Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

Soybean isoflavones inhibit tumor necrosis factor-alpha-induced apoptosis and the production of interleukin-6 and prostaglandin E2 in osteoblastic cells

The effects of individual soybean isoflavones, genistein (4',5,7-trihydroxyisoflavone) and daidzein (4',7-dihydroxyisoflavone), on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis and the production of local factors in osteoblastic cells has been investigated. Soybean isoflavones increased DNA synthesis and the number of viable cells. When cells were treated with TNF-alpha, the number of viable cells dose-dependently decreased. The decrease in cell number caused by TNF-alpha treatment was due to apoptosis, which was confirmed by TUNEL and cell death ELISA analyses. Soybean isoflavones inhibited apoptosis of osteoblastic cells subjected to TNF-alpha treatment. MC3T3-E1 osteoblastic cells secrete interleukin-6 (IL-6), interleukin-1beta (IL-1beta), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) constitutively, but at low levels. Soybean isoflavones had no effect on the constitutive production of these local factors. When cells were treated with TNF-alpha (10(-10)M), the production of IL-6 and PGE(2), but not that of IL-1beta and NO, significantly increased. Treatment with soybean isoflavones (10(-5)M), in the presence of TNF-alpha (10(-10)M), for 48 h inhibited production of IL-6 and PGE(2), suggesting the antiresorptive action of soy phytoestrogen may be mediated by decreases in these local factors. The findings of this study thus suggest that soybean isoflavones may promote the function of osteoblastic cells and play an important role in bone remodeling.     

Effects of genistein on expression of bone markers during MC3T3-E1 osteoblastic cell differentiation

In this in vitro study, the hypothesis that the beneficial effects of dietary genistein on bone are through the modulation of the bone marker synthesis by osteoblastic MC3T3-E1 cells was tested, and the possible roles of estrogen receptors in the actions of genistein on osteoblastic cells were also examined. Interleukin-6 production was decreased 40% to 60% in osteoblastic cells treated with genistein from either day 8-16 or day 12-16, at dietarily achievable concentrations (10(-10) to 10(-8) M) (P<0.05). The mRNA expression of osteoprotegerin increased about 140% in cells treated from with genistein day 4-8 at a concentration of 10(-8) M (P<0.05). The ratio of estrogen receptor-alpha to beta expression increased 10-fold from day 0 to 12 of culture (P<0.05). Correlating with this time-dependent variation in estrogen receptor expression, treatments of 17beta-estradiol and genistein had opposite dose patterns on the ratio of estrogen receptor-alpha to beta expression following treatment from day 4 to 6 compared to from day 0 to 2. The addition of ICI-182,780, an estrogen receptor blocker, reduced the inhibitory effect of genistein on IL-6 production by 30-50%. In summary, these findings suggest that the beneficial skeletal effects of genistein, at dietarily achievable levels, appear to be mediated, at least in part, by interleukin-6 and osteoprotegerin, and estrogen receptors play important roles in the inhibition of interleukin-6 synthesis by genistein in osteoblastic MC3T3-E1 cells.     

Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro

Menaquinone-7, which is vitamin K₂ (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10^–7–10^–5 M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10^–6 M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10^–7–10^–5 M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.     

Arginine vasopressin increases the rate of protein synthesis in isolated perfused adult rat heart via the V1 receptor

The effect of daidzein on cortical bone in vitro was investigated. Femoral-diaphyseal tissues obtained from elderly female rats were cultured for 24 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplementation with antibiotics and bovine serum albumin. The experimental cultures contained 10-7 to 10-5 M daidzein. The presence of daidzein (10-6 and 10-5 M) caused a significant increase of alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in bone tissues. This effect was equal to that of genistein (10-6 and 10-5 M). Daidzein (10-5 M) or genistein (10-5 M)-induced increase of calcium content and alkaline phosphatase activity in bone tissues was completely prevented by cycloheximide (10-6 M), an inhibitor of protein synthesis. Anabolic effect of daidzein and genistein on bone components was equal to that of 17-estradiol (10-8 M). The effect of isoflavohoids was not enhanced by the addition of 17-estradiol. The combination of daidzein and genistein did not have an additive effect. These findings indicate that daidzein has an anabolic effect on bone metabolism in tissue culture in vitro, and that this effect is equal to genistein effect. Isoflavonoids may stimulate bone formation and mineralization.     

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.     

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.     

Human purified interleukin-1 inhibits DNA synthesis and cell growth of osteoblastic cell line (MC3T3-E1), but enhances alkaline phosphatase activity in the cells

We examined the effects of human purified interleukin-1 (IL-1) on DNA synthesis, cell growth, and alkaline phosphatase activity in the osteoblastic cell line MC3T3-E1 under both preconfluent and confluent culture conditions. Addition of IL-1 to the cells markedly inhibited their DNA synthesis and growth over the range 1-10 U/ml. Such significant inhibitory effects were observed in cells cultivated in 1 or 5% fetal calf serum (FCS)-containing alpha modification Eagle's medium (alpha-MEM), but not in alpha-MEM containing 10% FCS. In contrast, alkaline phosphatase activity was enhanced significantly by IL-1 in the cell line cultivated in 1% FCS-containing alpha-MEM. These results demonstrate that human purified IL-1 is effective in inducing the differentiation of osteoblastic cell MC3T3-E1.     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.     

Vitamin K2 modulates proliferation and function of osteoblastic cells in vitro.

A human osteosarcoma cell line, HOS TE85 cells, and a mouse osteoblastic cell line, MC3T3-E1 cells, were cultured for 3 days in a medium containing various concentrations of menaquinone-4 (vitamin K2). As a result, the proliferation of HOS cells was suppressed by vitamin K2 in a dose dependent manner up to 56% of control by 10(-7)M of vitamin K2 and that of MC3T3-E1 cells was suppressed to 84% of control by 10(-6)M of vitamin K2. Vitamin K2 increased alkaline phosphatase activity in both kinds of cells. Warfarin counteracted the effect of vitamin K2 on osteoblastic cell proliferation. Our results show that vitamin K2 modulates proliferation and function of osteoblastic cells by some mechanisms including gamma-carboxylation system.     

Effect of transferrin on alkaline phosphatase activity and collagen synthesis in osteoblastic cells derived from newborn mouse calvaria

The effect of transferrin was tested on osteoblastic cells (clone MC3T3-E1) cultured in serum-free medium containing 1% bovine serum albumin (BSA). Transferrin (Tf) stimulated increases of protein content and protein synthesis, but not of DNA content and cell number, in the cells. This protein also increased alkaline phosphatase activity and collagen synthesis in combination with 1% BSA. Actinomycin D and cycloheximide inhibited alkaline phosphatase activity induced by Tf, suggesting that Tf may enhance de novo synthesis of the enzyme. These results indicate that Tf may be involved in differentiation of osteoblastic cells, but not in their proliferation, in vitro.     

The phytoestrogens daidzein and genistein enhance the insulin-stimulated sulfate uptake in articular chondrocytes

Clinical observations have suggested a relationship between osteoarthritis and a changed estrogen metabolism in menopausal women. Phytoestrogens have been shown to ameliorate various menopausal symptoms. Proteoglycans (PG) consisting of low and high sulfated glycosaminoglycans (GAG) are the main components of articular cartilage matrix, and their synthesis is increased by insulin in growth plate cartilage. We have investigated whether GAG synthesis and sodium [35S]sulfate incorporation in female bovine articular chondrocytes are affected by daidzein, genistein, and/or insulin. For comparative purposes, estradiol incubations were performed. Articular chondrocytes were cultured in monolayers at 5% O2 and 5% CO2 in medium containing serum for 7 days followed by the addition of 10(-11) M-10(-4) M daidzein, genistein, 17beta-estradiol, or 5 microg/ml insulin in a serum-free culture phase of 2 days. Photometrically analyzed GAG synthesis was significantly suppressed by high doses (10(-5) M-10(-4) M) of daidzein, genistein, and 17beta-estradiol. Although insulin raised the sodium [35S]sulfate uptake significantly, different concentrations of daidzein, genistein, or 17beta-estradiol showed no significant effects. However, the stimulating effect of insulin on sulfate incorporation was enhanced significantly after preincubation of cells with 10(-11) M-10(-5) M daidzein or 10(-9) M-10(-5) M genistein but not by 17beta-estradiol. In view of the risks of long-term estrogen replacement therapy, further experiments should clarify the potential benefit of phytoestrogens and insulin in articular cartilage metabolism.     

Zinc Upregulates the Expression of Osteoprotegerin in Mouse Osteoblasts MC3T3-E1 Through PKC/MAPK Pathways

Zinc is an essential element for bone formation; however, its role in osteoblast has not been well understood. In the present study, we hypothesized that zinc could increase osteogenetic function by stimulating osteoblast proliferation and osteoprotegerin (OPG) activity. To test this hypothesis, osteoblastic MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 10, 30, 50, 70, 110, 130, and 150 μM) for 24 and 48 h. 3-[4,5-dimethylthiazol-2-y]-2,5-diphenyltetrazolium bromide assay showed that cell proliferation was significantly stimulated with 50 μM zinc treatment. Furthermore, under the same treatment condition, OPG expression was significantly increased as evidenced by the results of RT-PCR and ELISA. However, the zinc-induced OPG expression was significantly attenuated when MC3T3-E1 cells were co-treated with either protein kinase C (PKC) inhibitor, GF109203X, or the Inhibitor of mitogen-activated extracellular signal-regulated kinase 1 (MEK1), PD98059. Moreover, OPG expression was further increased when MC3T3-E1 cells were treated with PMA (the activator of protein of kinase C) in the presence of zinc. These results suggested that zinc would increase osteogenic function by stimulating PKC and MAPK signaling pathways.     

IGF-I and MAP kinase involvement in the stimulatory effects of LNCaP prostate cancer cell conditioned media on cell proliferation and protein synthesis in MC3T3-E1 osteoblastic cells

Bone metastases from prostate cancer cause abnormal new bone formation, however, the factors involved and the pathways leading to the response are incompletely defined. We investigated the mechanisms of osteoblast stimulatory effects of LNCaP prostate carcinoma cell conditioned media (CM). MC3T3-E1 osteoblastic cells were cultured with CM from confluent LNCaP cells. LNCaP CM stimulated MAP kinase, cell proliferation (3H-thymidine incorporation), and protein synthesis (14C-proline incorporation) in the MC3T3-E1 cells. The increases in cell proliferation and protein synthesis were prevented by inhibition of the MAP kinase pathway. IGF-I mimicked the effects of the CM on the MC3T3-E1 cells and inhibition of IGF-I action decreased the LNCaP CM stimulation of 3H-thymidine and 14C-proline incorporation and MAP kinase activity. The findings indicate that IGF-I is an important factor for the stimulatory effects of LNCaP cell CM on cell proliferation and protein synthesis in osteoblastic cells, and that MAP kinase is a component of the signaling pathway for these effects.     

Modulatory effects of luteolin on osteoblastic function and inflammatory mediators in osteoblastic MC3T3-E1 cells

The effects of luteolin on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were investigated. Luteolin (1microM) caused a significant elevation of collagen content, alkaline phosphatase (ALP) activity, and osteocalcin secretion in the cells (P<0.05). The effect of luteolin in increasing collagen content and ALP activity was completely prevented by the presence of 10(-6)M cycloheximide and 10(-6)M tamoxifen, suggesting that luteolin's effect results from a newly synthesized protein component and might be partly involved in estrogen action. We then examined the effect of luteolin on the 3-morpholinosydnonimine (SIN-1)-induced production of oxidative stress markers [nitric oxide (NO) and prostaglan E(2) (PGE(2))] and cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)] in osteoblasts. Luteolin (1 and 10microM) decreased the SIN-1-induced production of NO, PGE(2), TNF-alpha, and IL-6 in osteoblasts. These results suggest that inflammatory mediators can be regulated by luteolin stimulating osteoblastic function.