Apoptosis In Osteoblastic Cells Research Articles

Increased Lipid Oxidation Causes Oxidative Stress, Increased Peroxisome Proliferator-activated Receptor-γ Expression, and Diminished Pro-osteogenic Wnt Signaling in the Skeleton

Loss of bone mass with advancing age in mice is because of decreased osteoblast number and is associated with increased oxidative stress and decreased canonical Wnt signaling. However, the underlying mechanisms are poorly understood. We report an age-related increase in the lipid oxidation product 4-hydroxynonenal (4-HNE) as well as increased expression of lipoxygenase and peroxisome proliferator-activated receptor-gamma (PPARgamma) in the murine skeleton. These changes together with decreased Wnt signaling are reproduced in 4-month-old mice bearing a high expressing allele of the lipoxygenase Alox15. The addition of 4-HNE to cultured osteoblastic cells increases oxidative stress, which in turn diverts beta-catenin from T-cell-specific transcription factors to Forkhead box O (FoxO) transcription factors, thereby attenuating the suppressive effect of beta-catenin on PPARgamma gene expression. Oxidized lipids, acting as ligands of PPARgamma, promote binding of PPARgamma2 to beta-catenin and reduce the levels of the latter, and they attenuate Wnt3a-stimulated proliferation and osteoblast differentiation. Furthermore, oxidized lipids and 4-HNE stimulate apoptosis of osteoblastic cells. In view of the role of oxidized lipids in atherogenesis, the adverse effects of lipoxygenase-mediated lipid oxidation on the differentiation and survival of osteoblasts may provide a mechanistic explanation for the link between atherosclerosis and osteoporosis.

Simvastatin attenuates TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Simvastatin is a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor known as a stimulator for bone formation via promoting osteoblast differentiation and mineralization. This study was designed to examine the role of simvastatin on TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured in DMEM essential medium supplemented with 10% fetal bovine serum and antibiotics, and 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate cell viability. A trypan blue exclusion assay was used to determine the level of cytotoxicity. Hoechst staining for cells treated with TNF-α was visualized under a microscope equipped with an epifluorescence illumination. Apoptosis of MC3T3-E1 cells was further examined by flow cytometric analysis after Annexin V/7-AAD double staining. MC3T3-E1 cells exposed to low concentrations (10−10–10−7 M) of simvastatin increased in both growth and viability. In contrast, high concentrations of simvastatin (10−6–10−5 M) have no effect on growth and viability of MC3T3-E1 cells. TNF-α-induced decrease in cell viability and growth were antagonized by simvastatin in a concentration-dependent manner. MC3T3-E1 cells treated with TNF-α at 10 ng/ml showed typical pyknotic fragmented nuclei and apoptotic bodies assessed by Hoechst staining. Moreover, Annexin V/7-AAD double staining indicated that MC3T3-E1 cells treated with 10 ng/ml of TNF-α resulted in a significant increase of the proportion of apoptotic cells up to 39.5% of total cell population, and the proportion of apoptotic cells were dose-dependently decreased after simvastatin treatment (10−10 – 10−7 M); the inhibitory rates were approximately 31.34, 48.5, 52.11, and 65.73%, respectively. Our data demonstrate that simvastatin increases growth and viability of osteoblastic cells and decreases TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells.

Arsenic induces cell apoptosis in cultured osteoblasts through endoplasmic reticulum stress

Osteoporosis is characterized by low bone mass resulting from an imbalance between bone resorption by osteoclasts and bone formation by osteoblasts. Therefore, decreased bone formation by osteoblasts may lead to the development of osteoporosis, and rate of apoptosis is responsible for the regulation of bone formation. Arsenic (As) exists ubiquitously in our environment and increases the risk of neurotoxicity, liver injury, peripheral vascular disease and cancer. However, the effect of As on apoptosis of osteoblasts is mostly unknown. Here, we found that As induced cell apoptosis in osteoblastic cell lines (including hFOB, MC3T3-E1 and MG-63) and mouse bone marrow stromal cells (M2-10B4). As also induced upregulation of Bax and Bak, downregulation of Bcl-2 and dysfunction of mitochondria in osteoblasts. As also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosolic-calcium levels. We found that As increased the expression and activities of glucose-regulated protein 78 (GRP78) and calpain. Transfection of cells with GRP78 or calpain siRNA reduced As-mediated cell apoptosis in osteoblasts. Therefore, our results suggest that As increased cell apoptosis in cultured osteoblasts and increased the risk of osteoporosis.

Activated T lymphocytes induce apoptosis of osteoblastic cells derived from stromal cell line st2

Activated T lymphocytes induce apoptosis of osteoblastic cells derived from stromal cell line st2

Estrogen regulation of apoptosis in osteoblasts

Dysregulated apoptosis is a critical failure associated with prominent degenerative diseases including osteoporosis. In bone, estrogen deficiency has been associated with accelerated osteoblast apoptosis and susceptibility to osteoporotic fractures. Hormone therapy continues to be an effective option for preventing osteoporosis and bone fractures. Induction of apoptosis in G-292 human osteoblastic cells by exposure to etoposide or the inflammatory cytokine TNF-α promoted acute caspase-3/7 activity and this increased activity was inhibited by pretreatment with estradiol. Etoposide also increased the expression of a battery of apoptosis-promoting genes and this expression was also inhibited by estradiol. Among the apoptotic genes whose expression was inhibited by estradiol was ITPR1, which encodes the type 1 InsP3R. InsP3Rs are intracellular calcium channels and key proapoptotic mediators. Estradiol via estrogen receptor β1 suppresses ITPR1 gene transcription in G-292 cells. These analyses suggest that an underlying basis of the beneficial activity of estrogens in combating osteoporosis may involve the prevention of apoptosis in osteoblasts and that a key event in this process is the repression of apoptotic gene expression and inhibition of caspase-3/7.

Expression of Foxo transcription factors during osteoblast differentiation and cell death

Foxo transcription factors regulate the differentiation of adipocytes and myoblasts. However less is known regarding their role in the differentiation of other mesenchyme‐derived cell types such as osteoblasts and chondrocytes. Here we examined the expression levels of Foxo protein and mRNA during osteo‐ and chondrogenesis in vitro. Levels of Foxo1a and Foxo4 protein remained relatively unchanged throughout osteoblast differentiation of MC3T3 cells. Low levels of Foxo3a protein were detected in undifferentiated MC3T3 cells however, Foxo3a increased 1 day after the onset of differentiation. Foxo3a mRNA and protein levels also increased following BMP2 treatment of mouse primary limb bud micromass cultures. Since Foxo proteins play critical roles in mediating cell survival following oxidative stress, we assayed the expression of Foxo proteins following H2O2 treatment. MC3T3 apoptosis was detected by caspase 3 cleavage in cells treated H2O2 and resulted in a decrease in Foxo1a. Finally the affect of modulating Sirt1 activity and Foxo protein function, on survivial of MC3T3‐E1 cells was examined. H2O2‐mediated apoptosis increased in MC3T3‐E1 cells when treated with the Sirt1 agonists, while treatment with Sirt1 inhibitors decreased apoptosis. Together these findings indicate the Foxo3a and Foxo1a participate specific processes governing differentiation and apoptosis of osteoblast cells in vitro.

Glucocorticoid induces apoptosis of osteoblast cells through the activation of glycogen synthase kinase 3β

Glucocorticoids (GCs), which play an important role in the normal regulation of bone remodeling, are widely used as anti-inflammatory and chemotherapeutic agents. However, continued exposure to GCs results in osteoporosis, which is partially due to apoptosis of osteoblasts and osteocytes. To understand the mechanism of how GCs induce cell death in osteoblasts, we examined apoptotic effects of dexamethasone (Dex), GC, on MC3T3-E1 osteoblast cells. Results revealed that Dex-induced apoptosis was inhibited by a GC receptor antagonist, mifepristone, and a general caspase inhibitor, Z-VAD-fmk, indicating that Dex induces apoptosis of MC3T3-E1 cells through the pathways involved in GC receptor and caspase. Glycogen synthase kinase 3beta (GSK3beta) is known to participate in apoptosis signaling in MC3T3-E1 cells. Dex activated both GSK3beta and p38-mitogen-activated protein kinase (MAPK). The inhibition of GSK3beta by inhibitor (LiCl) or small interference RNA (siRNA) decreased apoptosis. In contrast, the inhibition of p38-MAPK by inhibitor (SB203580) or siRNA did not decrease, but increase apoptosis. These results suggest that Dex-mediated apoptosis of osteoblasts is facilitated by GSK3beta, but prevented by p38-MAPK.

Serum from children with polyarticular juvenile idiopathic arthritis (pJIA) inhibits differentiation, mineralization and may increase apoptosis of human osteoblasts “in vitro”

We examined the effects of polyarticular juvenile idiopathic arthritis (pJIA) serum on proliferation, differentiation, mineralization, and apoptosis of human osteoblast cells (hOb) in culture. The hOb were cultured with 10% serum from active pJIA and healthy controls (CT) and were tested for DNA synthesis, alkaline phosphatase (AP) activity, osteocalcin (OC) secretion, calcium levels, caspase 3 activity, and DNA fragmentation. None of the patients had used glucocorticoids for at least 1 month before the study, or any other drug that can affect bone mineral metabolism. Human inflammatory cytokine levels (IL-6, IL-8, IL-10, IL-1beta, TNF-alpha, and IL-12p70) were measured in pJIA and CT sera. Low levels of AP activity was observed in pJIA cultures compared with CT cultures (67.16 +/- 53.35 vs 100.11 +/- 50.64 mumol p-nitrophenol/h(-1) mg(-1) protein, P = 0.008). There was also a significant decrease in OC secretion (9.23 +/- 5.63 vs 12.82 +/- 7.02 ng/mg protein, P = 0.012) and calcium levels (0.475 +/- 0.197 vs 0.717 +/- 0.366 mmol/l, P = 0.05) in pJIA hOb cultures. No difference was observed in cell proliferation (323.56 +/- 108.23 vs 328.91 +/- 88.03 dpm/mg protein, P = 0.788). Osteoblasts cultured with JIA sera showed lower levels of DNA and increased fragmentation than osteoblasts cultured with CT sera. pJIA sera showed higher IL-6 values than CT (21.44 +/- 9.31 vs 3.58 +/- 2.38 pg/ml, P < 0.001), but no difference was observed related to IL-8, IL-10, IL-1beta, TNF-alpha, and IL-12p70 between pJIA and controls. This study suggests that serum from children with pJIA inhibits differentiation, mineralization and may increase apoptosis of hOb cultures, and inflammatory cytokines such as IL-6 might be a mechanism in this find. These results may represent an alternative therapeutic target for prevention and treatment of bone loss in JIA.

Ghrelin antagonizes MPTP-induced neurotoxicity to the dopaminergic neurons in mouse substantia nigra

Ghrelin, a stomach-derived hormone which induces growth hormone release and promotes positive energy balance, has been reported to inhibit cell apoptosis in endotheliocytes, osteoblasts and cardiocytes. Recent evidence has shown that ghrelin can also inhibit neuronal apoptosis of the hypothalamus and the hippocampus. However, little is known about the effects of ghrelin on the substantia nigra pars compacta (SNpc) neurons in which ghrelin's receptor, growth hormone secretagogue receptor (GHSR)-1a, is highly expressed. In the present study, we investigated whether ghrelin could protect nigral dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice. We observed that ghrelin, acting through GHS-R 1a, inhibited MPTP-induced dopaminergic neuronal loss in the SNpc as well as dopamine depletion in the striatum. Ghrelin could also reverse the down-regulated the expression of Bcl-2, up-regulated the expression of Bax, and caspase-3 activation caused by MPTP. This study demonstrated that ghrelin might be a potential protector of dopaminergic neurons in a therapeutic strategy for Parkinson's disease.

15-Deoxy-Δ 12,14-prostaglandin J 2 induces apoptosis via JNK-mediated mitochondrial pathway in osteoblastic cells

The cyclopentenone prostaglandin 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2) induces apoptosis in various cell types. However, the underlying mechanism of 15d-PGJ 2-induced apoptosis is not fully understood. The present study was undertaken to determine the molecular mechanism by which 15d-PGJ 2 induces apoptosis in MC3T3-E1 mouse osteoblastic cells. 15d-PGJ 2 caused a concentration- and time-dependent apoptotic cell death. 15d-PGJ 2 induced a transient activation of ERK1/2 and sustained activation of JNK. 15d-PGJ 2-induced cell death was prevented by the JNK inhibitor SP6001, but not by inhibitors of ERK1/2 and p38. JNK activation by 15d-PGJ 2 was blocked by antioxidants N-acetylcysteine (NAC) and GSH. 15d-PGJ 2 caused ROS generation and 15d-PGJ 2-induced cell death was prevented by antioxidants, suggesting involvement of ROS generation in 15d-PGJ 2-induced cell death. 15d-PGJ 2 triggered the mitochondrial apoptotic pathway indicated by enhanced Bax expression, loss of mitochondrial membrane potential, cytochrome c release, and caspase-3 activation. The JNK inhibitor blocked these events induced by 15d-PGJ 2. Taken together, these results suggest that the 15d-PGJ 2 induces cell death through the mitochondrial apoptotic pathway dependent of ROS and JNK activation in osteoblastic cells.

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.

Adrenomedullin is anti‐apoptotic in osteoblasts through CGRP1 receptors and MEK‐ERK pathway

Adrenomedullin (ADM) has been shown to mediate multifunctional responses in cell culture and animal system such as regulation of growth and apoptosis. ADM stimulates the proliferation of osteoblasts in vitro and promotes bone growth in vivo. The ability of ADM to influence osteoblastic cell number through inhibition of apoptosis has not yet been studied. To address this question we have investigated its effect on the apoptosis of serum-deprived osteoblastic cells using mouse MC3T3-E1 cells which express both ADM and ADM receptors. Treatment with ADM significantly blunted apoptosis, evaluated by caspase-3 activity, DNA fragmentation quantification and annexin V-FITC labeling. This effect was abolished by the subtype-1 CGRP receptor antagonist, CGRP(8-37). Both ADM and its specific receptor antagonist, the (22-52) ADM fragment exhibited a similar anti-apoptotic effect. Thus, our data suggest that ADM exerts anti-apoptotic effects through CGRP1 receptors. This was substantiated by a similar protective effect of CGRP on MC3T3-E1 cells apoptosis. Accordingly, neutralization of endogenous ADM by a specific antibody enhanced apoptosis. Finally, the selective inhibitor of MAPK kinase (MEK), PD98059, abolished the apoptosis protective effect of ADM and prevented ADM activation of ERK1/2. These data show that ADM acts as a survival factor in osteoblastic cells via a CGRP1 receptor-MEK-ERK pathway, which provides further understanding on the physiological function of ADM in osteoblasts.

Quercetin accelerates TNF-α-induced apoptosis of MC3T3-E1 osteoblastic cells through caspase-dependent and JNK-mediated pathways

The bioflavonoid, quercetin, is believed to inhibit bone loss by regulating many systemic and local factors including hormones and cytokines. However, our previous findings revealed that quercetin did not inhibit but facilitate the tumor necrosis factor (TNF)-α-mediated apoptosis of MC3T3-E1 osteoblastic cells. Therefore, this study was carried out to examine the cellular mechanisms for how quercetin accelerates TNF-α-mediated apoptosis, and to determine whether the accelerating effect of quercetin is a general effect in osteoblastic cells. Quercetin promoted the TNF-α-induced apoptosis of MC3T3-E1 cells through both the mitochondrial-mediated and caspase-dependent mechanisms. Quercetin also augmented the TNF-α-mediated apoptosis by activating c-Jun N-terminal kinase (JNK) with the attendant activation of activator protein-1, where the nuclear translocation of c-Jun protein appeared to be a critical event responsible for the accelerating action of quercetin. However, TNF-α-mediated apoptosis and its acceleration by quercetin were not observed in primary osteoblasts. These results strongly suggest that quercetin accelerates TNF-α-mediated apoptosis of osteoblasts through caspase-dependent and JNK-mediated pathways, and that the cellular responses of osteoblasts to TNF-α and/or quercetin might differ according to their origins.

Differential Effects of TGF-?? Isoforms on Murine Fetal Dural Cells and Calvarial Osteoblasts

Proteins within the transforming growth factor (TGF)-beta family play a central role in both normal and pathologic calvarial morphogenesis. Previous work has suggested differential functions of the TGF-beta isoforms in these processes. Little is known, however, about effects of TGF-betas on the underlying dura. Furthermore, studies on the effects of TGF-beta isoforms on osteoblasts have been conflicting. The purpose of this study was to determine the effect of TGF-beta isoforms, specifically TGF-beta1 and TGF-beta3, on fetal calvarial osteoblast and dural cell differentiation, proliferation, and apoptosis. Primary cultures of fetal calvarial osteoblasts and dural cells were established from embryonic day-18 CD-1 mice. Cells were treated for 48 hours with TGF-beta1 or TGF-beta3. Northern blot analysis, cell counts, and apoptosis assays were performed. In dural cells, TGF-beta1 stimulated the expression of early osteodifferentiation genes and resulted in a slight decrease in cell number and no effect on apoptosis. Similar results were observed in osteoblasts. TGF-beta3 had little or no effect on the genes studied in both cell types but resulted in increased apoptosis and concomitant decreases in cell number in both cell types. This study demonstrates that dural cells respond to TGF-beta and that this response is isoform-specific. TGF-beta1 stimulates osteodifferentiation of previously uncommitted cells in the dura. It also stimulates early events in bone matrix deposition and has little effect on late markers of bone differentiation in osteoblasts and dural cells. Both isoforms result in decreases in cell number. TGF-beta3 results in greater decreases in cell number and isoform-specific stimulation of apoptosis in both dural cells and calvarial osteoblasts.

Calyculin A induces apoptosis and stimulates phosphorylation of p65NF-κB in human osteoblastic osteosarcoma MG63 cells

Previously, we reported that okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, induced apoptosis in human osteoblastic cells. However, it is not clear whether calyculin A, another inhibitor of protein phosphatases, would induce apoptosis in human osteoblastic cells and if so, which mechanisms are involved and whether the phosphorylation status of NF-kappaB could be affected by the treatment with calyculin A. In this report, we demonstrate that calyculin A induced apoptosis in MG63 cells, as judged by WST-8 assay, nuclear fragmentation, and DNA ladder formation. Expression of PTEN, FasL, and FasR mRNA was stimulated by calyculin A treatment in MG63 cells. Calyculin A also enhanced the phosphorylation level of NF-kappaB, as judged from the results of Western blot analysis and an in vitro dephosphorylation assay. Western blot analysis with anti-phospho-p65NF-kappaB antibody disclosed that the NF-kappaB was phosphorylated on serine 536 in cytosol and translocated into nucleus with calyculin A-treatment. The phosphorylation status of p65NF-kappaB was further confirmed by using the phosphorylation site-mutated p65NF-kappaB gene transfected into HEK293 cells. Unlike TNF-alpha, calyculin A treatment did not degraded IkappaBalpha within 10 min, while it degraded IkappaBalpha at 2-h treatment. Our findings indicate that calyculin A elicit phosphorylation of NF-kappaB on serine 536 in MG63 cells, resulting in the translocation of phospho-NF-kappaB to the nucleus, thereby promoting transcriptional activity of NF-kappaB-related genes.

ApoE Gene Deficiency Enhances the Reduction of Bone Formation Induced by a High-Fat Diet Through the Stimulation of p53-Mediated Apoptosis in Osteoblastic Cells

Osteoblast apoptosis increased in the tibias of apoE(-/-) mice fed with a high-fat diet, decreasing bone formation. The expression of p53 mRNA in marrow adherent cells increased. LDL or oxidized LDL increased apoptosis in the calvarial cells of apoE(-/-) mice. The increase in p53-mediated apoptosis is apparently related to a high-fat diet-induced osteopenia in apoE(-/-) mice. The effects of high-fat loading and the apolipoprotein E (apoE) gene on bones have not been elucidated. We hypothesized that apoE gene deficiency (apoE(-/-)) modulates the effects of high-fat loading on bones. We assessed this hypothesis using wildtype (WT) and apoE(-/-) mice fed a standard (WTS and ApoES groups) or a high-fat diet (WTHf and ApoEHf groups). The concentration of serum lipid levels and bone chemical markers were measured. Histomorphometry of the femurs was performed using microCT and a microscope. Bone marrow adherent cells from the femurs were used for colony-forming unit (CFU)-fibroblastic (CFU-f) assay and mRNA expressions analysis. The apoptotic cells in the tibias were counted. TUNEL fluorescein assay and Western analysis were performed in cultures of calvarial cells by the addition of low-density lipoprotein (LDL) or oxidized LDL. In the ApoEHf group, the values of cortical bone volume and trabecular and endocortical bone formation of the femurs decreased, and urinary deoxypyridinoline increased. Subsequent analysis revealed that the number of apoptotic cells in the tibias of the ApoES group increased, and more so in the ApoEHf group. The ratio of alkaline phosphatase-positive CFU-f to total CFU-f was decreased in the ApoEHf group. p53 mRNA expression in adherent cells of the apoE(-/-) mice increased and had a significantly strong positive correlation with serum LDL. TUNEL fluorescein assay of osteoblastic cells revealed an increase of apoptotic cells in the apoE(-/-) mice. The number of apoptotic cells in the apoE(-/-) mice increased with the addition of 100 microg/ml LDL or oxidized LDL. The p53 protein expression in apoE(-/-) cells exposed to 100 microg/ml LDL or oxidized LDL increased. We concluded that apoE gene deficiency enhances the reduction of bone formation induced by a high-fat diet through the stimulation of p53-mediated apoptosis in osteoblastic cells.

Fidgetin-Like 1 Gene Inhibited by Basic Fibroblast Growth Factor Regulates the Proliferation and Differentiation of Osteoblasts

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.

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-α. 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.

Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways

We have previously shown that diabetes significantly enhances apoptosis of osteoblastic cells in vivo and that the enhanced apoptosis contributes to diabetes impaired new bone formation. A potential mechanism is enhanced apoptosis stimulated by advanced glycation end products (AGEs). To investigate this further, an advanced glycation product, carboxymethyl lysine modified collagen (CML-collagen), was injected in vivo and stimulated a 5-fold increase in calvarial periosteal cell apoptosis compared to unmodified collagen. It also induced apoptosis in primary cultures of human or neonatal rat osteoblastic cells or MC3T3-E1 cells in vitro. Moreover, the apoptotic effect was largely mediated through RAGE receptor. CML-collagen increased p38 and JNK activity 3.2- and 4.4-fold, respectively. Inhibition of p38 and JNK reduced CML-collagen stimulated apoptosis by 45% and 59% and by 90% when used together ( P < 0.05). The predominant apoptotic pathway induced by CML-collagen involved caspase-8 activation of caspase-3 and was independent of NF-κB activation. When osteoblastic cells were exposed to a long-term low dose incubation with CML-collagen, there was a higher degree of apoptosis compared to short-term incubation. In more differentiated osteoblastic cultures, apoptosis was enhanced even further. These results indicate that advanced glycation end products, which accumulate in diabetic and aged individuals, may promote apoptosis of osteoblastic cells and contribute to deficient bone formation.

Cytokine-induced nitric-oxide-dependent apoptosis in mouse osteoblastic cells: Involvement of p38MAP kinase

The apoptotic signalling induced by pro-inflammatory cytokines was examined in mouse osteoblastic MC3T3-E1 cells. Annexin-V/propidium iodine double-staining analysis demonstrated that the combination of tumour necrosis factor-alpha, interleukin-1beta and interferon-gamma caused cell death in osteoblastic cells mediated by apoptosis, not necrosis. Treatment with these cytokines resulted in potent enhancement of inducible nitric-oxide synthase (iNOS) mRNA and nitric-oxide (NO) in the cells. A specific inhibitor of p38 mitogen-activated protein (MAP) kinase, i.e. SB203580, dose dependently inhibited the induction of iNOS mRNA, its enzyme product, NO and DNA fragmentation (as an apoptosis index) in the cytokine-treated cells (P<0.05). In contrast, PD98059, a specific inhibitor of MEK that acts immediately upstream of classic MAP kinase, had no effect on the induction of iNOS, NO or DNA fragmentation in the cells. These results demonstrate that this cytokine-induced apoptosis in mouse osteoblastic cells was mediated by a p38MAP-kinase-dependent iNOS system.