OB-6 Cells Research Articles

Atorvastatin Upregulates microRNA-186 and Inhibits the TLR4-Mediated MAPKs/NF-κB Pathway to Relieve Steroid-Induced Avascular Necrosis of the Femoral Head.

Steroid-induced avascular necrosis of the femoral head (SANFH) is caused by the death of active components of the femoral head owing to hormone overdoses. The use of lipid-lowering drugs to prevent SANFH in animals inspired us to identify the mechanisms involving Atorvastatin (Ato) in SANFH. However, it is still not well understood how and to what extent Ato affects SANFH. This study aimed to figure out the efficacy of Ato in SANFH and the underlying molecular mechanisms. After establishment of the SANFH model, histological evaluation, lipid metabolism, inflammatory cytokines, oxidative stress, apoptosis, and autophagy of the femoral head were evaluated. The differentially expressed microRNAs (miRs) after Ato treatment were screened out using microarray analysis. The downstream gene and pathway of miR-186 were predicted and their involvement in SANFH rats was analyzed. OB-6 cells were selected to simulate SANFH in vitro. Cell viability, cell damage, inflammation responses, apoptosis, and autophagy were assessed. Ato alleviated SANFH, inhibited apoptosis, and promoted autophagy. miR-186 was significantly upregulated after Ato treatment. miR-186 targeted TLR4 and inactivated the MAPKs/NF-κB pathway. Inhibition of miR-186 reversed the protection of Ato on SANFH rats, while inhibition of TLR4 restored the protective effect of Ato. Ato reduced apoptosis and promoted autophagy of OB-6 cells by upregulating miR-186 and inhibiting the TLR4/MAPKs/NF-κB pathway. In conclusion, Ato reduced apoptosis and promoted autophagy, thus alleviating SANFH via miR-186 and the TLR4-mediated MAPKs/NF-κB pathway.

FGF23 protects osteoblasts from dexamethasone-induced oxidative injury.

Dexamethasone (DEX) can exert a cytotoxic effect on cultured osteoblasts. The current study explored the potential osteoblast cytoprotective effect of fibroblast growth factor 23 (FGF23). In OB-6 human osteoblastic cells and primary murine osteoblasts, FGF23 induced phosphorylation of the receptor FGFR1 and activated the downstream Akt-S6K1 signaling. FGF23-induced FGFR1-Akt-S6K phosphorylation was largely inhibited by FGFR1 shRNA, but augmented with ectopic FGFR1 expression in OB-6 cells. FGF23 attenuated DEX-induced death and apoptosis in OB-6 cells and murine osteoblasts. Its cytoprotective effects were abolished by FGFR1 shRNA, Akt inhibition or Akt1 knockout. Conversely, forced activation of Akt inhibited DEX-induced cytotoxicity in OB-6 cells. Furthermore, FGF23 activated Akt downstream nuclear-factor-E2-related factor 2 (Nrf2) signaling to alleviate DEX-induced oxidative injury. On the contrary, Nrf2 shRNA or knockout almost reversed FGF23-induced osteoblast cytoprotection against DEX. Collectively, FGF23 activates FGFR1-Akt and Nrf2 signaling cascades to protect osteoblasts from DEX-induced oxidative injury and cell death.

Four-octyl itaconate activates Nrf2 cascade to protect osteoblasts from hydrogen peroxide-induced oxidative injury

Four-octyl itaconate (4-OI) is the cell-permeable derivative of itaconate that can activate Nrf2 signaling by alkylating Keap1’s cysteine residues. Here, we tested the potential effect of 4-OI on hydrogen peroxide (H2O2)-induced oxidative injury in osteoblasts. In OB-6 cells and primary murine osteoblasts, 4-OI was able to activate Nrf2 signaling cascade and cause Keap1–Nrf2 disassociation, Nrf2 protein stabilization, cytosol accumulation, and nuclear translocation. 4-OI also augmented antioxidant-response element reporter activity and promoted expression of Nrf2-dependent genes (HO1, NQO1, and GCLC). Pretreatment with 4-OI inhibited H2O2-induced reactive oxygen species production, cell death, and apoptosis in osteoblasts. Furthermore, 4-OI inhibited H2O2-induced programmed necrosis by suppressing mitochondrial depolarization, mitochondrial cyclophilin D-ANT1 (adenine nucleotide translocase 1)-p53 association, and cytosol lactate dehydrogenase release in osteoblasts. Ectopic overexpression of immunoresponsive gene 1 (IRG1) increased endogenous itaconate production and activated Nrf2 signaling cascade, thereby inhibiting H2O2-induced oxidative injury and cell death. In OB-6 cells, Nrf2 silencing or CRISPR/Cas9-induced Nrf2 knockout blocked 4-OI-induced osteoblast cytoprotection against H2O2. Conversely, forced Nrf2 activation, by CRISPR/Cas9-induced Keap1 knockout, mimicked 4-OI-induced actions in OB-6 cells. Importantly, 4-OI was ineffective against H2O2 in Keap1-knockout cells. Collectively, 4-OI efficiently activates Nrf2 signaling to inhibit H2O2-induced oxidative injury and death of osteoblasts.

MiR-107 inhibition upregulates CAB39 and activates AMPK-Nrf2 signaling to protect osteoblasts from dexamethasone-induced oxidative injury and cytotoxicity.

To human osteoblasts dexamethasone (DEX) treatment induces significant oxidative injury and cytotoxicity. Inhibition of CAB39 (calcium binding protein 39)-targeting microRNA can induce CAB39 upregulation, activating AMP-activated protein kinase (AMPK) signaling and offering osteoblast cytoprotection. Here we identified a novel CAB39-targeting miRNA: the microRNA-107 (miR-107). RNA-Pull down assay results demonstrated that the biotinylated-miR-107 directly binds to CAB39 mRNA in OB-6 human osteoblastic cells. Forced overexpression of miR-107, by infection of pre-miR-107 lentivirus or transfection of wild-type miR-107 mimic, largely inhibited CAB39 expression in OB-6 cells and primary human osteoblasts. Contrarily, miR-107 inhibition, by antagomiR-107, increased its expression, resulting in AMPK cascade activation. AntagomiR-107 largely attenuated DEX-induced cell death and apoptosis in OB-6 cells and human osteoblasts. Importantly, osteoblast cytoprotection by antagomiR-107 was abolished with AMPK in-activation by AMPKα1 dominant negative mutation, silencing or knockout. Further studies demonstrated that antagomiR-107 activated AMPK downstream Nrf2 cascade to inhibit DEX-induced oxidative injury. Conversely, Nrf2 knockout almost abolished antagomiR-107-induced osteoblast cytoprotection against DEX. Collectively, miR-107 inhibition induced CAB39 upregulation and activated AMPK-Nrf2 signaling to protect osteoblasts from DEX-induced oxidative injury and cytotoxicity.

Circular RNA HIPK3 downregulation mediates hydrogen peroxide-induced cytotoxicity in human osteoblasts.

Hydrogen peroxide (H2O2) induces oxidative injury to human osteoblasts. The expression and potential function of circular RNA HIPK3 (circHIPK3) in H2O2-treated human osteoblasts were tested. We show that H2O2 significantly downregulated circHIPK3 in OB-6 cells and primary human osteoblasts. Furthermore, circHIPK3 levels were decreased in the necrotic femoral head tissues of dexamethasone-treated patients. In OB-6 osteoblastic cells and primary human osteoblasts, forced overexpression of circHIPK3 by a lentiviral construct alleviated H2O2-induced viability reduction, cell death and apoptosis. Contrarily, circHIPK3 silencing by targeted shRNA potentiated H2O2-induced cytotoxicity in OB-6 cells and primary human osteoblasts. Moreover, circHIPK3 downregulation by H2O2 induced miR-124 accumulation in OB-6 cells and primary human osteoblasts. On the contrary, miR-124 inhibition by transfection of the miR-124 inhibitor protected human osteoblasts from H2O2. Importantly, forced overexpression of miR-124 by transfection of the miR-124 mimic induced significant cytotoxicity in OB-6 cells and primary human osteoblasts. H2O2 downregulated miR-124’s targets, cyclin dependent kinase 6 and Rho-Associated Protein Kinase 1, in human osteoblasts. In conclusion circHIPK3 downregulation mediates H2O2-induced cytotoxicity in human osteoblasts.

PGK1 depletion activates Nrf2 signaling to protect human osteoblasts from dexamethasone

Activation of nuclear-factor-E2-related factor 2 (Nrf2) cascade can alleviate dexamethasone (DEX)-induced oxidative injury and death of human osteoblasts. A recent study has shown that phosphoglycerate kinase 1 (PGK1) inhibition/depletion will lead to Kelch-like ECH-associated protein 1 (Keap1) methylglyoxal modification, thereby activating Nrf2 signaling cascade. Here, in OB-6 osteoblastic cells and primary human osteoblasts, PGK1 silencing, by targeted shRNA, induced Nrf2 signaling cascade activation, causing Nrf2 protein stabilization and nuclear translocation, as well as increased expression of ARE-dependent genes (HO1, NQO1, and GCLC). Functional studies demonstrated that PGK1 shRNA largely attenuated DEX-induced oxidative injury and following death of OB-6 cells and primary osteoblasts. Furthermore, PGK1 knockout, by the CRISPR/Cas9 method, similarly induced Nrf2 signaling activation and protected osteoblasts from DEX. Importantly, PGK1 depletion-induced osteoblast cytoprotection against DEX was almost abolished by Nrf2 shRNA. In addition, Keap1 shRNA mimicked and nullified PGK1 shRNA-induced anti-DEX osteoblast cytoprotection. At last we show that PGK1 expression is downregulated in human necrotic femoral head tissues of DEX-taking patients, correlating with HO1 depletion. Collectively, these results show that PGK1 depletion protects human osteoblasts from DEX via activation of Keap1-Nrf2 signaling cascade.

MicroRNA-7 inhibition protects human osteoblasts from dexamethasone via activation of epidermal growth factor receptor signaling.

Sustained dexamethasone (Dex) treatment could induce secondary osteoporosis, osteonecrosis, or even bone fractures. Dex can induce potent cytotoxicity in cultured human osteoblasts. The aim of this study was to test the potential role of microRNA-7 (miR-7), which targets the epidermal growth factor receptor (EGFR), in Dex-treated human osteoblasts. In OB-6, hFOB1.19, and primary human osteoblasts, miR-7 depletion by a lentiviral antagomiR-7 construct (LV-antagomiR-7) increased EGFR expression and downstream Akt activation, protecting cells from Dex-induced viability reduction, cell death, and apoptosis. In contrast, forced overexpression of miR-7 by a lentiviral miR-7 construct (LV-miR-7) inhibited EGFR expression and Akt activation, potentiating Dex-induced cytotoxicity in OB-6, hFOB1.19, and primary human osteoblasts. EGFR is the primary target of miR-7 in human osteoblasts. Luciferase activity of the EGFR 3-untranslated region was enhanced by LV-antagomiR-7, but decreased by LV-miR-7 in OB-6 cells. Further, LV-antagomiR-7-induced osteoblast cytoprotection against Dex was abolished by the EGFR inhibitors AG1478 and PD153035. Moreover, neither LV-antagomiR-7 nor LV-miR-7 was functional in EGFR-KO OB-6 cells. We also show that miR-7 is upregulated in the necrotic femoral head tissues of Dex-administered patients, correlating with EGFR downregulation. Together, we conclude that miR-7 inhibition protects human osteoblasts from Dex via activation of EGFR signaling.

Isopsoralen ameliorates H2O2-induced damage in osteoblasts via activating the Wnt/β-catenin pathway.

Osteoporosis is a disease with a worldwide prevalence that involves a severe loss of bone mineral density and decreased microarchitecture, which increases the risk of bone fracture. The present study evaluated the effects of isopsoralen on osteoblastic OB-6 cells following hydrogen peroxide (H2O2)-induced damage and investigated the molecular mechanisms involved in this process. For in vitro experiments, OB-6 osteoblasts were treated with H2O2 or H2O2 + isopsoralen then the cell viability, apoptosis, reactive oxygen species (ROS) production and calcium accumulation were determined. Results demonstrated that treatment with H2O2 reduced cell viability, runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) expression levels, and calcium deposition, whilst markedly increasing cell apoptosis and ROS production. However, isopsoralen (1 µM) provided significant protection against H2O2-induced alterations in osteoblasts. In addition, isopsoralen effectively upregulated protein expression of tankyrase and β-catenin which are the main transductors of the Wnt/β-catenin pathway. Of note, the protective effects of isopsoralen against H2O2-induced damage were attenuated in OB-6 cells treated with tankyrase inhibitor XAV-939. In conclusion, the present findings provided evidence that isopsoralen attenuated oxidative stress-induced injury in osteoblasts via the Wnt/β-catenin signaling pathway.

Dexamethasone-induced cytotoxicity in human osteoblasts is associated with circular RNA HIPK3 downregulation

Dexamethasone (DEX) exerts potent cytotoxicity against cultured human osteoblasts. The current study examined the role of the circular RNA HIPK3 (circHIPK3) in the mechanism of cell death. We found that circHIPK3 expression was downregulated in DEX-treated human osteoblasts and circHIPK3 levels decreased in human necrotic femoral head tissues. In OB-6 osteoblastic cells and primary human osteoblasts ectopic overexpression of circHIPK3 potently suppressed DEX-induced apoptosis and programmed necrosis. Conversely, knockdown of circHIPK3by targeted siRNAs enhanced DEX-induced cytotoxicity in human osteoblasts. We further observed that microRNA-124 (miR-124), a key miRNA sponged by circHIPK3, accumulated following DEX treatment in OB-6 cells and primary osteoblasts. Confirming the role of miR-124 in DEX-induced cytotoxicity, miR-124 inhibitor attenuated cell death in human osteoblasts. Conversely, forced overexpression of miR-124 mimicked DEX-induced actions and induced cytotoxicity in human osteoblasts. We conclude that DEX-induced cytotoxicity in human osteoblasts is associated with circHIPK3 downregulation.

LncRNA EPIC1 protects human osteoblasts from dexamethasone-induced cell death

Dexamethasone (Dex) can induce injury to human osteoblasts. Long non-coding RNA (LncRNA) EPIC1 (Lnc-EPIC1) is a novel Myc-interacting LncRNA. Its effect on Dex-treated human osteoblasts is studied here. In OB-6 osteoblastic cells and primary human osteoblasts, treatment with Dex increased expression of Lnc-EPIC1. Its expression is also elevated in the necrotic femoral head tissues of Dex-taking patients. Ectopic overexpression of Lnc-EPIC1 inhibited Dex-induced apoptosis and programmed necrosis in OB-6 cells and primary human osteoblasts. Reversely, Lnc-EPIC1 silencing by targeted siRNA potentiated Dex-induced cytotoxicity. Myc is the target of Lnc-EPIC1 in osteoblasts. Exogenous overexpression of Myc protected OB-6 cells from Dex. Conversely, Myc knockout by CRISPR-Cas-9 method abolished Lnc-EPIC1-induced OB-6 cytoprotection against Dex. Together, Lnc-EPIC1 expression protects human osteoblasts from Dex possible via regulation of Myc.

MicroRNA-29a inhibition induces Gab1 upregulation to protect OB-6 human osteoblasts from hydrogen peroxide

The present study determines the role of the Gab1 in hydrogen peroxide (H2O2)-induced death of human osteoblasts. We show that Gab1 is required for H2O2-induced Akt activation to promote osteoblast survival. In OB-6 human osteoblasts, Gab1 silencing (by targeted-shRNA) or complete knockout (by CRISPR-Cas9 KO plasmid) largely attenuated Akt activation by H2O2. Gab1-depleted OB-6 cells were more vulnerable to H2O2. Conversely, forced over-expression of Gab1 by an adenovirus vector increased Akt activation to protect OB-6 cells from H2O2. Significantly, the anti-sense of microRNA-29a (“antagomiR-29a”) induced Gab1 expression to facilitate H2O2-induced Akt activation, which protected OB-6 cells from apoptosis. AntagomiR-29a was however ineffective in Gab1-deficient and Akt-inhibited OB-6 cells. Forced over-expression of miR-29a induced Gab1 downregulation to inhibit H2O2-induced Akt activation, causing enhanced OB-6 cell death. miR-29a-induced actions were abolished by an adenovirus constitutively-active Akt1 (Ad-caAkt1) in OB-6 cells. Together, microRNA-29a inhibition induces Gab1 upregulation and Akt activation to protect OB-6 osteoblasts from H2O2.

Activation of Nrf2 by MIND4-17 protects osteoblasts from hydrogen peroxide-induced oxidative stress.

MIND4-17 is a recently developed NF-E2-related factor 2 (Nrf2) activator, which uniquely causes Nrf2 disassociation from Keap1. Here, we showed that pretreatment with MIND4-17 significantly inhibited hydrogen peroxide (H2O2)-induced viability reduction of primary osteoblasts and OB-6 osteoblastic cells. Meanwhile, MIND4-17 inhibited both apoptotic and non-apoptotic osteoblast cell death by H2O2. MIND4-17 treatment induced Keap1-Nrf2 disassociation, causing Nrf2 stabilization, accumulation and nuclear translocation in osteoblasts, leading to transcription of several Nrf2-dependent genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), γ-glutamylcysteine synthetase modifier subunit (GCLM) and catalytic subunit (GCLC). Additionally, MIND4-17 largely attenuated H2O2-reactive oxygen species (ROS) production, lipid peroxidation and DNA damages. Nrf2 knockdown by targeted short hairpin RNA (shRNA) exacerbated H2O2-induced cytotoxicity in OB-6 osteoblastic cells, and nullified MIND4-17-mediated cytoprotection against H2O2. Meanwhile, Keap1 shRNA took over MIND4-17′s actions and protected OB-6 cells from H2O2. Together, MIND4-17 activates Nrf2 signaling and protects osteoblasts from H2O2.

MicroRNA-200a activates Nrf2 signaling to protect osteoblasts from dexamethasone.

Treatment with dexamethasone in human osteoblasts leads to oxidative stress and cell injures. NF-E2-related factor 2 (Nrf2) is a key anti-oxidant signaling. We want to induce Nrf2 activation via microRNA-mediated silencing its suppressor Keap1. Our results show that microRNA-200a (“miR-200a”) expression depleted Keap1, causing Nrf2 protein stabilization in OB-6 osteoblastic cells. Reversely, the miR-200a anti-sense led to Keap1 upregulation and Nrf2 degradation. miR-200a expression activated Nrf2 signaling, which inhibited dexamethasone-induced reactive oxygen species production and OB-6 cell death/apoptosis. Keap1 shRNA also activated Nrf2 and protected OB-6 cells from dexamethasone. Importantly, miR-200a was in-effective in Keap1-silenced (by shRNA) OB-6 cells. In the primary human osteoblasts, Keap1 silence by targeted-shRNA or miR-200a protected cells from dexamethasone. Significantly, miR-200a level was decreased in necrotic femoral head tissues, which was correlated with Keap1 mRNA upregulation. Together, miR-200a expression activates Nrf2 signaling and protects human osteoblasts from dexamethasone.

OSU53 Rescues Human OB-6 Osteoblastic Cells from Dexamethasone through Activating AMPK Signaling.

Excessive dexamethasone (Dex) application causes osteoblast cell death, which could lead to osteoporosis or osteonecrosis. AMP-activated protein kinase (AMPK) activation is shown to protect osteoblasts/osteoblastic cells from Dex. In this report, we tested the potential effect of OSU53, a novel AMPK activator, in Dex-treated osteoblastic cells. We show that OSU53 activated AMPK signaling in human OB-6 osteoblastic cells. Further, Dex-induced osteoblastic OB-6 cell death and apoptosis were largely attenuated with pre-treatment with OSU53. OSU53 was more efficient than other known AMPK activators (A-769662 and Compound 13) in protecting OB-6 cells against Dex. AMPK activation is required for OSU53-induced actions in OB-6 cells. AMPKα shRNA knockdown or dominant-negative mutation (dn-AMPKα T172A) almost completely blocked OSU53-induced AMPK activation and OB-6 cell protection against Dex. Further studies showed that OSU53 increased NADPH (nicotinamide adenine dinucleotide phosphate) activity and alleviated Dex-induced oxidative stress in OB-6 cells. Such effects by OSU53 were again almost abolished with AMPKα shRNA or dn-AMPKα in OB-6 cells. Together, these results demonstrate that OSU53 protects osteoblastic cells from Dex possibly via activating AMPK-dependent signaling.

Dual mTORC1/2 inhibition by INK-128 results in antitumor activity in preclinical models of osteosarcoma

Existing evidence has shown that mammalian target of rapamycin (mTOR) overactivation is an important contributor of osteosarcoma (OS) progression. Here, we studied the potential anti-OS activity of a potent mTOR kinase inhibitor: INK-128 (MLN0128). We demonstrated that INK-128 induced potent cytotoxic effects against several human OS cell lines (U2OS, MG-63 and SaOs-2), yet same INK-128 treatment was safe (non-cytotoxic) to OB-6 human osteoblastic cells and MLO-Y4 human osteocytic cells. INK-128 induced caspase-dependent apoptosis in OS cells, but not in MLO-Y4/OB-6 cells. The caspase-3 specific inhibitor (z-DVED-fmk) or the pan caspase inhibitor (z-VAD-fmk) dramatically attenuated INK-128-exerted cytotoxicity against OS cells. Molecularly, INK-128 inhibited activation of mTORC1 (S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 phosphorylation), without affecting AKT Thr-308 phosphorylation in U2OS cells. Significantly, AKT inhibition by MK-2206 (an AKT inhibitor), or AKT1/2 stable knockdown by targeted-shRNA, remarkably sensitized INK-128-induced activity in OS cells. In vivo, oral administration of INK-128 potently inhibited U2OS xenograft growth in severe combined immuno-deficient (SCID) mice. mTORC1/2 activation in xenograft tumors was also suppressed with INK-128 administration. In summary, we show that INK-128 exerts potent anti-OS activity in vitro and in vivo. INK-128 might be further investigated as a novel anti-OS agent.

Dexamethasone-induced apoptosis of osteocytic and osteoblastic cells is mediated by TAK1 activation

Increased apoptosis of osteoblasts and osteocytes is the main mechanism of glucocorticoid (GC)-induced osteonecrosis. In the current study, we investigated whether dexamethasone (Dex)-induced osteoblastic and osteocytic cell apoptosis is mediated through activation of transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), and whether TAK1 inhibition could promote survival opposing the deleterious effects of Dex. We found that TAK1 was activated by Dex in both osteocytic MLO-Y4 and osteoblastic OB-6 cells, which was prevented by two known anti-oxidants N-acetylcysteine (NAC) and ebselen. TAK1 inhibitors, including LYTAK1 and 5Z-7-oxozeaenol (57-OZ), inhibited Dex-induced apoptosis of MLO-Y4 and OB-6 cells. Meanwhile shRNA-mediated knockdown of TAK1 also suppressed Dex-induced damages to MLO-Y4 and OB-6 cells. On the other hand, exogenously over-expressing TAK1 enhanced Dex-induced MLO-Y4 and OB-6 cell apoptosis. At the molecular level, we found that TAK1 mediated Dex-induced pro-apoptotic Pyk2-JNK activation. Inhibition or silencing of TAK1 almost abolished Pyk2-JNK phosphorylations by Dex in MLO-Y4 and OB-6 cells. TAK1 over-expression, on the other hand, increased Dex's activity on Pyk2-JNK phosphorylations in above cells. We conclude that part of the pro-apoptotic actions of Dex on osteoblastic and osteocytic cells are mediated through TAK1 activation, and that inhibition of TAK1 might protect from GC-induced damages to osteoblasts and osteocytes.

Prevention of glucocorticoid induced-apoptosis of osteoblasts and osteocytes by protecting against endoplasmic reticulum (ER) stress in vitro and in vivo in female mice

Endoplasmic reticulum (ER) stress is associated with increased reactive oxygen species (ROS), results from accumulation of misfolded/unfolded proteins, and can trigger apoptosis. ER stress is alleviated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which inhibits protein translation allowing the ER to recover, thus promoting cell viability. We investigated whether osteoblastic cell apoptosis induced by glucocorticoids (GCs) is due to induction of ROS/ER stress and whether inhibition of eIF2α dephosphorylation promotes survival opposing the deleterious effects of GC in vitro and in vivo. Apoptosis of osteocytic MLO-Y4 and osteoblastic OB-6 cells induced by dexamethasone was abolished by ROS inhibitors. Like GC, the ER stress inducing agents brefeldin A and tunicamycin induced osteoblastic cell apoptosis. Salubrinal or guanabenz, specific inhibitors of eIF2α dephosphorylation, blocked apoptosis induced by either GC or ER stress inducers. Moreover, GC markedly decreased mineralization in OB-6 cells or primary osteoblasts; and salubrinal or guanabenz increased mineralization and prevented the inhibitory effect of GC. Furthermore, salubrinal (1mg/kg/day) abolished osteoblast and osteocyte apoptosis in cancellous and cortical bone and partially prevented the loss of BMD at all sites and the decreased vertebral cancellous bone formation induced by treatment with prednisolone for 28days (1.4mg/kg/day). We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells is mediated through ER stress, and that inhibition of eIF2α dephosphorylation protects from GC-induced apoptosis of osteoblasts and osteocytes in vitro and in vivo and from the deleterious effects of GC on the skeleton.

Soy protein isolate down‐regulates caveolin‐1 expression to suppress osteoblastic cell senescence pathways

It has been suggested that the beneficial effects of soy protein isolate (SPI) on bone quality are due to either stimulation of estrogenic signaling via isoflavones or through a novel and as yet uncharacterized nonestrogenic pathway. In our study, SPI-fed rat serum inhibited the osteoblastic cell senescence pathway. This effect was accompanied by stimulation of cell differentiation, proliferation, and significant restoration of replicative senescent bone marrow mesenchymal ST2 cells (passaged 30 times). These effects were reproduced in bone from 5-wk-old intact and 10-wk-old ovariectomized female rats fed SPI diets. Caveolin-1 and p53 expression was decreased in bone in SPI-fed, but not in 17β-estradiol (E2)-treated rats. In cell culture studies, membranous caveolin-1 and nuclear p53 expression was greater in replicative senescent ST2 cell cultures than in earlier passaged cells. SPI-fed rat serum significantly down-regulated both caveolin-1 and p53 in senescent and nonsenescent cells. Replicative senescent ST2 cells exhibited a strong association among caveolin-1, p53, and mouse double minute 2 homologue (mdm2), which was inhibited by SPI-fed rat serum. Overexpression of caveolin-1 in ST2 cells resulted in increased expression of p53 and p21, whereas, knockdown of caveolin-1 using shRNA led to increases in mdm2 and eliminated SPI-fed rat serum's effects on p53 and p21 expression. In contrast, manipulation of caveolin-1 expression did not affect the actions of E2 or isoflavones on p53 expression in either ST2 or OB6 cells. These results suggest that caveolin-1 is a mediator of nonestrogenic SPI effects on bone cells.-Zhang, J., Lazarenko, O. P., Blackburn, M. L., Badger, T. M., Ronis, M. J. J., Chen, J.-R. Soy protein isolate down-regulates caveolin-1 expression to suppress osteoblastic cell senescence pathways.

Mechanical properties of human amniotic fluid stem cells using nanoindentation

The aim of this study was to obtain nanomechanical properties of living cells focusing on human amniotic fluid stem (hAFS) cell using nanoindentation techniques. We modified the conventional method of atomic force microscopy (AFM) in aqueous environment for cell imaging and indentation to avoid inherent difficulties. Moreover, we determined the elastic modulus of murine osteoblast (OB6) cells and hAFS cells at the nucleus and cytoskeleton using force–displacement curves and Hertz theory. Since OB6 cell line has been widely used, it was selected to validate and compare the obtained results with the previous research studies. As a result, we were able to capture high resolution images through utilization of the tapping mode without adding protein or using fixation methods. The maximum depth of indentation was kept below 15% of the cell thickness to minimize the effect of substrate hardness. Nanostructural details on the surface of cells were visualized by AFM and fluorescence microscopy. The cytoskeletal fibers presented remarkable increase in elastic modulus as compared with the nucleus. Furthermore, our results showed that the elastic modulus of hAFS cell edge (31.6kPa) was lower than that of OB6 cell edge (42.2kPa). In addition, the elastic modulus of nucleus was 13.9kPa for hAFS cell and 26.9kPa for OB6 cells. Differences in cell elastic modulus possibly resulted from the type and number of actin cytoskeleton organization in these two cell types.

Induction of a program gene expression during osteoblast differentiation with strontium ranelate

Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized ( von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obs at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate.