoblastic MC3T3-E1 cells by hydroxyapatite nanoparticles through lysosomal and mitochondrial pathways

Hydroxyapatite nanoparticles (HAPs) cause apoptosis of osteoblastic MC3T3-E1 cells through oxidative stress-induced lysosomal and mitochondrial pathway.

Bazedoxifene Ameliorates Homocysteine-Induced Apoptosis and Accumulation of Advanced Glycation End Products by Reducing Oxidative Stress in MC3T3-E1 Cells.

Elevated plasma homocysteine (Hcy) level increases the risk of osteoporotic fracture by deteriorating bone quality. However, little is known about the effects of Hcy on osteoblast and collagen cross-links. This study aimed to investigate whether Hcy induces apoptosis of osteoblastic MC3T3-E1 cells as well as affects enzymatic and nonenzymatic collagen cross-links and to determine the effects of bazedoxifene, a selective estrogen receptor modulator, on the Hcy-induced apoptosis and deterioration of collagen cross-links in the cells. Hcy treatments (300 μM, 3mM, and 10mM) increased intracellular reactive oxygen species (ROS) production in a dose-dependent manner. Propidium iodide staining showed that 3 and 10mM Hcy induced apoptosis of MC3T3-E1 cells. Moreover, the activities of caspases-8, 9, and 3 were increased by 3mM Hcy. The detrimental effects of 3mM Hcy on apoptosis and ROS production were partly reversed by bazedoxifene and 17β estradiol. In addition, real-time PCR, immunostaining and Western blot showed that 300 μM Hcy decreased the expression of lysyl oxidase (Lox). Furthermore, 300μM Hcy increased extracellular accumulation of pentosidine, an advanced glycation end product. Treatment with bazedoxifene ameliorated Hcy-induced suppression of Lox expression and increase in pentosidine accumulation. These findings suggest that high-dose Hcy induces apoptosis of osteoblasts by increasing oxidative stress, and low-dose Hcy decreases enzymatic collagen cross-links and increases pentosidine accumulation, resulting in the deterioration of bone quality. Bazedoxifene treatment effectively prevents the Hcy-induced detrimental reactions of osteoblasts. Thus, bazedoxifene may be a potent therapeutic drug for preventing Hcy-induced bone fragility.

Autophagy protects osteoblasts from advanced glycation end products-induced apoptosis through intracellular reactive oxygen species.

Patients with type II diabetes are susceptible to fracture; however, these patients typically have normal bone mineral density. Thus, such fractures cannot be entirely explained by advanced glycation end products (AGEs)-induced osteoblast apoptosis. Autophagy is a molecular process allowing cells to degrade unnecessary or dysfunctional cellular organelles, and closely interacts with apoptosis. The aim of this study was to determine whether autophagy participated in the pathology of AGEs-treated osteoblasts, and the possible mechanism of such an involvement. Osteoblastic MC3T3-E1 cells were used. Autophagy was evaluated by detecting the level of LC3 via western blotting and immunofluorescence. p62/SQSTM1 expression was also assessed by western blotting. The autophagy inducer rapamycin (RA) and the autophagy inhibitor 3-methyladenine were used to determine whether autophagy has effect on AGEs-induced apoptosis. N-Acetylcysteine (NAC), reactive oxygen species (ROS) inhibitor, was used to determine whether ROS and mitochondrial damage were involved in autophagy regulation. The results showed that the autophagy level was increased in MC3T3-E1 cells treated with AGEs, as represented by an increase in both the total LC3 level and the LC3II/LC3I ratio, as well as a decrease in p62/SQSTMI expression. Further inducing autophagy by RA attenuated AGEs-induced apoptosis. The antioxidant NAC suppresses AGEs-induced autophagy in osteoblastic MC3T3-E1 cells. These results demonstrate that autophagy participates in the pathology of AGEs-treated osteoblasts, and may play a protective role in AGEs-induced apoptosis in osteoblastic MC3T3-E1 cells. ROS and mitochondrial damage are essential in upregulating AGEs-induced autophagy.

Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways.

Bezafibrate prevents palmitate-induced apoptosis in osteoblastic MC3T3-E1 cells through the NF-κB signaling pathway

Osteoporosis is a bone condition defined by low bone mass and increase of fracture risk due to imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Low bone mass is likely to be due to the alteration of the osteoclast and osteoblast lifespan through regulated apoptosis. Saturated fatty acid (SFA) intake is negatively associated with bone mineral density (BMD). Furthermore, SFA induces apoptosis in osteoblastic cell lines. Bezafibrate could increase bone mass in intact male rats principally through increasing periosteal bone formation. At present, it is unknown whether bezafibrate attenuates palmitate-induced apoptosis in MC3T3-E1 cells. In the present study, we found that palmitate stimulated the degradation of IκBα and NF-κB translocation, as well as up-regulation of NF-κB-mediated Fas expression in obsteoblastic MC3T3-E1 cells. Furthermore, the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) could restore palmitate-induced caspase-3 decrease and inhibit palmitate-induced cleaved caspase-3 increase. We observed that bezafibrate, a dual ligand for the peroxisome proliferator-activated receptors α (PPARα) and PPARδ, significantly attenuated the palmitate-induced cytotoxicity as determined by the MTT assay and inhibited the palmitate-induced apoptosis as determined by a flow cytometry assay using Annexin V-FITC/PI and assessment of the activity of caspase-3. Pre-treatment of bezafibrate prevented palmitate-induced NF-κB activation. Therefore, these findings indicate that bezafibrate inbibits palmitate-induced apoptosis via the NF-κB signaling pathway. Our results point to bezafibrate as a new strategy to attenuate bone loss associated with high fat diet beyond its lipid-lowering actions.

Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells

Ghrelin is a 28-amino-acid peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) that strongly stimulates the release of growth hormone at the hypothalamus and pituitary level. Although GHS-Rs are expressed in a variety of peripheral tissues, little is known about its effect on bone independent of GH/IGF-1 axis. This study was undertaken to investigate whether ghrelin exerts a direct effect on osteoblasts. We identified mRNA and protein expression of GHS-R in primary osteoblasts as well as a number of osteoblastic cell lines, including MC3T3-E1, ROS 17/2.8, UMR-106, MG63, and SaOS2 cells. Treatment of ghrelin (10 −11 to 10 −7 M) to MC3T3-E1 cells showed dose-dependent stimulation of proliferation, which was abrogated by treatment with [ d-Lys]-GHRP-6 (10 −3 M), a selective antagonist of the ghrelin receptor. Ghrelin activated ERK1/2 MAPK and pretreatment with MAPK kinase inhibitors, PD98059 attenuated the ghrelin-induced cell proliferation. Ghrelin also inhibited TNFα-induced apoptosis and suppressed caspase-3 activation that occurs in response to TNFα as well as during in vitro differentiation process. Moreover, ghrelin treatment enhanced in vitro osteoblast differentiation as evidenced by matrix mineralization, alkaline phosphatase activity, and osteoblast-specific gene expression. These results suggest that ghrelin promotes proliferation and differentiation and inhibits apoptosis of osteoblasts.

Calbindin-D28k Is Expressed in Osteoblastic Cells and Suppresses Their Apoptosis by Inhibiting Caspase-3 Activity

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.