Stimulative effects of (22E,24R)-ergosta-7,22-diene-3beta,5alpha,6beta-triol from fruiting bodies of Tricholoma auratum,on a mouse osteoblastic cell line,MC3T3-E1

We screened the differentiation-inducing activities of 39 mushroom extracts from Akita prefecture, Japan, on the mouse osteoblastic cell line, MC3T3-E1. Sixteen phosphate buffered saline (PBS), 8 boiled PBS, 14 ethanol and 12 methanol extracts induced alkaline phosphatase (ALP) activities, an indicator of MC3T3-E1 cell differentiation. The enzyme activities were markedly induced by extracts of Tricholoma auratum, and we isolated the active compound from methanol extracts of this mushroom. Physical data for the isolated active compound were identical to those for (22E,24R)-ergosta-7,22-diene-3beta,5alpha,6beta-triol (1). 1 induced ALP activities of MC3T3-E1 cells and promoted cell proliferation. To investigate the relationships between the chemical structure and differentiation-inducing activity of the compound, ALP-inducing activities of MC3T3-E1 cells by 1, ergosterol (2), ergocalciferol (3), cholesta-3beta3,5alpha6beta-triol (4), 7-dehydrocholesterol (5) and cholecalciferol (6) were tested. The enzyme activities of MC3T3-E1 cells were increased 3.0-fold by 10 microM 1 and 2.4-fold by 10 microM 4. However, 2, 3, 5 and 6 did not induce MC3T3-E1 cell ALP activity at 0.1-10 microM. These results suggested that the OH groups at C-5 and/or C-6 of 1 and 4 played an important role in their differentiation-inducing activities on MC3T3-E1 cells. Furthermore, 1 suppressed induction of MC3T3-E1 cell apoptosis by serum starvation.     

xy9902对MC3T3-E1细胞的增殖和分化作用

目的研究化合物xy9902对成骨细胞MC3T3-E1的增殖和分化的影响,并初步探讨其机制。方法采用MTT比色法测定化合物对成骨细胞MC3T3-E1的增殖作用;通过硝基苯磷酸盐法测定细胞内碱性磷酸酶(A lkaline phosphatase,ALP)活性的变化,观察化合物对细胞分化的影响;用放射性配体结合法考察化合物与雌激素受体(Estrogen ic recep-tor,ER)的亲和力。结果化合物xy9902对成骨细胞有促增殖和促分化作用,这一作用可被tamoxifen阻断;xy9902与ER有亲和力,对ERα和ERβ的IC50分别为8.45×10-6mol.L-1和1.66×10-6mol.L-1。结论化合物xy9902对成骨细胞有促增殖和促分化作用,其作用机制可能与ER激动有关。     

Chemical constituents of Acanthus ilicifolius L. and effect on osteoblastic MC3T3E1 cells

A new coumaric acid derivative called acancifoliuside (1) and six known compounds as acteoside (2), isoacteoside (3), acanthaminoside (4), (+)-lyoniresinol 3a-O-beta-glucopyranoside (5), (-)-lyoniresinol (6), and alpha-amyrin (7), were isolated from the methanolic extract of the leaves of Acanthus ilicifolius L. (Acanthaceae). Their structures were determined by spectroscopic methods and a comparison with the spectral data reported in the literature. The effects of the compounds on the function of osteoblastic MC3T3-E1 cells were tested. Compounds 2, 3, and 5 (30 microM) increased the growth and differentiation of osteoblasts significantly (P<0.05), indicating that A. ilicifolius leaves may help prevent osteoporosis.     

染料木素促成骨样细胞增殖作用及其机制研究

目的观察染料木素(genistein)对成骨样细胞MC3T3-E1细胞增殖及骨形成蛋白-2(BMP-2)、β-连环素(β-catenin)表达的影响。方法培养成骨样细胞MC3T3-E1,加入不同浓度的染料木素(1×10-10、1×10-9、1×10-8、1×10-7、1×10-6和1×10-5mol·L-1),以1×10-9mol·L-1雌激素(β-estradiol,E2)作为阳性对照组,采用MTT比色试验法和碱性磷酸酶(ALP)活性检测法,观察染料木素作用后MC3T3-E1的增殖及分化情况,以Westernblot方法检测成骨样细胞中BMP-2和β-catenin蛋白的表达。结果染料木素可促进MC3T3-E1细胞增殖,染料木素作用后细胞的ALP值也明显升高,呈浓度依赖性;染料木素可增加BMP-2和β-catenin表达,且呈浓度依赖性。结论染料木素促进MC3T3-E1细胞增殖与分化,可通过调节BMP-2和Wnt/β-catenin信号通路而影响成骨样细胞的活性。     

地黄活性成分梓醇对小鼠成骨细胞MC3T3-E1增殖、分化和矿化的影响

目的检测地黄活性成分梓醇对成骨细胞株MC3T3-E1细胞增殖、分化和矿化的影响。方法制备不同浓度地黄活性成分梓醇提取液。以小鼠成骨细胞株MC3T3-E1作为药物筛选的细胞模型;用MTT法测定不同浓度的梓醇溶液的促细胞增殖作用;采用ALP活性和骨钙素定量检测分别观察不同浓度的梓醇溶液的促细胞分化作用;以Vonkos-sa钙化染色法了解不同浓度的梓醇溶液的促细胞钙化作用。结果梓醇在1×10-7～1×10-9mol·L-1浓度范围内培养24及48h促进成骨细胞株MC3T3-E1细胞增殖。梓醇在浓度1×10-5～1×10-6mol·L-148及72h提高成骨细胞株MC3T3-E1细胞内碱性磷酸酶的活性。梓醇在浓度1×10-5～1×10-6mol·L-1培养8及12d时能明显促进成骨细胞MC3T3-E1骨钙素合成和分泌。梓醇在浓度1×10-5～1×10-6mol·L-1培养19d时成骨细胞株MC3T3-E1细胞的矿化结节(mineralized bone nodular structure,MBNS)数目增多。结论梓醇可以提高成骨细胞株MC3T3-E1增殖和分化能力,梓醇可能是地黄治疗骨质疏松作用的活性成分之一。     

A new norlupane triterpene from the leaves of <Emphasis Type="Italic">Acanthopanax koreanum</Emphasis> increases the differentiation of osteoblastic MC3T3-e1 cells

A new norlupane-triterpene, 3α,11α-dihydroxy-20,23-dioxo-30-norlupane-28-oic acid (1), and four known compounds; 3α,11α-dihydroxy-23-oxo-lup-20(29)-en-28-oic acid (2), 3α,11α-dihydroxylup-20(29)-en-28-oic acid (3), ent-kaur-16-en-19-oic acid (4), and betulabuside B (5) were isolated from the methanolic extract of the leaves of Acanthopanax koreanum. Their chemical structures were elucidated by mass, 1D- and 2D-NMR spectroscopy. To investigate the bioactivities of these compounds, their effects on the differentiation of osteoblastic MC3T3-E1 cells were tested. Compounds 3 and 4 significantly increased osteoblastic cell growth and differentiation as assessed by the MTT assay and collagen content, respectively (p < 0.05). Compounds 1 and 5 significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of osteoblastic cell differentiation as assessed by the alkaline phosphatase activity. These results suggest that the compounds isolated from A.koreanum leaves have a direct stimulatory effect on bone formation in vitro and may contribute to the prevention for osteoporosis.     

New anthracene glycosides from <Emphasis Type="Italic">Rhodomyrtus tomentosa</Emphasis> stimulate osteoblastic differentiation of MC3T3-E1 cells

Two new anthracene glycosides (1, 2) were isolated from aerial parts of Rhodomyrtus tomentosa, along with three known compounds (3–5). The structures of two new compounds were established to be 4,8,9,10-tetrahydroxy-2,3,7-trimethoxyanthracene-6-O-β-D-glucopyranoside (1) and 2,4,7,8,9,10-hexahydroxy-3-methoxyanthracene-6-O-α-L-rhamnopyranoside (2) based on spectroscopic and chemical methods. Among them, compound 1, 2, and 5 significantly (P<0.05) increased the alkaline phosphatase activity, collagen synthesis, and mineralization of the nodules of MC3T3-E1 osteoblastic cells compared to those of the control, respectively.     

Protective effect of tetrahydroxystilbene glucoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells

Oxidative stress can induce apoptosis and decrease activities of osteoblasts. 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from a Chinese herb Polygonum multiflorum Thunb. To evaluate the protective effect provided by TSG to osteoblastic MC3T3-E1 cells, the cells were pretreated with TSG for 24h before being treated with 0.3mM hydrogen peroxide (H(2)O(2)) for 24 h, then some markers of osteoblast function and oxidative damage of the cells were examined. Our data demonstrated that TSG significantly (P< 0.05) increased cell survival, alkaline phosphatase (ALP) activity, calcium deposition, and the mRNA expression of ALP, collagen I (COL-I) and osteocalcin (OCN) in the presence of H(2)O(2). In addition, TSG decreased the production of receptor activator of nuclear factor-κB ligand (RANKL), interleukin-6 (IL-6), intracellular reactive oxygen species and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells induced by H(2)O(2). Taken together, these results demonstrated that the protective effect provided by TSG to osteoblastic MC3T3-E1 cells was mediated, at least in part, via inhibition of the release of bone-resorbing mediators and oxidative damage of the cells. Our results indicated that TSG may be effective in providing protection against osteoporosis associated with oxidative stress.     

Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells.     

Myricetin, a naturally occurring flavonoid, prevents 2-deoxy-D-ribose induced dysfunction and oxidative damage in osteoblastic MC3T3-E1 cells

Myricetin, a naturally occurring flavonoid, was investigated to determine whether it could protect osteoblasts from 2-deoxy-d-ribose induced dysfunction and oxidative damage in the MC3T3-E1 cells. MC3T3-E1 cells were incubated with 2-deoxy-d-ribose and/or myricetin, and markers of osteoblast function and oxidative damage were examined. Compared with control incubation, 2-deoxy-d-ribose significantly (P<0.05) inhibited alkaline phosphatase (ALP) activity, collagen content, and calcium deposition at the concentration of 20 mM. Cellular malondialdehyde (MDA), protein carbonyl, and advanced oxidation protein products contents were significantly (P<0.05) increased in the presence of 2-deoxy-d-ribose (20 mM). Myricetin significantly (P<0.05) increased cell survival, ALP activity, collagen, osteocalcin, osteoprotegerin, and calcium deposition and decreased MDA, protein carbonyl, and advanced oxidation protein products contents of osteoblastic MC3T3-E1 cells in the presence of 20 mM 2-deoxy-d-ribose. These results demonstrate that myricetin attenuates 2-deoxy-d-ribose induced damage, suggesting that myricetin may be a useful dietary supplement for minimizing oxidative injury in diabetes related bone diseases.     

Expression of core binding factor alpha1 up-regulated by IGF-I, GM-CSF, and EGF through MAPK pathway in MC3T3-E1 and C2C12 cells

AIM: To study the regulating function and mechanism of insulin-like growth factor-I (IGF-I), granulocyte-macrophage colony-stimulating factor (GM-CSF), and epidermal growth factor (EGF) on murine core binding factor alpha1 (Cbfalpha1) gene expression. METHODS: Luciferase reporter gene method and RT-PCR technique were used to examine the effects of these growth factors on the promoter activity and mRNA expression of Cbfalpha1 gene in MC3T3-E1 and C2C12 cells. RESULTS: IGF-I (from 1 nmol/L to 1 micromol/L), GM-CSF (100 nmol/L), and EGF (1 micromol/L) increased the luciferase expression in MC3T3-E1 cells (P<0.05). And mitogen-activated protein kinase (MAPK) inhibitor, PD 98059 (10 micromol/L), completely blocked IGF-1, GM-CSF, and EGF-induced expression of Cbfa1 promoter activity (P<0.01). In C2C12 cells, IGF-I (from 1 nmol/L to 10 micromol/L), GM-CSF (100 nmol/L and 1 micromol/L), and EGF (100 nmol/L) enhanced the expression of luciferase reporter plasmid driven by mCbfalpha1 promoter (P<0.05). Addition of PD 98059 also blocked the stimulatory effects of these growth factors on Cbfalpha1 promoter activity (P<0.01). Moreover, Cbfalpha1 mRNA expression was significantly increased after treatment with IGF-I (1 nmol/L, 100 nmol/L), GM-CSF (100 nmol/L, 1 micromol/L), and EGF (1 micromol/L, 100 nmol/L) in MC3T3-E1 and C2C12 cells, respectively (P<0.05). These stimulatory effects of IGF-I, GM-CSF, and EGF on Cbfalpha1 mRNA expression were abolished by PD 98059. CONCLUSION: IGF-I, GM-CSF, and EGF could increase the promoter activity and the mRNA expression of murine Cbfalpha1 gene in MC3T3-E1 and C2C12 cells. These stimulatory effects might be mediated by activating the intracellular MAPK-dependent signaling pathway.     

Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways.     

Overexpression of QM induces cell differentiation and mineralization in MC3T3-E1

It has been reported that QM was highly expressed by cells isolated from epiphyseal cartilage as opposed to proliferative chondrocytes. In vitro investigation of the expression of QM revealed higher QM expression in nonmineralizing osteoblast and pericyte cultures as compared with mineralizing cultures. These evidences suggest that QM may play an essential role in cell differentiation before mineralization. However, our research results showed that QM overexpression in MC3T3-E1 enhanced cell differentiation and mineralization. In this study, alkaline phosphatase (ALP) activity and nodule mineralization were increased in MC3T3-E1 from QM overexpression cultures relative to normal expression QM cultures. RT-PCR revealed upregulation of the marker genes type I collagen, ALP, osteocalcin, osterix and BMP-2 and a slight decrease of a negative regulator osteopontin. These results suggest that the increasing of QM expression could stimulate osteoblast differentiation and mineralization in MC3T3-E1.     

镁锌合金对成骨细胞增殖的影响

目的研究镁锌合金对前成骨细胞MC3T3-E1细胞增殖的影响,探讨镁锌合金成为一种新型骨科内植物材料的可行性。方法在镁锌合金金属片上接种前成骨细胞MC3T3-E1,采用MTT法及碱性磷酸酶（ALP）检测试剂盒等方法检测MC3T3-E1细胞在材料表面的增殖活性,用左旋聚乳酸对照组。结果培养第1,3天后,镁锌合金表面成骨细胞增殖与对照组没有差异（P〉0.05）,第5、7、9后,镁锌合金表面成骨细胞增殖明显高于对照组（P〈0.01）。结论镁锌合金能够促进MC3T3-E1细胞的增殖,可以初步认为具有良好的生物相容性。     

Apigenin increases osteoblastic differentiation and inhibits tumor necrosis factor-alpha-induced production of interleukin-6 and nitric oxide in osteoblastic MC3T3-E1 cells

Many plant-derived substances have estrogenic activities. Due to their ability to bind the estrogen receptor, these compounds have the potential to counteract the deleterious effects of estrogen deficiency on bone. In this study, the effects of apigenin on the function of osteoblastic MC3T3-E1 cells and the production of local factors in osteoblasts were investigated. Apigenin (0.01 microM) significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of alkaline phosphatase (ALP) activity and collagen content in the cells (P < 0.05). The effect of apigenin in increasing ALP activity and collagen content was completely prevented by the presence of 10(-6) M cycloheximide and 10-6 M tamoxifen, suggesting that apigenin's effect results from a newly synthesized protein component and might be partly involved in estrogen action. Locally derived mediators in bone play a crucial role in the regulation of bone remodeling, i.e., bone formation and bone resorption processes. The effect of apigenin on the TNF-alpha-induced production of IL-6 and nitric oxide (NO) in osteoblasts was examined. Treatment with apigenin (10 microM) decreased the TNF-alpha-induced production of IL-6 and NO in osteoblasts. Taken together, these results suggest that apigenin may represent new pharmacological tools for the treatment of osteoporosis.     

罗格列酮对体外培养小鼠前成骨细胞系MC3T3-E1生长与分化的影响研究

目的:研究罗格列酮对体外培养小鼠前成骨细胞系MC3T3-E1生长与分化的影响。方法:以小鼠MC3T3-E1作为药物筛选的细胞模型,分别加入1、2、5、10μmo·lL-1罗格列酮干预,MTT法测定细胞活性并计算生长抑制率;流式细胞术检测细胞凋亡率;免疫组化法分析细胞中促凋亡因子Bax和抗凋亡因子Bcl-2蛋白的表达;测定细胞中分化指标碱性磷酸酶(ALP)的活性。设立只加入培养基处理的空白对照组。结果:与空白对照组比较,罗格列酮各浓度组生长抑制率、细胞凋亡率升高(P均<0.05),Bax表达增强、Bcl-2表达减弱、ALP活性下降(P均<0.05),且呈浓度依赖性。结论:罗格列酮可抑制MC3T3-E1细胞生长及分化,并诱导其凋亡;而Bax/Bcl-2可能参与凋亡的发生,并可能起关键调控作用。     

Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

Aim:

To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.

Methods:

MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.

Results:

Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation.

Conclusion:

Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.     

A comparative study of histamine activities on differentiation of osteoblasts and osteoclasts

The effects of histamine and its receptor antagonists on mouse bone marrow cells (MBMC) and MC3T3-E1 cells were studied to elucidate the precise molecular mechanisms underlying histamine activities in the respective cell types. The studied parameters were osteoclast differentiation and expressions of receptor activator of nuclear factor kappaB ligand (RANKL), histamine receptors (HR), and osteoblast differentiation markers. The osteoclastogenesis was assessed by TRAP-dye method. Expressions of RANKL, HR and the osteoblast differentiation markers were evaluated by RT-PCR analysis. In MBMC, 1 microM histamine doubled the number of osteoclast-like cells in a dose-dependent manner. Expressions of RANKL peaked at histamine concentrations of 1 microM and 0.1 microM in MBMC and MC3T3-E1, respectively. H(1)R antagonist, but not H(2)R antagonist, inhibited RANKL expressions induced by histamine in MC3T3-E1. Histamine induced expressions of cell differentiation markers in MC3T3-E1, but not in MBMC, under the conditions that RANKL expressions were induced by histamine in both types of cells. These results indicate the following: (1) Histamine induction of osteoclastogenesis is mediated by RANKL expressed via H(1)R, but not via H(2)R in mouse osteoblast-like cells; (2) and the major target of histamine action is the RANKL-RANK signaling pathway in osteocytes. This observation is consistent with the traditionally recognized histamine action of bone resorption at the osteoclast site.     

Stimulatory effect of Daidzein in osteoblastic MC3T3-E1 cells

Daidzein is a natural isoflavone found in Leguminosae. The effect of daidzein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured in a serum-free medium for 48 hr in the presence of daidzein (10(-7)-10(-5) M). Daidzein (10(-6) and 10(-5) M) caused a significant elevation of protein content, alkaline phosphatase activity, and DNA content in cells; those increases were about 1.4-, 1.5-, and 2.0-fold, respectively. The ability of daidzein (10(-5) M) to increase protein content, alkaline phosphatase activity, and DNA content in cells was prevented completely by the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, indicating that the isoflavone has a stimulatory effect on cellular protein synthesis. The anabolic effect of daidzein (10(-6) or 10(-5) M) in cells was not distinguishable from that of genistein (10(-6) or 10(-5) M). Meanwhile, cell protein showed an additive effect of 17beta-estradiol and daidzein, but their effects on alkaline phosphatase were not additive. Moreover, the effect of daidzein (10(-5) M) in elevating cellular protein content and alkaline phosphatase activity was inhibited completely by the presence of tamoxifen (10(-6) M), suggesting that the effect of the isoflavone is mediated partly through estrogen action. This study demonstrates that daidzein has an anabolic effect in osteoblastic MC3T3-E1 cells.