C-fos expression of osteoblast-like MC3T3-E1 cells induced either by cooling or by fluid flow.

Fluid flow is one of the most potent mechanical stimulators for bone cells. Recent reports suggest that primary cilia as well as ion channels and related molecules play roles in the flow detection by kidney epithelial cells. We asked if there is any commonality between kidney and bone cells in flow detection. Primary cilia of cultured osteoblastic cell line MC3T3-E1 were detected by fluorescence staining of acetylated alpha-tubulins. The cells responded to fluid flow, generated by manual rocking of flasks, and expressed c-fos gene. Moreover, the cells were found to respond not only the flow but also cooling of the culture fluid, with a simple technique to keep temperature precisely. It was suggested that bone and kidney cells might share certain similarity in flow detection mechanisms.     

Simvastatin promotes osteoblast differentiation and mineralization in MC3T3-E1 cells

The cholesterol-lowering drug, simvastatin, is a pro-drug of a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor and inhibits cholesterol synthesis in humans and animals. In addition, the bone effects of statins including simvastatin are being studied. We assessed the effects of simvastatin on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. Simvastatin enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the statin was observed at relatively low doses (significant at 10(-8) M and maximal at 10(-7) M). Northern blot analysis showed that the statin (10(-7) M) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. Simvastatin (10(-7) M) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 14 and 22 of culture. These results indicate that simvastatin has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis.     

Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells

Glucocorticoids are widely used as anti-inflammatory and chemotherapeutic agents. However, prolonged use of glucocorticoids leads to osteoporosis. This study was designed to examine the mechanism of dexamethasone (DEX)-induced apoptosis in murine osteoblastic MC3T3-E1 cells. Total RNA was extracted from MC3T3-E1 cells treated with 10(-7) M DEX for 6 h. DEX exerted a variety of effects on apoptotic gene expression in osteoblasts. Ribonuclease protection assays (RPA) revealed that DEX upregulated mRNA levels of caspases-1, -3, -6, -8, -11, -12, and bcl-XL. Western blot analysis showed enhanced processing of these caspases, with the appearance of their activated enzymes 8 h after DEX treatment. In addition, DEX also induced the activation of caspase-9. DEX elevated the levels of cleaved poly(ADP-ribose) polymerase and lamin A, a caspase-3 and a caspase-6 substrate, respectively. Expression of bcl-XL protein level was upregulated by DEX. Cytochrome c release was detected in the cytosol of DEX-treated cells. Furthermore, caspase-3 enzyme activity was elevated by 2-fold after DEX treatment for 7 h. Finally, early apoptotic cells were detected in cells treated with DEX for 3 h. Our results demonstrate that DEX-induced apoptosis involves gene activation of a number of caspases.     

Osteogenic role of endosomal chloride channels in MC3T3-E1 cells

PHR protein family consists of C. elegan Rpm-1/Drosophila Highwire/Zebrafish Esrom/Mouse Phr-1/Human Pam. Esrom is required for correct neurites exiting the paused state at intermediate targets as well as pteridine synthesis. This study reports the identification and characterization of two novel Esrom splice variants, named splice variants 2 (splicing out 5′ 24 bp of exon 17) and 3 (splicing out 5′ 24 bp of exons 17 and 18). Polypeptides encoded by 5′ 24 bp of exons 17 and 18 are part of basic amino-acid-rich region inside Esrom RCC1-like domain (RLD). These two splice variants maintain the whole protein reading frame and alternative exons usage patterns are conserved with mammal. At different developmental stages and adult zebrafish tissues, abundances of these splice variants are different. Importantly, by yeast two-hybrid screen and confocal colocalization analysis, it was found that alternative splicing of exon 18 regulates Esrom RLD interaction with kinesin family member 22 and G protein beta-subunit 1. Taken together, these results suggest that Esrom RLD functions are regulated by alternative splicing at temporal and spatial-specific manner.     

Expression of lysyl oxidase isoforms in MC3T3-E1 osteoblastic cells

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.     

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.     

Chemotactic responses of osteoblastic MC3T3-E1 cells toward zinc chloride

Although zinc (Zn) is known to participate in bone formation, its exact role in the remodeling of this tissue has not been fully clarified. The present study was designed to investigate whether Zn has a role at the resorptive sites in vitro. We investigated the migration of osteoblastic MC3T3-E1 cells in response to Zn using a Boyden chamber assay. Exposure of MC3T3-E1 cells to Zn stimulated the migration of MC3T3-E1 cells. Checkerboard analysis revealed that the migration of MC3T3-E1 cells toward Zn was a directional (chemotaxis) rather than a random (chemokinesis) motion. Pretreatment of MC3T3-E1 cells with pertussis toxin completely blocked the chemotactic response of cells to Zn, indicating that it is mediated by G-protein-coupled receptors. Because the bone is one of the major Zn storage sites, we suggest that Zn released from bone-resorptive sites plays an important role in the recruitment of osteoblasts and bone renewal.     

Apocynin stimulates osteoblast differentiation and inhibits bone-resorbing mediators in MC3T3-E1 cells

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. In the present study, the effect of apocynin on the function of osteoblastic MC3T3-E1 cells was studied. Apocynin caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content, and mineralization in the cells (P < 0.05). Antimycin A (AMA), which inhibits complex III of the electron transport system, has been used as a reactive oxygen species (ROS) generator in biological systems. We exposed cultured osteoblastic MC3T3-E1 cells to AMA with or without pretreatment with apocynin. Apocynin significantly (P < 0.05) increased cell survival, calcium deposition, and osteoprotegerin release and decreased the production of ROS and osteoclast differentiation inducing factors such as TNF-α, IL-6, and receptor activator of nuclear factor-kB ligand (RANKL) in the presence of AMA. These results demonstrate that apocynin can protect osteoblasts from mitochondrial dysfunction-induced toxicity and may have positive effects on skeletal structure.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells

Abstract

Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca²⁺ chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca²⁺ release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction.     

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.     

The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts

Fluid flow due to loading in bone is a potent mechanical signal that may play an important role in bone adaptation to its mechanical environment. Previous in vitro studies of osteoblastic cells revealed that the upregulation of cyclooxygenase-2 (COX-2) and c-fos induced by steady fluid flow depends on a change in actin polymerization dynamics and the formation of actin stress fibers. Exposing cells to dynamic oscillatory fluid flow, the temporal flow pattern that results from normal physical activity, is also known to result in increased COX-2 expression and PGE₂ release. The purpose of this study was to determine whether dynamic fluid flow results in changes in actin dynamics similar to steady flow and to determine whether alterations in actin dynamics are required for PGE₂ release. We found that exposure to oscillatory fluid flow did not result in the development of F-actin stress fibers in MC3T3-E1 osteoblastic cells and that inhibition of actin polymerization with cytochalasin D did not inhibit intracellular calcium mobilization or PGE₂ release. In fact, PGE₂ release was increased threefold in the polymerization inhibited cells and this PGE₂ release was dependent on calcium release from the endoplasmic reticulum. This was in contrast to the PGE₂ release that occurs in normal cells, which is independent of calcium flux from endoplasmic reticulum stores. We suggest that this increased PGE₂ release involves a different molecular mechanism perhaps involving increased deformation due to the compromised cytoskeleton. mechanotransduction; cell mechanics     

Cadmium stimulates prostaglandin E2 synthesis in osteoblast-like cells, MC3T3-E1

The effect of Cd on prostaglandin E2 production in osteoblasts was studied using cloned osteoblast-like cells, MC3T3-E1, which were established from new-born mouse calvaria. Treatment of the cells with Cd caused a dose- (0-10 microM) and time- (0-24 h) dependent increase in the release of prostaglandin E2 from the cells into the culture medium. A lag time of 4 h was required for the onset of the phenomenon. The release of [14C]arachidonic acid from prelabeled cell membrane was little influenced by the Cd treatment, while conversion of [14C]arachidonic acid to prostaglandin E2 by the homogenate of the cells treated with Cd was enhanced as compared to that by untreated cells. The stimulatory effect of Cd on prostaglandin E2 production was abolished in the presence of cycloheximide (100 ng/ml). By Western blot analysis with polyclonal rabbit anti-cyclooxygenase antibody, it was revealed that Cd treatment augmented the amount of immunoreactive cyclooxygenase in the cells. These results strongly suggest that Cd stimulates prostaglandin E2 production through the induction of cyclooxygenase in MC3T3-E1 cells. This effect of Cd may be involved in the mechanisms of Cd-induced bone injury.     

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.     

Nephronectin expression is regulated by SMAD signaling in osteoblast-like MC3T3-E1 cells

Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin α8β1. We previously demonstrated that Npnt expression was suppressed by TGF-β through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-β type I receptor (TGF-β R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-β signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts.