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.     

Competition between c-fos and 1,25(OH)2 vitamin D3 in the transcriptional control of type I collagen synthesis in MC3T3-E1 osteoblastic cells

Interaction between c-fos and 1,25(OH)₂ vitamin D₃ (VD) on the type I collagen synthesis was studied. VD inhibited collagen synthesis and type I collagen mRNA expression in MC3T3-E1 osteoblastic cells. In contrast, VD reversed the inhibition of collagen synthesis and mRNA expression of the c-fos transfectants that overexpressed c-fos gene to a comparable level as those of the control transfectants. The gel shift assay showed that vitamin D receptor (VDR) complex binding to vitamin D responsive element (VDRE) was inhibited under constitutively expressed c-fos gene, suggesting that c-fos gene product, c-Fos, may inhibit the binding of VDR complex to VDRE by making a c-Fos-VDR complex. The result suggests the existence of a fine tuning between c-fos and VD in the bone metabolism which may be relevant to the pathogenesis of rheumatoid bone lesion. © 1995 Wiley-Liss, Inc.     

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.     

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.     

Synthesis of colony-stimulating factor (CSF) and differentiation-inducing factor (D-factor) by osteoblastic cells, clone MC3T3-E1

The role of osteoblasts in inducing the proliferation and differentiation of bone marrow cells was examined. Conditioned medium obtained from mouse osteoblastic cell (MC3T3-E1) cultures stimulated colony formation of mouse bone marrow cells (CSF) and differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells (D-factor). The CSF activity increased time dependently in parallel with the increase of alkaline phosphatase activity during the culturing of the MC3T3-E1 cells. The activity of the D-factor attained a maximum on days 12 - 15 and decreased thereafter. Both the CSF and the D-factor were eluted in a range of 25,000 to 67,000 daltons on gel filtration. The fraction containing both factors exhibited bone-resorbing activity. These results suggest that osteoblasts are involved in bone resorption at least in part by enhancing the proliferation and differentiation of osteoclast progenitors.     

Bordetella bronchiseptica dermonecrotizing toxin stimulates protein synthesis in an osteoblastic clone, MC3T3-E1 cells

Abstract The effects of Bordetella bronchiseptica dermonecrotizing toxin on protein synthesis in an osteoblastic clone, MC3T3-E1 cells, were investigated. The rate of protein synthesis in the serum-starved cells was increased by the toxin after a latent period of about 4 h, and reached 2.5 times that of the control 24 h after addition of toxin. The toxin raised the level of protein synthesis even in actively proliferating cells. The stimulatory effect of the toxin on protein synthesis occurred earlier than other toxic events so far reported, such as the stimulation of DNA synthesis and the inhibition of osteoblastic differentiation, and was apparently dependent on the toxin concentrations over the range 0.05 ng ml⁻¹ to 6.0 ng ml⁻¹. Therefore, the stimulatory effect of the toxin on protein synthesis could be useful in determining the mode of action of the toxin.     

Prostaglandin E2 stimulates DNA synthesis by a cyclic AMP-independent pathway in osteoblastic clone MC3T3-E1 cells

The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. PGE2 at 2 micrograms/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose-related fashion after a constant lag time, the maximal effect being at 2-10 micrograms/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 microgram/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2',5'-dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevation of cAMP content was rapidly depressed by the stimulated degradation system. Consequently, the stimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.     

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.     

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.     

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.     

Prostaglandin E2 stimulates collagen and non-collagen protein synthesis and prolyl hydroxylase activity in osteoblastic clone MC3T3-E1 cells

We investigated the stimulative effect of prostaglandin E2 (PGE2) on an osteoblastic cell line, clone MC3T3-E1, in serum-free medium. PGE2 elevated collagen and non-collagen protein syntheses in a dose-related fashion up to 2 micrograms/ml, the maximal increases being 2- and 3-fold, respectively, over that in the control. Its stimulative effect was evident as early as 12 h. PGE2 slightly increased DNA content, but its effect was less than that on collagen and non-collagen protein syntheses. Moreover, PGE stimulated an increase in prolyl hydroxylase activity with a maximal effect at 1-2 micrograms/ml, the activity being 15-fold over that of the control. These results strongly indicate that PGE2 directly enhances total protein synthesis including that of collagen in osteoblasts in vitro, suggesting its direct effect on bone formation in vivo as well.     

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.     

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.     

Glabridin protects osteoblastic MC3T3-E1 cells against antimycin A induced cytotoxicity

Mitochondrial dysfunction, particularly respiratory chain disruption, is often responsible for aging-related bone diseases. In this study, the protective effects of glabridin, an isoflavan isolated from licorice root, against pharmacological inhibition of the respiratory chain were studied using osteoblastic MC3T3-E1 cells treated with antimycin A, which inhibits complex III of the electron transport system. Glabridin restored mitochondrial membrane potential dissipation, ATP loss, inactivation of complex IV, intracellular calcium elevation, and cytochrome c release that was induced by antimycin A treatment. This compound also prevented cell death. These results imply that glabridin protects osteoblasts from antimycin A-induced cell death via improved mitochondrial function. Glabridin scavenged ROS and mitochondrial superoxide anions generated by antimycin A. In addition, glabridin prevented antimycin A-induced nitrotyrosine increase and thioredoxin reductase inactivation, suggesting that glabridin may be useful for protecting mitochondria against a burst of oxidative stress. Since phosphoinositide 3-kinase (PI3K) and cAMP-response element-binding protein (CREB) signaling is known to be pro-survival, we determined whether PI3K and CREB activation is associated with the cytoprotective effects of glabridin in the MC3T3-E1 cells. Glabridin restored antimycin A-induced inactivation of PI3K and CREB, suggesting that PI3K and CREB-dependent pathways may be involved in glabridin-induced cytoprotective responses. Our study demonstrates that glabridin reduces mitochondrial dysfunction induced during aging, and could significantly prevent osteoblast damage in osteoporotic patients.