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INTRODUCTION
The role of estrogens and estrogen-like molecules, including isoflavones, in regulating the bone cell activities is essential for understanding the etiology and treatment of post-menopausal osteoporosis. Isoflavonoids, which are found abundant in soybeans, are a group of phytochemicals that show affinity for the estrogen receptor11,22). Isoflavonoids have been shown to be effective in preventing bone loss in ovariectomized rats and postmenopausal women17). Moreover, genistein was found to inhibit the bone loss caused by an estrogen deficiency in mice, and an analogue of coumestrol has been reported to increase the activity of ALP (an osteoblastic phenotype marker) in osteoblast-like cells9). These results suggest that isoflavones prevent bone loss and promote bone formation. In addition, it has been reported that isoflavones participate in the apoptosis and differentiations of several cell types6,8,24). Unlike estrogen, the consumption of isoflavones has been suggested to reduce the risk of breast and prostate cancer12).
Phytoestrogens have the potential to maintain bone health and delay or prevent osteoporosis, and several studies have examined the dose-dependent effects on bone and their modes of action4,14,23). Some reports concluded that the mechanism of action underlying the prevention of bone loss by isoflavones appears to differ from that of estrogen and the selective estrogen receptor modulators5,15). Whilst bothestrogen and raloxifene prevent bone loss by reducing bone resorption, the isoflavones are antiresorptive only at chronic high doses. Therefore, they have been reported to either inhibit or not affect the bone turnover rate. Moreover, neither estrogen nor raloxifene blocks the elevated bone resorption rates induced by an ovariectomy or menopause5). On the other hand, certain isoflavones, including genistein, have been shown to modestly stimulate the osteoblast characteristics, such as, the synthesis of total proteins and mineralized matrix deposition in immature and mature osteoblast cell cultures26). Hence, while isoflavone treatment may prevent bone loss, in part, by modulating the balance between bone formation and resorption, the mechanism underlying the enhanced bone formation after an isoflavone treatment in vivo is unknown.
C2C12 cells were derived from mouse myoblasts, and have the potential to differentiate into osteoblasts. Therefore, they are composed of committed osteoblastic precursor cells3). Many studies have examined the conversion of C2C12 cells into an osteoblast lineage when mediated by BMP2,10,27). It was hypothesized that C2C12 cells might provide useful information about the effects of isoflavones and BMP-4 on osteoblast differentiation. This study examined the in vitro effects of daidzein, genistein, or equol and/or BMP using C2C12 cells to determine the bioactivity of isoflavones in the presence or absence of BMP-4 on the bone metabolism.
MATERIALS AND METHODS
1. Cell culture and chemicals
The low passage mouse pluripotential mesenchymal precursor line, C2C12, was purchased from American Type Culture Collection (Manassas, VA). BMP-4 was obtained from R & D Systems (Minneapolis, MN) and was prepared as a stock solution (1 mg/ml) and stored at 4℃. The isoflavones (daidzein, genistein and Equol) was purchased from PKC Biochemicals (Woburn, MA) and was reconstituted in dimethyl sulfoxide (DMSO) at a 1000× stock concentration of 5 and 50 mM, respectively. The solutions were stored at -20℃. The cells were maintained in Dulbecco's modified Eagle medium (DMEM, Irvine Scientific, Santa Ana, CA) containing 20% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA), supplemented with 50 units/ml penicillin and 50 µg/ml streptomycin (Mediatech Mediatech, Herndon, VA).
2. ALP activity
For the differentiation assays, the C2C12 cells were plated at an initial density of ca. 5×104 cells/cm2. BMF-4 at 1, 3, or 10 ng/ml (final concentration) was added alone or in combination with 5 or 50 µM (final concentration) of either daidzein, genistein, or equol in 2% DMEM medium. The medium was renewed after 3 days.
The substrate used for the ALP assays was purchased from Sigma Chemical (St. Louis, MO). The ALP activity was visualized as follows. The medium was aspirated from the C2C12 cells cultured in 12 well plates, and the cells were then rinsed with phosphate-buffered saline (PBS), formaldehyde fixed for 10 min, and incubated in 300 µl of a Western blue stabilized substrate for ALP. The reaction was quenched after 30 min by removing the reaction buffer and washing once with PBS. The ALP activity was determined by comparing the colors of the scanned images.
The ALP activity was quantified using a previously described protocol. The medium was aspirated from the osteoblasts cultured in 12 well plates. The cells were rinsed with PBS and sonicated for 3×10 secs in cold lysis buffer (50 mM Tris (pH 7.5), 0.1% Triton X-100). The ALP activity in the cleared supernatant was assayed using reverse transcriptase (RT) in a reaction buffer containing 0.1 M 2 amino-2 methyl-1 propanol and 2 mM MgCl2 at pH 10.5, for 30 min using p-nitrophenyl phosphate (Sigma Chemical, St. Louis, MO). The reactions were quenched by adding 0.1 M NaOH, and the activities were determined by measuring the light absorbance at 410 nm and comparing results with standard p-nitrophenol solutions and the appropriate blank. The results are expressed as micromoles of p-nitrophenol produced per min per mg of protein. The ALP level was normalized to the protein concentration using Bicinchoninic Acid (BCA) Protein Assay Reagent (Pierce Chemical, Rockford, IL) that was determined spectrophotometrically at 562 nm and compared with the standard protein solutions.
3. Analysis of gene expression using the Illumina mouse MAC array
The total RNA was isolated from a 10 cm template after 96 hours of culture with daidzein. RNAeasy kits (QIAGEN, Valencia, CA) were used, and the RNA concentrations were quantified by measuring the absorbance at 260 nm. The labeled probes were prepared by reverse transcription of 5 µg of RNA in the presence of dATP (PerkinElmer, Wellesley, MA). The probes were hybridized to Illumina arrays (Sentrix mouse Ref-8 Expression Bead Chips, Illumina, Inc., San Diego, CA) overnight. After thorough washing according to the manufacturer's protocol, the raw data was obtained. The scans were performed using an Applied Biosystems 1700 chemiluminescent Microarray Analyzer version 1.1.0. The differences in themicroarray intensities were normalized and grouped using Avadis Prophetic 3.3 version (Strand Genomics Pvt. Ltd., USA) by Quartile normalization, the 2-fold change method, and K-means cluster (Euclidean method/Complete linkage). The data was then entered into a database and analyzed graphically.
RESULTS
1. ALP staining
1) C2C12 osteoblast differentiation induced by daidzein and BMP-4
Daidzein dose-dependently induced C2C12 osteoblast differentiation in the presence of various BMP-4 concentrations. The C2C12 cell ALP activity was significantly higher after treatment with 50 µM daidzein than after 5 µM daidzein, as evidenced by the significantly higher level of ALP staining. In contrast to the cells treated with daidzein plus BMP-4, the cells treated with daidzein alone showed little color development (Fig. 1).
2) C2C12 osteoblast differentiation induced by genistein and BMP-4
Treatment with genistein plus BMP-4 exhibited mild ALP activity at 5 µM genistein but there was significant induction at 50 µM. C2C12 cells treated with genistein alone showed only slight color changes.
3) Effect of equol on C2C12 differentiation
The addition of 5 µM to 50 µM equol dose-dependently enhanced the ALP staining intensity compared with BMP-4 alone. As was observed with the other two isoflavones, equol alone was ineffective in inducing C2C12 differentiation, as evidenced by the weak blue staining.
4) Quantitative changes in ALP activity
In order to quantify the ALP activity induced by isoflavones, the C2C12 myoblasts were treated separately with the isoflavones or with BMP-4. As shown in Fig. 2 (A-C), 50 uM of daidzein, equol or genistein synergistically induced ALP activity at BMP-4 concentrations of 1-10 ng/ml. In particular, daidzein appeared to induce ALP activity efficiently at the lower BMP-4 concentrations (i.e., 1 to 3 ng/ml)(Fig. 2).
2. Analysis of gene expression using Illumina mouse MAC arrays
This array technique was used to examine the RNA samples from the C2C12 cells treated with daidzein. The genes associated with the ECM glycoproteins, e.g., Leprel1, Nid1, Efemp1 and Ltbp3, were down-regulated, whereas Mfap3 and Fbln1 were up-regulated. The genes associated with the ECM linker proteins, e.g., Lamc1, Lama2, Lama5 were down-regulated. The genes associated with the ECM structural proteins, e.g., Col5a1, Col4a2, Col1a2 and Col8a1 were also down-regulated (Fig. 3). Among the other ECM associated genes, e.g., Mmp13 and Mmp3 were up-regulated but Mmp9 and Pcolce2 were down-regulated.
DISCUSSION
Osteogenin and the related BMPs are members of the TGF-β supergene family that are involved in a number of physiological events including embryonic development and skeletal tissue maintenance and repair20). In vitro, BMPs induce the differentiation of various types of cells to the osteoblastic lineage. These cells include undifferentiated mesenchymal cells, bone marrow stromal cells, and preosteoblasts. Moreover, BMP-2, 4, 6, 7, and 9 have been reported to significantly induce ALP activity in preosteoblastic C2C12 cells10).
An association between the ectoenzyme ALP and matrix vesicles during osteoblast differentiation and mineralization is required for ester phosphate hydrolysis at the sites of mineralization in order to provide ionic phosphate for calcium-phosphate formation1). Previous studies, using the ALP inhibitor levamisole, have reported the importance of ALP in the commitment of bone preosteoblasts to mineralization13). Indeed, it has been suggested that intracellular ALP participates in the regulation of cellular differentiation19). In the present study, ALP was used as a marker of osteoblastic differentiation of C2C12 cells. The results showed that the ALP activity increased appreciably by a treatment with the isoflavones plus BMP-4 whereas the isoflavones administered alone had no significant effect on osteoblastic differentiation. This suggests that isoflavones play a role in bone metabolism through growth factors such as BMP-4. Isoflavones are known weak agonist for estrogen receptors, while estrogen acts as an antiresorptive agent by inhibiting osteoclasts rather than by acting as a strong promoter of osteoblast production. However, the presence of isoflavones becomes meaningful when and where there are growth factors like BMPs. The action of isoflavones on bone development is expected to be more apparent in settings where the production of endogenous estrogens and/or BMPs are decreasing, such as in ovariectomized mice and/or heterozygotes of estrogen/BMPs.
Considerable attention has been given to the expression of specific gene products in osteoblasts16,25) but the comprehensive profiles of the changes in intracellular gene expression during differentiation to the osteoblast phenotype were not accessible before the introduction of gene array technology. Previous studies have shown that cells can be induced to differentiate to osteoblasts under a variety of conditions. In this study, gene array technology was used to chart the expressional changes in hundreds of gene during the osteoblastic differentiation of C2C12 mouse myoblasts. Gene expression was analyzed using a mouse gene array panel, which included a large number of genes encoding the structural ECM proteins. The results showed that the expression of many genes was altered in a seemingly unstructured manner. However, the up-regulation of Mmp13 and the down-regulation of Col4a2, Col5a1 and Mmp9 might bemeaningful in terms of osteoblastic differentiation. Along with MMP-14, MMP-9 (92-kDa gelatinase; gelatinase B) and MMP-13 (collagenase-3) have been reported to be the most abundant proteinases that regulate cellular migration, changes in the extracellular matrix and apoptosis in growth plate cartilage17). On the other hand, Mmp2 knock-out (-/-) mice have been reported to exhibit opposing bone phenotypes due to an impaired osteocytic canalicular networkand decreased bone mineral density in the limb and trunk bones but increased bone volume in the calvariae7). Type IV collagens including Col4a1 are the major components of the basement membrane in blood vessels, and are expressed ubiquitously in the basement membranes during the early developmental stages. Type V collagen (Col5), in addition to types I and XXIV collagens, is known to be a component of mineralized bone and corneal, whereas types II, XI, and XXVII collagen are the components of cartilage21). Although these MMPs and collagens are widely perceived to be essential structural and functional components during bone and cartilage development, the contribution of individual gene expression is unclear. Therefore, studies using knock-out mice with single or multiple genes deleted will be needed. Overall, these findings suggest that daidzein may affect osteoblastic differentiation in part by regulating the expression of the ECM associated proteins at the transcription level. However, further study will be needed to determine the mechanism whereby isoflavones affects osteoblast differentiation.
CONCLUSION
In summary, results of this study provide evidence that isoflavones weakly induce the expression of the osteoblastic marker, ALP, in C2C12 cells (a pre-osteoblastic cell line), particularly in the presence of BMP-4. This suggests that isoflavones may act with growth factors such as BMP-4 during the bone metabolism. Mouse illumina expression chip arrays showed that the expression of a subset of ECM associated genes are modulated by daidzein but the significance of these changes in the osteogenic process has yet to be determined. Overall these results suggest that isoflavones cause expressional changes in the ECM associated proteins that are known to play key roles in the osteogenic process, at the gene level. Comparative animal studies on bone development, bone density, and extracellular bone mineralization in mice fed daidzein with/without BMP should provide a deeper understanding of the effects of isoflavones on bone homeostasis.
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