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Keywords:

  • osteoprogenitor cells;
  • osteoblast;
  • adipocyte;
  • soy phytoestrogens;
  • genistein

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The soy phytoestrogen daidzein has biphasic dose responses, but the underlying mechanisms are not yet clear. Transcriptional and biochemical data show that PPARs, in addition to ERs, are molecular targets of daidzein, which divergently regulates osteogenesis and adipogenesis. Dose responses are the result of a balance among PPARs and between ERs and PPARs.

Introduction: Soy phytoestrogens have been used for the purposes of treatment and prevention of osteoporosis. Biphasic dose responses of daidzein, one of the main soy phytoestrogens, have long been recognized, but the underlying molecular mechanisms of action are not yet clear.

Materials and Methods: Mouse bone marrow cells and mouse osteoprogenitor KS483 cells that concurrently differentiate into osteoblasts and adipocytes were cultured. Biochemical measurement of alkaline phosphatase (ALP) activity, RT-PCR, and gene reporter assays were used in this study.

Results: Daidzein, one of the major soy phytoestrogens, had biphasic effects on osteogenesis and adipogenesis. Daidzein stimulated osteogenesis (ALP activity and nodule formation) and decreased adipogenesis (the number of adipocytes) at concentrations below 20 μM, whereas it inhibited osteogenesis and stimulated adipogenesis at concentrations higher than 30 μM. When estrogen receptors (ERs) were blocked by ICI182,780, daidzein-induced effects were not biphasic. A decrease in osteogenesis and an increase in adipogenesis were observed at the concentrations higher than 20 and 10 μM, respectively. In addition to ERs, daidzein transactivated not only peroxisome proliferator-activate receptor γ (PPARγ), but also PPARα and PPARδ at micromolar concentrations. Activation of PPARα had no direct effects on osteogenesis and adipogenesis. In contrast, activation of PPARδ stimulated osteogenesis but had no effects on adipogenesis, whereas PPARγ inhibited osteogenesis and stimulated adipogenesis. Transfection experiments show that an activation of PPARα or PPARγ by daidzein downregulated its estrogenic transcriptional activity, whereas activation of PPARδ upregulated its estrogenic transcriptional activity. Activation of ERα or ERβ by daidzein downregulated PPARγ transcriptional activity but had no influence on PPARα or PPARδ transcriptional activity.

Conclusions: Daidzein at micromolar concentrations concurrently activates different amounts of ERs and PPARs, and the balance of the divergent actions of ERs and PPARs determines daidzein-induced osteogenesis and adipogenesis.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Genistein (5,7,4′-trihydroxyisoflavone) and daidzein (7,4′-trihydroxyisoflavone) are two main phytoestrogens found in soy products, and they have attracted much attention among public and scientific communities.(1–3) Substantial evidence shows that these soy phytoestrogens have protective effects against a variety of disorders including osteoporosis, cardiovascular disease, cancer, hyperlipidemia, diabetes, menopausal symptoms, and various forms of chronic renal disease.(4–7) They are considered to be used as an alternative for estrogen replacement therapy because both genistein and daidzein bind to estrogen receptor α and β (ERα and ERβ, respectively) and have estrogenic effects.(2,8,9) Furthermore, these substances exert antiestrogenic effects in a variety of cell cultures and animal models.(4,10,11) Some studies have shown that their underlying molecular mechanisms are caused by an activation of ERs.(11–13) In contrast to these similarities, genistein and daidzein also have differential effects. For example, genistein is a potent inhibitor for protein tyrosine kinase, DNA typoisomerases I and II, and ribosomal S6 kinase, whereas daidzein is not.(14–16) It has been shown that, although both inhibited growth of human bladder cancer cells in a similar dose-dependent way, genistein inhibited cdc2 kinase activity and therefore induced G2-M phase cell cycle arrest, whereas daidzein only induced apoptosis without altering cell cycle distribution.(17) These similarities and differences indicate that their underlying molecular mechanisms are not yet clear. Moreover, soy phytoestrogens have been used as food supplements for the purposes of treatment and prevention of osteoporosis.(18–20) It is therefore necessary to find and understand their underlying molecular mechanisms with regard to bone remodeling.

Using mouse osteoprogenitor KS483 cells, which concurrently differentiate into osteoblasts and adipocytes,(21) we recently showed that peroxisome proliferator-activate receptor (PPARγ), a ligand-activated transcription factor essential for regulating adipocyte differentiation, is a new molecular target of genistein.(22) Because genistein-stimulated PPARγ activity may result from its enzyme inhibiting effects, it would be very interesting to see whether daidzein, an inactive form of genistein, could activate PPARγ. Moreover, PPARs include three isoforms: α, γ, and δ.(23–25) Some ligands of PPARγ can also activate other PPAR isoforms,(26–28) and these different isoforms have differential effects on adipogenesis. It has been shown, for example, that activation of PPARγ prompt adipogenesis,(27,29,30) whereas activation of PPARδ did not influence adipogenesis.(31) It is necessary to test whether soy phytoestrogens can activate the other PPAR isoforms and whether these other isoforms differentially contribute to osteogenesis and adipogenesis of osteoprogenitor cells induced by soy phytoestrogens. Furthermore, it would be very interesting to know whether different isoforms of PPARs influence estrogenic transcriptional activity in a similar way and whether ERs exert similar effects on different isoforms of PPARs.

To study molecular mechanisms of soy phytoestrogens, we used daidzein as a model compound and tested whether it activates PPARγ, PPARα, and PPARδ and whether this activation influences osteogenesis and adipogenesis of KS483 cells. Here we show that daidzein-induced osteogenesis and adipogenesis are determined by the balance between ERs and PPARs.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Cell cultures and assays

The methods of cell culture and assays have been described before. (21,22) In brief, KS483 cells were cultured in phenol red free α-MEM supplemented with 10% FBS (Gibco BRL Life Technologies, Breda, The Netherlands) and penicillin/streptomycin. For the experiments, these cells were seeded in 12-well plates at a density of 15,000 cells/cm2 using 10% charcoal-stripped FBS as supplement. Osteogenic culture medium were maintained by adding ascorbic acid (50 μg/ml) and β-glycerophosphate (10 mM) after 4 or 11 days of seeding, respectively. Cells were continuously exposed to daidzein 1 day after plating until the end of the experiment at day 18. Alkaline phosphatase (ALP) activity was kinetically determined using p-nitrophenylphosphate as a substrate at pH 10.5 and reading the optical density at 405 nm. DNA was measured by the method of an enhancement of fluorescence using Hoechst 33258 (Sigma, St Louis, MO, USA) binding to DNA. Adipocytes were stained with Oil-red-O. Numbers of nodules and adipocytes were objectively counted under a light microscope. Expression of mRNA of PPARs was done by RT-PCR, and the primers used were described in the literature.(21,32)

Transient gene expression assays in KS483 cells

Transfection and luciferase reporter assays were carried out as described(22) with some modifications. Briefly, KS83 cells were seeded into 24-well plates. After 24 h, they were transfected using a lipid-based FuGENE 6 transfection reagent according to manufacturer (Roche, Basel, Switzerland). For each triplicate of samples, 100 ng luciferase reporter and 100 ng Renilla plasmid were applied together with the expression vectors coding for ERα, ERβ, PPARα, PPARγ, and PPARδ or equal amounts of DNA in each transfection. The transfection medium was changed after 16 h into the different treatment medium as indicated. After 48 h, cells were washed twice with PBS, lysed in passive lysis buffer and treated according to manufacturer's instructions (Promega, Leiden, The Netherlands). Luciferase activity was measured and expressed as fold induction ± SE, which was corrected for transfection efficiency by Renilla values.

Statistics

Data are presented as means ± SE. Differences between groups were accepted at p < 0.05, which were assessed by one-way ANOVA or related test using software Instat (GraphPad, San Diego, CA, USA).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Osteogenesis and adipogenesis

Osteogenesis

KS483 cells, cultured in osteogenic medium supplemented with charcoal-stripped serum, can concurrently differentiate into both osteoblasts and adipocytes and form mineralized nodules.(21,22) We used this cell culture system to study the effects of daidzein on osteogenesis and adipogenesis. Daidzein had a clear biphasic effect on osteogenesis of KS483 cells. It dose-dependently stimulated ALP activity (Fig. 1A) and nodule formation (Fig. 1B) at the concentrations between 1 and 20 μM, with the maximum stimulation at 20 μM. In contrast, it dose-dependently decreased ALP activity and nodule formation at concentrations higher than 40 μM. Similar biphasic dose responses were observed in mouse bone marrow cell cultures, with the stimulation of ALP activity at concentrations between 1 and 10 μM and the inhibition of ALP activity at concentrations of 20 μM or higher. These data show that daidzein affects osteogenesis not only in KS483 cells but also in bone marrow cells in a biphasic way, which is similar to that of estrogen as well as genistein.(21,22) To study whether daidzein-regulated osteogenesis is ER-mediated, we used the specific antiestrogenic compound ICI182,780, which has been shown to block ER-mediated effects in KS483 cells.(21) ICI182,780 alone had no effects on both ALP activity and nodule formation. However, it decreased daidzein-stimulated effects on ALP activity and nodule formation to control levels at daidzein concentrations between 1 and 10 μM. Interestingly, it decreased daidzein-stimulated osteogenesis to the levels lower than controls at daidzein concentration of 20 μM. It further decreased daidzein-inhibited osteogenesis at daidzein concentrations higher than 30 μM. KS483 cells exposed to daidzein at concentrations higher than 75 μM, alone or in combination with ICI182,780, could not form nodules. In summary, our results strongly suggest that daidzein-regulated effects on osteogenesis were partly ER-mediated.

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Figure FIG. 1. Osteogenesis and adipogenesis. Osteoprogenitor KS483 cells were cultured in 12-well plates in medium containing 10% charcoal-stripped serum and continuously exposed to different concentrations of daidzein alone or in combination with antiestrogenic compound ICI182,780 (0.5 μM) for 18 days. (A) Cellular ALP activity, (B) number of nodules, and (C) number of adipocytes were quantified. Each value is the mean ± SE of the results from three different wells and is representative of results from at least five different experiments. Significant differences (*p < 0.05) between control and treatment are indicated.

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Adipogenesis

In contrast to osteogenesis, daidzein decreased the number of adipocytes at concentrations between 1 and 20 μM, whereas it dose-dependently increased the number of adipocytes at the concentrations of 30 μM or higher (Fig. 1C). Moreover, these biphasic dose responses were also observed in mouse bone marrow cell cultures with a decreased number of adipocytes at the concentrations between 1 and 10 μM and an increased number of adipocytes at the concentration of 20 μM. These data further show that daidzein, similar to genistein(22) but different from estrogen,(21) influences adipogenesis of KS483 cells as well as bone marrow cells in a biphasic way. Interestingly, when ER-mediated effects were completely blocked by ICI182,780, the biphasic pattern of daidzein-regulated adipogenesis was changed. As shown in Fig. 1C, ICI182,780 alone had no effects on adipogenesis but can block the inhibitory effects induced by daidzein at the concentration of 1 μM. Moreover, ICI182,780 increased the number of adipocytes inhibited by daidzein at concentrations of 10 and 20 μM to the levels higher than controls. In addition, ICI182,780 could further potentiate daidzein-increased number of adipocytes when daidzein concentrations were higher than 30 μM. We have shown previously that adipogenesis of KS483 cells under control conditions was associated with PPARγ activity.(21) Our results here suggest that daidzein-induced estrogenic effects counteracted daidzein-induced PPARγ effects.

Daidzein activates PPARs

Expression of PPARs in KS483 cells

All isoforms of PPARs were detected in KS483 cells. As shown in Fig. 2A, mRNA expression of PPARα, PPARγ1, and PPARδ was not changed during 21 days of cell culture (Fig. 2A). In contrast, mRNA expression of PPARγ2 increased in the first 14 days of cell culture and then decreased thereafter.(21)

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Figure FIG. 2. PPARs in KS483 cells. (A) Constant mRNA expression of PPARα, PPARγ1, and PPARδ in KS483 cells was detected by RT-PCR during 21 days of cell culture. Specific ligands of PPARs influence osteogenesis (B and D, ALP activity) and adipogenesis (C and E, the number of adipocytes). These cells were cultured in 12-well plates and exposed for 18 days to Wy14643 (B and C) or carbaprostacyclin (D and E). Each value is the mean ± SE of the results from three different wells and is representative of results from three different experiments. (C) Significant differences (*p < 0.05) are indicated compared with control.

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We used specific ligands of PPARs to investigate the function of PPARs in osteogenesis and adipogenesis of KS483 cells. Compared with controls, PPARα agonist Wy14643 at concentrations of 1 and 10 μM did not change ALP activity or the number of adipocytes. However, it decreased ALP activity and increased the number of adipocytes at a concentration of 100 μM (Figs. 2B and 2C), indicating an activation of PPARγ and the effects are similar to those of ciglitazone, a specific PPARγ agonist.(22) Compared with controls, PPARδ agonist carbaprostacyclin increased ALP activity at the concentrations of 0.1 and 1 μM, but had no influence on the number of adipocytes. However, this stimulation of osteogenesis was decreased, and the number of adipocytes was increased at the concentration of 10 μM (Figs. 2D and 2E), indicating PPARγ activity; this was confirmed by our transcriptional results (data not shown). We also tested carbaprostacyclin at concentrations higher than 10 μM and observed an more pronounced increase in adipogenesis but a decrease in osteogenesis of KS483 cells (data not shown). Clearly, these results showed divergent effects of PPARs on osteogenesis and adipogenesis of KS483 cells: PPARα influenced neither osteogenesis nor adipogenesis; PPARγ downregulated osteogenesis and upregulated adipogenesis(22); PPARδ upregulated osteogenesis but had no influence on adipogenesis. At high concentrations, agonists of PPARs activated their different isoforms, which contribute to their action on osteogenesis and adipogenesis of KS483 cells.

Daidzein activates PPARs

Our results strongly suggest that daidzein activated PPARγ. We transiently transfected KS483 cells with a consensus PPRE luciferase reporter construct and exposed these cells to daidzein. Daidzein dose-dependently increased PPRE-luc activity at concentrations between 5 and 50 μM, with a maximum fold induction of 1.7 ± 0.05 at 50 μM. When these transfected KS483 cells were exposed to PPAR agonists, Wy14643 (10 μM), ciglitazone (30 μM), or carbaprostacyclin (1 μM), the fold induction of PPRE-luc was 1.7 ± 0.13, 1.66 ± 0.08, and 1.8 ± 0.11, respectively. Moreover, we transiently transfected KS483 cells with a consensus PPRE luciferase reporter construct together with vectors encoding PPARγ2, PPARα, or PPARδ and exposed these cells to daidzein. As shown in Fig. 3, daidzein at concentrations higher than 1 μM transactivated all isoforms of PPARs. Transcriptional activity of PPARα was activated to the highest level at daidzein concentrations of 10 and 20 μM and decreased thereafter (Fig. 3A). PPARγ activity, however, was increased in a dose-dependent way (Fig. 3B). Similar to PPARα, daidzein increased PPARδ activity dose-dependently and reached the highest level at the concentrations between 20 and 40 μM and then decreased (Fig. 3C). An activation of PPARs was confirmed with several reporter constructs including ACO-luc and DRE-luc,(33) indicating that an activation of PPARs by daidzein is independent of constructs used. Furthermore, we tested, using luciferase assays, the daidzein-induced PPAR activity in several breast cancer cell lines including MCF7, T47D, and MDA-MD-231, and the results are consistent with the data in RAW 264.7 cells.(34) Our results show that daidzein transcriptionally activates PPARs.

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Figure FIG. 3. Daidzein activates PPARs. KS483 cells were seeded into 24-well plates that were transiently transfected with consensus PPRE-luc reporter plasmid, a constitutive Renilla reporter, and vectors encoding PPARα, PPARγ2, or PPARδ. These cells were exposed to various concentrations of daidzein for 48 h. Our results showed that daidzein at micromolar concentrations transactivated (A) PPARα, (B) PPARγ, and (C) PPARδ. Each value is the mean ± SE of the results from three different wells and is representative of results from three different experiments. Significant differences (*p < 0.05) are indicated.

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Estrogenic and antiestrogenic effects

To further show daidzein-induced estrogenic activity in KS483 cells, we transiently transfected KS483 cells with a consensus ERE-luc reporter construct and exposed these cells to daidzein. As shown in Fig. 4, daidzein increased ERE-luc activity at micromolar concentrations, with maximal stimulation at a concentration of 10 μM (Fig. 4A). The estrogenic transcriptional activity of daidzein at 100 μM was comparable with that of E2 (10−8 M). Combination of daidzein (10, 20, or 100 μM) and E2 had similar estrogenic transcriptional activity as daidzein alone at the same concentrations (data not shown). Moreover, combination of daidzein (10, 20, or 100 μM) and E2 had also similar effects on ALP activity as daidzein alone at the same concentrations. For example, daidzein (100 μM), alone or in combination with E2 (10−8 M), decreased ALP activity to a level lower than controls, indicating an antiestrogenic effects (Fig. 4B). We have shown that E2 could inhibit adipogenesis in KS483 cells.(21) However, E2 could not block adipogenesis induced by daidzein at the concentration of 100 μM (Fig. 4C). These data strongly suggest that daidzein-activated estrogenic effects were less potent than those of daidzein-induced PPARγ effects. In other words, PPARγ may play a dominant role at daidzein concentration of 100 μM.

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Figure FIG. 4. Estrogenic and antiestrogenic effects. KS483 cells were seeded into 24-well plates that were transiently transfected with ERE-luc reporter plasmid and a constitutive Renilla reporter and exposed to various concentrations of daidzein for 48 h. (A) Daidzein increased ERE-luc activity. (B) When these cells were cultured in 12-well plates and exposed to daidzein (100 μM) alone or in combination with E2 (10−8 M) for 18 days, daidzein had antiestrogenic effects on cellular ALP activity. (C) Moreover, the number of adipocytes induced by daidzein (100 μM) was not blocked by E2. Each value is the mean ± SE of the results from three different wells and is representative of results from three different experiments. Significant differences (*p < 0.05) are indicated. C, control; E, E2 (10−8 M); D, daidzein (100 μM).

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ERs and PPARs

Daidzein at micromolar concentrations concurrently activated ERs and PPARs. Activated ERs and PPARs may influence each other at the transcriptional level. To study this, we transiently transfected vectors expressing PPARα, PPARγ2, or PPARδ along with an ERE-luc construct and exposed KS483 cells to daidzein. As shown in Fig. 5, PPARγ, as well as PPARα, decreased daidzein-induced ERE-luc reporter activity. In contrast, transient transfection of PPARδ potentiated daidzein-induced ERE-luc activity. These results suggest that PPARs had differential effects on estrogenic transcriptional activity. Both PPARα and PPARγ contribute to the antiestrogenic effects of daidzein.

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Figure FIG. 5. Effects of PPARα, PPARγ, or PPARδ on estrogenic transcriptional activity. KS483 cells were seeded into 24-well plates and transiently transfected with an equal amount of DNA containing an ERE-luc reporter plasmid, a constitutive Renilla reporter, and expression plasmids encoding PPARα, PPARγ2, or PPARδ. These cells were exposed to various concentrations of daidzein for 48 h, and ERE-luc activity was measured. Cotransfection of PPARα or PPARγ downregulates daidzein-induced estrogenic transcriptional activity, whereas cotransfection of PPARδ upregulates daidzein-induced estrogenic transcriptional activity. Each value is the mean ± SE of the results from three different wells and is representative of results from at least three different experiments. Significant differences (*p < 0.05) are indicated.

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We then tested whether an activation of ERs by daidzein can alter the transcriptional activity of PPARs. We transiently transfected KS483 cells with a consensus PPRE luciferase reporter construct together with vectors encoding PPARα, PPARδ, or PPARγ2 and vectors expressing ERα or ERβ. As shown in Fig. 6, cotransfection of ERα or ERβ decreased PPARγ transcriptional activity (Fig. 6B) but had no influence on PPARα (Fig. 6A) or PPARδ transcriptional activity (Fig. 6C). In summary, our results indicate that an activation of ERs by daidzein had differential effects on the transcriptional activities of PPARs.

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Figure FIG. 6. Effects of ERα or ERβ on PPAR-mediated transcriptional activity. KS483 cells were seeded into 24-well plates and transiently transfected with equal amounts of DNA containing a PPRE-luc reporter plasmid, a constitutive Renilla reporter, and an expression vector encoding (A) PPARα, (B) PPARγ2, or (C) PPARδ with or without an ERα or ERβ expression plasmid. These cells were exposed to various concentrations of daidzein for 48 h, and PPRE-luc activity was measured. Each value is the mean ± SE of the results from three different wells and is representative of results from at least three different experiments. Significant differences (*p < 0.05) are indicated.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Emerging evidence shows that soy phytoestrogens play a beneficial role in the prevention and treatment of chronic diseases such as cardiovascular diseases, osteoporosis, cancer, obesity, and diabetes.(6,13,35) However, their molecular targets are poorly understood. ERs have been proposed as their molecular targets, but these beneficial health effects cannot be solely explained by ER-mediated pathway.(16) Our results indicate that, PPARs, in addition to ERs, are the critical molecular targets of phytoestrogens.

Similar to E2, daidzein transcriptionally activates the ER-mediated pathway, resulting in a stimulation of osteogenesis and an inhibition of adipogenesis. Our results that daidzein at concentrations below 20 μM increased osteogenesis and inhibited adipogenesis indicate a dominant role of its estrogenic activity. Although the effects of daidzein above 30 μM on osteogenesis and adipogenesis were opposite to those of E2, these antiestrogenic effects of daidzein did not rule out its estrogenic activity. This estrogenic activity was indeed confirmed by our transcriptional data. Furthermore, by using the specific antiestrogenic compound ICI182,780, we demonstrated that the estrogenic activity of daidzein at all tested concentrations plays a role in stimulation of osteogenesis and inhibition of adipogenesis.

One of the most important findings of this study is that daidzein transcriptionally activated not only PPARγ, but also PPARα and PPARδ, which are the critical targets of many chronic diseases. These findings indicate that activation of PPARs by soy phytoestrogens is independent of their enzyme-inhibiting ability. The divergent effects of different isoforms of PPARs contribute to daidzein-induced osteogenesis and adipogenesis of KS483 cells.

Using specific ligands of PPARs, we showed that activation of different isoforms of PPARs has divergent effects on osteogenesis and adipogenesis of KS483 cells. For example, Wy14643, a ligand of PPARα, transcriptionally activated PPARα to a similar level at the concentrations between 1 and 100 μM. It did not activate PPARγ at the concentrations of 1 and 10 μM. At these two concentrations, it has no influence on both osteogenesis and adipogenesis, which is consistent with the observations in PPARα-null mice.(36) Similarly, activation of PPARδ by carbaprostacyclin at concentrations of 0.1 and 1 μM increased osteogenesis and had no influence on adipogenesis. Our results of PPARδ on adipogenesis are in line with the observations in mice as well as in 3T3-L1 adipocyte differentiation.(31) However, the results of these specific ligands of PPARs at high concentrations indicate that the balance among different isoforms of activated PPARs determined osteogenesis and adipogenesis of KS483 cells. Indeed, we observed that Wy14643 at 100 μM and carbaprostacyclin at 10 μM transactivated all isoforms of PPARs (data not shown), which is consistent with the observations in other cell lines.(26) We and others have shown that activation of PPARγ downregulated osteogenesis and upregulated adipogenesis.(22,32,37) Our data on decreased osteogenesis and increased adipogenesis influenced by Wy14643 and carbaprostacyclin at these high concentrations reflect PPARγ activity.

Our results showed that an activation of PPARs by daidzein influenced both osteogenesis and adipogenesis. Several lines of evidence support this conclusion. First, we observed daidzein increased PPARγ transcriptional activity in a dose-related way. In parallel, daidzein at concentrations higher than 30 μM decreased osteogenesis and increased adipogenesis in a dose-dependent way. An upregulation of adipogenesis by daidzein at concentrations higher than 30 μM indicates that PPARγ plays a dominant role in the determination of the fate of these osteoprogenitor cells. These results are in line with the observations in mesenchymal progenitor UAMS-33 cells.(38) Second, daidzein at the concentrations of 10 and 20 μM increased osteogenesis and inhibited adipogenesis, showing a dominant role of its estrogenic effects. However, the transcriptional data and ICI182,780 experiments indicated that PPARγ was functional at these concentrations. Third, daidzein increased PPARδ transcriptional activity. As an activation of PPARδ increased osteogenesis, daidzein-activated PPARδ may increase osteogenesis. The finding that ICI182,780 decreased daidzein-stimulated osteogenesis to a control level suggests that the stimulatory effects of PPARδ counteract the inhibitory effects of PPARγ on osteogenesis induced by daidzein at the concentration of 10 μM. Moreover, daidzein had the highest estrogenic activity at the concentration of 10 μM in KS483 cells. However, the maximum stimulatory effects of daidzein on osteogenesis were observed at the concentration of 20 μM, which was the concentration of the highest PPARδ transcriptional activity. Clearly, our results suggest that an activation of PPARδ by daidzein contribute to its positive effects on osteogenesis.

In addition to direct effects of ERs and PPARs on osteogenesis and adipogenesis, we showed that ERs and PPARs influenced each other at the transcriptional level in a differential way. The cross-talk between ERs and PPARs may further contribute to the effects of daidzein on osteogenesis and adipogenesis. For example, PPARα did not have direct effects on osteogenesis and adipogenesis. However, it downregulated daidzein-induced estrogenic transcriptional activity. As a result, an activation of PPARα contributed to the antiestrogenic effects of daidzein. Similarly, PPARγ downregulated daidzein-induced estrogenic transcriptional activity. Our results showed that both PPARα and PPARγ contribute to the antiestrogenic effects of daidzein. Because some phytoestrogens such as genistein have enzyme-inhibiting activities, the mechanisms of antiestrogenic action have been explained by an inhibition of enzyme activity. Here we used daidzein, an inactive form of genistein, to show that this is not the case. Different from PPARα or PPARγ, we showed that PPARδ upregulated daidzein-induced estrogenic transcriptional activity. Both estrogenic and PPARδ transcriptional activities decreased at high concentrations of daidzein. A decrease in osteogenesis and an increase in adipogenesis at high concentrations of daidzein suggest that the effects of this cross-talk between ERs and PPARδ on osteogenesis and adipogenesis are limited. Clearly, our data showed that there are cross-talks between ERs and PPARs, which may contribute to daidzein-induced osteogenesis and adipogenesis. The cross-talks between ERs and PPARs have been reported recently in uterine leiomyoma and MCF-7 breast cancer cells.(39,40) PPARα-RXR heterodimers could block ER binding to the ERE of the promoter and inhibit ER-mediated transcriptional activity.(39) Inhibition of PPAR transactivation by ER could be mediated through competition for common coactivators or increased corepressors. However, the underlying molecular mechanism remains unclear.

We demonstrated that daidzein concurrently activated transcriptional factors, ERs and PPARs and these transcriptional factors influenced each other. As a result, the balance of divergent actions of these transcriptional factors determine the biological effects of daidzein on osteogenesis and adipogenesis. As demonstrated by ICI182,780, this balance was indeed observed at all tested concentrations. Based on our biochemical and transcriptional data, we conclude that the amount of activated ERs or PPARs was determined by the dose of daidzein. As a result, the biological effects of daidzein were concentration-dependent. It is worth noting that the balance between ERs and PPARs may not be the only molecular mechanism responsible for daidzein action. It has been shown, for example, that soy phytoestrogens enhance bone formation by increasing the transcription of the BMP-2 gene in mouse mesenchymal stem cells(41)or by enhancing synthesis of BMP-2 in primary osteoblastic cells isolated from newborn Wistar rats.(42) Other possible molecular mechanisms of action of soy phytoestrogens are currently under investigation in our laboratory.

We show that PPARs, in addition to ERs, are new targets of phytoestrogens. As shown in this study, not only PPARγ but also PPARα and PPARδ directly influences osteogenesis and adipogenesis in a divergent way. Interestingly, they differentially influence estrogenic activity induced by daidzein at the transcriptional level. Moreover, our data suggest that a cross-talk exists bi-directionally between ERs and PPARs, which may further contribute to the effects on osteogenesis and adipogenesis. Because daidzein activated ERs and PPARs to different levels, which are dose-dependent, a complex balance among PPARs themselves and between ERs and PPARs determines the biological effects of daidzein on osteogenesis and adipogenesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

This study was financially supported by NUMICO Research B.V.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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