Relationship between alkaline phosphatase levels,osteopontin expression,and mineralization in differentiating MC3T3-E1 osteoblasts

We are using viral oncogene probes to study the pathways by which osteoblast-specific gene expression is induced in ascorbic acid-treated MC3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directly to key cellular regulators and, as a result, represses tissue specific gene expression and blocks differentiation in a wide variety of cell types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family. The p300 family appears to be the primary target for E1A-mediated repression of tissue-specific gene expression in a variety of cell types. We have generated MC3T3-E1 cell lines that stably express either the wild-type 12S product or a mutant that targets p300/CBP, but not the pRB family. Using these constructs to dissect osteoblast differentiation, we found that targeting of p300/CBP appears to be sufficient to repress alkaline phosphatase expression, although a low but functional level of expression can be maintained if the pRB family is not targeted as well. Induction of alkaline phosphatase expression and activity can be dissociated from expression of late-stage markers such as osteocalcin and osteopontin. Surprisingly, cell lines exhibiting severe repression of alkaline phosphatase activity differentiate to a mineral-secreting phenotype much like normal MC3T3-E1 cells. Osteopontin induction is dependent on at least a minimal level of alkaline phosphatase activity, although it is not dependent on induction of alkaline phosphatase at the RNA level. If alkaline phosphatase is supplied exogenously, osteopontin expression can be induced in conditions in which endogenous alkaline phosphatase is severely repressed. J. Cell. Biochem. 68:269–280, 1998. © 1998 Wiley-Liss, Inc.     

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.     

Inductive effects of prostaglandins on alkaline phosphatase in osteoblastic cells, clone MC3T3-E1

The effects of prostaglandins (PGs) on the induction of alkaline phosphatase (ALP) were investigated in osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. Prostaglandin E2 (PGE2) stimulated ALP activity in the cells in a dose-dependent fashion with a maximal effect which was about twice that in the control cells at concentrations of 100-500 ng/ml. Actinomycin D and cycloheximide inhibited the stimulative effect of PGE2 on ALP activity in the cells. PGE2-induced and native ALPs in the cells were of the same type as that in adult mouse calvaria, being heat-labile, L-homoarginine- and levamisole-sensitive, and L-phenylalanine-insensitive. Isobutyl methylxanthine (IBMX), a cAMP phosphodiesterase inhibitor, stimulated the inductive effect of PGE2 on ALP activity at 0.1 mM, at which concentration IBMX alone had little effect on the activity. PGE2 also increased the intracellular cAMP content in a dose-dependent fashion with a maximal effect at 100 ng/ml. PGE1, PGF1 alpha, and PGF2 alpha (primary PGs like PGE2) increased the activity. Our present results suggest that PGs stimulate the differentiation of osteoblasts and are involved in bone formation in vivo, as well as in bone resorption.     

Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts.

In serum-deprived MC3T3-E1 osteoblasts, mechanical stimulation caused by mild (287 x g) centrifugation induced a 10-fold increase in mRNA levels of the proto-oncogene, c-fos. Induction of c-fos was abolished by the cAMP-dependent protein kinase inhibitor H-89, suggesting that the transient c-fos mRNA increase is mediated by cAMP. Down-regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c-fos induction, suggesting that TPA-sensitive isoforms of PKC are not responsible for c-fos up-regulation. In addition, 287 x g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8-fold (P<0. 005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c-fos expression via a cAMP-mediated mechanism, we asked whether the increase in c-fos mRNA was due to centrifugation-induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c-fos by mechanical stimulation. We conclude that c-fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c-fos is not necessarily dependent on the action of newly synthesized PGE2.     

Boron regulates mineralized tissue-associated proteins in osteoblasts (MC3T3-E1)

The aim of this study was to determine the effects of boron (B) on the cell-survival, proliferation, mineralization and mRNA expression of mineralized tissue-associated proteins. Additionally, determination of the effects of B on the BMP-4, -6 and -7 protein levels of pre-osteoblastic cells (MC3T3-E1) was also intended. The effects of B (pH 7.0) concentrations (0, 0.1, 1, 10, 100, 1000, 2000, 4000, 8000 and 10,000 ng/ml) on the survival of the cells were evaluated at 24 and 96 hrs with MTT assay. To evaluate the proliferation in long term, MC3T3-E1 cells were treated with different concentrations of B (0, 0.1, 1, 10, 100 and 1000 ng/ml) and were counted on days 2, 5, and 14. While in short term, decreased cell survival rate was observed at 1000 ng/ml and above, at long term no statistically significant difference was detected in different B concentrations applied. Slight decreases at the proliferation of the B-treated groups were determined on days 5 and 14 but one-way analysis of variance revealed that the difference was statistically insignificant. In mineralization assay, increased mineralized nodules were apparently observed in B treatment (1 and 10 ng/ml concentrations) groups. Based on quantitative RT-PCR results, remarkable regulation in favor of osteoblastic function for Collagen type I (COL I), Osteopontin (OPN), Bone Sialoprotein (BSP), Osteocalcin (OCN) and RunX2 mRNA expressions were observed in B treatment groups in comparison with untreated control groups. Increased BMP-4, -6 and -7 protein levels were detected at 0.1, 1, 10 and 100 ng/ml B concentrations. Results of the study suggest that at the molecular level B displays important roles on bone metabolism and may find novel usages at the regenerative medicine.     

Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

Mechanical stimulation of bone induces new bone formation invivo and increases the metabolic activity and gene expression ofosteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission ofmechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts causedreorganization of actin filaments into contractile stress fibers andinvolved recruitment of₁-integrins and -actinin tofocal adhesions. Fluid shear also increased expression of two proteinslinked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and theearly response gene product c-fos. Inhibition of actin stress fiberdevelopment by treatment of cells with cytochalasin D, by expression ofa dominant negative form of the small GTPase Rho, or by microinjectioninto cells of a proteolytic fragment of -actinin that inhibits-actinin-mediated anchoring of actin filaments to integrins at theplasma membrane each blocked fluid-shear-induced gene expression inosteoblasts. We conclude that fluid shear-induced mechanical signalingin osteoblasts leads to increased expression of COX-2 and c-Fos througha mechanism that involves reorganization of the actin cytoskeleton.Thus Rho-mediated stress fiber formation and the -actinin-dependentanchorage of stress fibers to integrins in focal adhesions may promotefluid shear-induced metabolic changes in bone cells.

     

Resistance to pyrazofurin and 6-azauridine in normal MC3T3-E1 murine osteoblasts

Stable variants resistant to pyrazofurin (PF) and 6-azauridine (AZUrd) were serially selected in increasing drug concentrations from an MC3T3-E1 nontumorigenic murine osteoblastic cell line. Monophosphates of both AZUrd and PF competitively inhibit orotidine-5'-monophosphate decarboxylase (ODCase) activity of the UMP synthase multifunctional enzyme. When compared to the wild type cells, the AZUrdr and PFr lines were 3000- and 10,000-fold more resistant, respectively. Flow cytometry indicated tetraploidy in wild type cells and a reduction of DNA content in both resistant cell lines. DNA dot blot analysis showed no amplification of the gene coding for UMP synthase in either AZUrdr or PFr cells. Measurements of UMP synthase showed a 6-fold higher activity in AZUrdr cells and no significant difference in PFr cells as compared to wild type. Sensitivity to 5-fluorouracil was increased in the AZUrdr line as opposed to PFr and normal cell lines, indicating an increased orotate phosphoribosyltransferase activity in the AZUrdr cells. In comparison to wild type cells, PFr cells were 100-fold resistant to 6-methylmercaptopurine riboside, suggesting a lack of adenosine kinase activity. The control and AZUrdr cells showed equal sensitivity to 5-fluorouridine, thus indicating unchanged uridine kinase levels. While PFr cells were not cross-resistant to AZUrd, the AZUrdr cells were cross-resistant to PF. These results indicate the possibility of an altered ODCase active site. Although amplification of unrelated sequences cannot be excluded, our findings show that bone tetraploid, nontumorigenic cells acquire drug resistance through mechanisms other than the amplification of a target gene and that this resistance is accompanied by the partial loss of a chromosomal complement.     

Continuous hypergravity alters the cytoplasmic elasticity of MC3T3-E1 osteoblasts via actin filaments

Osteoblasts are sensitive to altered gravity conditions, displaying changes in RNA and protein expression, proliferation, and differentiation; however, the effect of hypergravity on the mechanical properties of osteoblasts remains unclear. In this study, atomic force microscopy (AFM) was used to evaluate the effect of hypergravity on the elasticity of osteoblasts. We demonstrate that a continuous hypergravitational environment increased the elasticity of the cytoplasm, but not the nuclei zone, of MC3T3-E1 osteoblasts. Actin filaments, but not microtubules, dominated in the increased elasticity. These findings provide new insights on cellular gravity-sensing mechanisms.     

Stimulation of differentiation in sodium-dependent vitamin C transporter 2 overexpressing MC3T3-E1 osteoblasts

Sodium-dependent vitamin C transporter (SVCT) 2 facilitates reduced ascorbic acid (AA) transport in MC3T3-E1 osteoblasts. Our previous studies suggested that Zn-induced osteoblast differentiation and Ca2+-, PO4(3-)-stimulated osteopontin (OPN) expression might result from their up-regulation effect on SVCT2 expression and AA uptake. Here, we investigated the role of SVCT2 on osteoblast differentiation by using SVCT2-overexpressing cells. Two clones of SVCT2-introduced cells overexpressed SVCT2 mRNA by 2.8- and 3.1-fold those of control cells, which resulted in obvious increase of AA uptake by 2.1- and 2.4-fold in Vmax with no change in Km. Alkaline phosphatase activity, hydroxyproline content significantly increased in SVCT2-overexpressing cells, and the induction of OPN mRNA was through up-regulation of OPN promoter activity by SVCT2 overexpression. Moreover, SVCT2-overexpressing cells exhibited more ability to promote mineralization and increase calcium deposition under the stimulation of 5 mM beta-glycerophosphate. These findings indicate that SVCT2 stimulates osteoblast differentiation and mineralization.     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

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     

Effect of 1,25-dihydroxyvitamin D3 on alkaline phosphatase activity and collagen synthesis in osteoblastic cells, clone MC3T3-E1

A stimulative effect of 1,25-dihydroxyvitamin D3 was tested on osteoblastic cells, clone MC3T3-E1, cultured in serum-free medium with 0.1% bovine serum albumin. This steroid increased alkaline phosphatase activity in a dose-related fashion. The steroid also stimulated dose-dependently collagen and non-collagen protein syntheses, their maximal effects being observed at 12 and 24 h, respectively. The incorporation of [3H]-proline into collagen or non-collagen protein in cells exposed to this steroid for 12 h was 2.9 or 1.9-fold over that of control cultures, respectively. These results strongly indicate the stimulative effects of 1,25-dihydroxyvitamin D3 on the differentiation of osteoblasts in vitro.     

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.     

Regulation of osteoblast differentiation by Nurr1 in MC3T3-E1 cell line and mouse calvarial osteoblasts

The orphan nuclear receptor Nurr1 is primarily expressed in the central nervous system. It has been shown that Nurr1 is necessary for terminal differentiation of dopaminergic (DA) neurons in ventral midbrain. The receptor, however, is also expressed in other organs including bone, even though the role of Nurr1 is not yet understood. Therefore, we investigated the role of Nurr1 in osteoblast differentiation in MC3T3-E1 cells and calvarial osteoblasts derived from Nurr1 null newborn pups. Our results revealed that reduced Nurr1 expression, using Nurr1 siRNA in MC3T3-E1 cells, affected the expression of osteoblast differentiation marker genes, osteocalcin (OCN) and collagen type I alpha 1 (COL1A1), as measured by quantitative real-time PCR. The activity of alkaline phosphatase (ALP), another osteoblast differentiation marker gene, was also decreased in Nurr1 siRNA-treated MC3T3-E1 cells. In addition, Nurr1 overexpression increased OCN and COL1A1 expression. Furthermore, consistent with these results, during osteoblast differentiation, the expression of osteoblast marker genes was decreased in primary cultured mouse calvarial osteoblasts derived from Nurr1 null mice. Collectively, our results suggest that Nurr1 is important for osteoblast differentiation.     

Murine recombinant leukemia inhibitory factor modulates inhibitory effect of 1,25 dihydroxyvitamin D3 on alkaline phosphatase activity in MC3T3-E1 cells

We demonstrated murine leukemia inhibitory factor (mLIF) mRNA in osteoblastic MC3T3-E1 cells, but not mLIF in their conditioned medium. Recombinant mLIF had an inhibitory effect on alkaline phosphatase (ALP) activity, but not on DNA synthesis, in these mLIF-free cells. This inhibitory effect was not prostaglandin E2 dependent. mLIF also modulated the inhibitory effect of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] on ALP activity, partly via down regulation of 1,25(OH)2D3 binding sites. These results suggest that LIF may play a role in regulating osteoblast differentiation.     

Alignment and proliferation of MC3T3-E1 osteoblasts in microgrooved silicone substrata subjected to cyclic stretching

Previous studies have shown that many types of cells align in microgrooves in static cultures. However, whether cells remain aligned and also proliferate in microgrooves under stretching conditions has not been determined. We grew MC3T3-E1 osteoblasts in deformable silicone dishes containing microgrooves oriented in the stretch direction. We found that with or without 4% stretching, cells aligned in microgrooves of all sizes, with the groove and ridge widths ranged from 1 to 6microm, but the same groove depth of about 1.6microm. In addition, actin cytoskeleton and nuclei became highly aligned in the microgrooves with and without 4% cyclic stretching. To further examine whether MC3T3-E1 osteoblasts proliferate in microgrooves with cyclic stretching, we grew the cells in six-well silicone dishes containing microgrooves in three wells and smooth surfaces in other three wells. After 4% cyclic stretching for 3, 4, and 7 days, we found that cell numbers in the microgrooves were not significantly different (p>0.05) from those on the smooth surface (p>0.05). Taken together, these results show that MC3T3-E1 osteoblasts can align and proliferate in microgrooves with 4% cyclic stretching. We suggest that the silicone microgrooves can be a useful tool to study the phenotype of MC3T3-E1 osteoblasts under controlled substrate strains. The silicone microgrooves can also be useful for delivering defined substrate strains to other adherent cells in cultures.