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To whom correspondence should be addressed: Dept. of Endocrinology and Metabolic Diseases (C4-R), Leiden University Medical Center, Albinusdreef 2, 2300 RC, Leiden, The Netherlands. Tel.: 0031-71-5263075; Fax: 0031-71-5248136
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The principal soy phytoestrogen genistein has an array of biological actions. It binds to estrogen receptor (ER) α and β and has ER-mediated estrogenic effects. In addition, it has antiestrogenic effects as well as non-ER-mediated effects such as inhibition of tyrosine kinase. Because of its complex biological actions, the molecular mechanisms of action of genistein are poorly understood. Here we show that genistein dose-dependently increases estrogenic transcriptional activity in mesenchymal progenitor cells, but its biological effects on osteogenesis and adipogenesis are different. At low concentrations (≤1 μm), genistein acts as estrogen, stimulating osteogenesis and inhibiting adipogenesis. At high concentrations (>1 μm), however, genistein acts as a ligand of PPARγ, leading to up-regulation of adipogenesis and down-regulation of osteogenesis. Transfection experiments show that activation of PPARγ by genistein at the micromolar concentrations down-regulates its estrogenic transcriptional activity, while activation of ERα or ERβ by genistein down-regulates PPARγ transcriptional activity. Genistein concurrently activates two different transcriptional factors, ERs and PPARγ, which have opposite effects on osteogenesis or adipogenesis. As a result, the balance between activated ERs and PPARγ determines the biological effects of genistein on osteogenesis and adipogenesis. Our findings may explain distinct effects of genistein in different tissues.
peroxisome proliferator-activated receptor-γ
17β-estradiol, MEM, minimum essential medium
mitogen-activated protein kinase
In recent years, soy phytoestrogens have attracted wide attention due to their potential beneficial effects on some common medical disorders (
), have been shown to differentiate into both osteoblasts and adipocytes. Using this cell line, we recently showed that 17β-estradiol (E2) stimulates osteogenesis and concurrently inhibits adipogenesis in these precursor cells (
). Whether the phytoestrogen genistein has similar effects is unknown.
In the present study, we examined the effects of genistein on osteogenesis and adipogenesis and explored its molecular mechanisms of action. Our results show that genistein, in addition to its estrogenic activity, activates PPARγ, resulting in a down-regulation of osteogenesis and an up-regulation of adipogenesis. This action is concentration-dependent. Our data show that the balance between activated ERs and PPARγ determines the biological effects of genistein.
Cell Cultures and Assays
The methods for cell culture have been described before (
). In brief, KS483 cells and mouse bone marrow cells were cultured in phenol red-free α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (Invitrogen) or 15% fetal bovine serum (for mouse bone marrow), 50 μg/ml ascorbic acid, 10 mm β-glycerophosphate, and 10−8m dexamethasone (only for mouse bone marrow). Cells were continuously exposed to genistein 1 day after plating until the end of the experiment at day 21. Assays for ALP activity and DNA content, mRNA expression by RT-PCR, and Oil-Red-O staining for adipocytes were performed as described previously (
). Binding buffer consisted of 10 mm Tris/HCl, pH 8.2, containing 50 mm KCl and 1 mm dithiothreitol. Membrane preparations (5 μg/ml) were incubated for 180 min at 4 °C in the presence of [3H]rosiglitazone (BRL49653, Amersham Biosciences) (10 nm) and the tested compounds. Nonspecific binding was defined using an excess of unlabelled rosiglitazone (10 μm). Incubation was terminated by the addition of ice-cold 50 mm Tris/HCl buffer, pH 7.4, followed by rapid filtration under reduced pressure through Whatman GF/C filter plates presoaked with ice-cold buffer, followed by three successive washes with the same buffer. Radioactivity was measured in a TopCount apparatus (Packard). The receptor preparation used during these experiments presented a Bmax of 49 pmol/mg proteins and a Kd of 5.58 nm for [3H]rosiglitazone. Genistein was solubilized in Me2SO and diluted to the appropriate working concentrations (100 μm-0.1 nm).
Transient Gene Expression Assays in KS483 Cells
The estrogen-responsive reporter gene construct (2XERE-TATA-luc), which contains two copies of a consensus estrogen response element, and the empty control TATA-luc plasmids were kindly provided by Dr. E. Kalkhoven and Dr. M. G. Parker. The peroxisome proliferator-responsive element (3XPPRE-tk-luc) containing three copies of a consensus peroxisome proliferator-responsive element and the human PPARγ2 constructs were kind gifts from Dr. J. Auwerx. The luciferase reporter construct (5XPPRE-TATA-luc) contained five copies of a consensus PPRE and a TATA box and were provided by Dr. M. Karperien. The pT-109 FARE PPRE construct was a kind gift from Dr. K. van der Lee and Dr. M. van Bilsen. The ACO-luc PPRE construct was kindly supplied by Dr. K. W. Kinzler and Dr. B. Vogelstein. The human ERα construct was kindly provided by Dr. G. Kuiper. KS483 cells were seeded into 24-well plates. After 24 h, they were transfected using a lipid-based FuGENE 6 transfection reagent according to the manufacturer (Roche Molecular Biochemicals). For each triplicate of sample, 100 ng of luciferase reporter and 500 ng of β-galactosidase expression vector were applied. The transfection medium was changed after 16 h into the different medium as indicated. After 48 h, cells were washed twice with PBS, lysed in PBS containing 1% Triton X-100 and sonicated. Luciferase activity was measured and expressed as fold induction ± S.E., which was corrected for transfection efficiency using β-galactosidase activity.
Data are presented as means ± S.E. Differences between groups were accepted at p < 0.05, which were assessed by one-way analysis of variance or related test using software Instat.
As shown in Fig. 1, genistein added to cultures of KS483 cells had a clear biphasic effect on osteogenesis, similar to that of E2 (
). At concentrations from 0.1 to 10 μm, genistein stimulated ALP activity, nodule formation, and calcium deposition, with a maximal effect at 1 μm. In contrast, at concentrations of 25 μm or higher, genistein inhibited ALP activity, nodule formation and Ca2+ deposition. These changes were paralleled by mRNA expression of the osteoblastic markers, Cbfa1, osteocalcin, and PTH/PTHrP receptor that, relative to control, were increased by 1 μm genistein and decreased by 25 μm (Fig. 1). Similar stimulatory and inhibitory effects of genistein on bone formation were also observed in mouse bone marrow cell cultures (Fig. 2). In those cultures, genistein stimulated ALP activity and Ca2+deposition at concentrations between 0.1 and 10 μm, whereas it inhibited osteogenesis at concentrations of 25 μm or higher. These data demonstrate that genistein affects osteogenesis of progenitor cells in a biphasic way; namely, it increases osteogenesis at low concentrations and inhibits osteogenesis at high concentrations.
Genistein had also a biphasic effect on adipogenesis, which was, however, different to that of E2(
). At low concentrations between 0.1 and 1 μm, it decreased adipocyte numbers, while at higher concentrations (>10 μm) it stimulated adipogenesis (Fig. 3A). The effects of genistein on adipogenesis were paralleled by changes in mRNA expression of the adipocyte markers, PPARγ2, aP2, and lipoprotein lipase (Fig. 3B). Adipogenic responses of mouse bone marrow cells to different doses of genistein are shown in Fig. 3C. Mouse bone marrow cultures treated with genistein concentrations of 25 μm or higher did not reach confluence, and there were no adipocytes during the cultures. However, compared with control an increase in adipocyte numbers was observed at the concentration of 10 μm, whereas a decrease in adipocyte numbers was found at the concentrations of 0.1 and 1 μm. These data show that genistein affects adipogenesis of progenitor cells in a biphasic way,i.e. an inhibition of adipogenesis at low concentrations and a stimulation of adipogenesis at high concentrations.
ER-dependent and ER-independent Effects of Genistein
Both ER-dependent and ER-independent effects were observed in KS483 cells treated with different concentrations of genistein (Fig. 4). At a concentration of 1 μm, the effects were mediated by ERs because stimulation of ALP activity and inhibition of adipogenesis were both blocked by 1 μm ICI 164,382, a specific antiestrogen. In contrast, at higher concentrations of genistein the effects observed were ER-independent because ICI 164.382 at concentrations from 0.01 to 100 μm did not affect the action of genistein on osteogenesis or adipogenesis. In addition, E2 (10−10m to 10−5m) did not reverse the effects of genistein at 25 μm on osteogenesis or adipogenesis. These data suggest that the action of genistein at low concentrations is likely ER-mediated, whereas its effects at high concentrations are not ER-mediated.
Activation of PPARγ
We transiently transfected KS483 cells with a luciferase reporter construct containing five copies of a consensus PPRE inserted in front of a TATA box together with expression plasmids encoding human PPARγ2. PPRE-luc reporter activity was measured after incubation of transfected cell cultures with different doses of genistein. As shown in Fig5A, genistein in the micromolar range increased PPRE-luc reporter activity dose-dependently. Furthermore, in the same concentration range, genistein increased PPRE-luc reporter activity in ER-positive and ER-negative breast cancer cell lines, T47D and MDA-MD-231, respectively (not shown). These results were confirmed with three other reporter constructs including the PPARγ response element ACO-luc (
). Thus, genistein transcriptionally activates PPRE-luc reporter activity independent of the cell lines and constructs used.
To determine whether genistein activates PPARγ through direct interaction with this receptor, we performed a membrane-bound PPARγ binding assay. Genistein had a measurable Ki of 5.7 μm (Fig. 5B), which is comparable to that of the known PPARγ ligands (
). We have checked whether genistein bound competitively with [3H]rosiglitazone to the same PPARγ site. Indeed the dissociation constant (Kd) of [3H]rosiglitazone in saturation experiments in the presence of a high dose of genistein was significantly reduced as compared with that in the absence of genistein. The maximal number of sites labeled was not altered. These data demonstrate that both genistein and [3H]rosiglitazone bind to the same PPARγ site (data not shown). Therefore, genistein can interact directly with the PPARγ ligand-binding domain and thus act as a PPARγ ligand.
Balance between Activated ERs and PPARγ
As both the antiestrogenic effects and the activation of PPARγ were increased by micromolar concentrations of genistein, we tested whether activation of PPARγ is involved in the antiestrogenic action. When KS483 cells were treated for 18 days either with a specific PPARγ agonist ciglitazone, genistein, E2, or a combination of genistein and E2, a decrease in ALP activity was observed with all treatments, except for E2 alone that increased ALP activity (Fig. 6A). When, however, we transiently transfected KS483 cells with a luciferase reporter construct containing two copies of a consensus ERE inserted in front of a TATA box and exposed these cells to different concentrations of genistein, a dose-related increase of ERE-luc reporter activity was observed at a concentration between 0.1 and 50 μm (Fig. 6B). Furthermore, the estrogenic potency of genistein at the micromolar range was greater than that of E2(10−8m), and anti-estrogenic effects of genistein were not observed (Fig. 6C). The lack of an antiestrogenic effect in the gene reporter assays could be due to low amount of endogenous PPARγ2 in KS483 cells during the first 5 days (
), the period in which the gene reporter assays were performed. To investigate this further, we transiently transfected vectors expressing human PPARγ2 or empty vector along with a ERE-luc construct and exposed KS483 cells to genistein. The transient co-transfection of PPARγ2 resulted in a decrease of ERE-luc reporter activity at high genistein concentrations. Taken together, our data show that the antiestrogenic effects of genistein are due to an activation of PPARγ2, leading to down-regulation of ER-mediated transcriptional activity and osteogenesis.
The question that arises is whether activation of ERs by genistein can also alter the transcriptional regulation of PPARγ. To investigate this, we transiently transfected vectors expressing human ERα or ERβ or empty vector along with a PPARγ2 construct and a PPRE-luc construct. Co-transfection of ERα (Fig. 7A) or ERβ (Fig. 7B) decreased PPRE-luc reporter activity in KS483 cells treated with different concentrations of genistein. Interestingly, genistein at 1 μm suppressed PPRE-luc reporter activity to a level lower than that of controls, while at 10 μm it suppressed it to the control level in the presence of sufficient levels of ERs. In contrast, PPRE-luc reporter activity was higher than the control levels at 50 μm genistein of and was not influenced by the levels of ERα or ERβ (Fig. 7).
We show here that PPARγ is a molecular target for genistein. At the micromolar range, genistein binds to and transactivates PPARγ, leading to a decrease of osteogenesis and an increase in adipogenesis. In addition, genistein dose-dependently transactivates ERs, resulting in an up-regulation of osteogenesis and a down-regulation of adipogenesis. Moreover, activation of ERs by genistein could down-regulate PPARγ transcriptional activity and vice versa. The balance between the activation of ERs and PPARγ is concentration-related. As a result, the biological effects,i.e. osteogenesis and adipogenesis, vary according to the concentrations of genistein (Fig. 8). Our findings can explain the previously reported diverse actions of genistein in different tissues.
At low concentrations (≤1 μm), genistein has ER-dependent effects on osteogenesis and adipogenesis; the effects are similar to those of E2 (
). At high concentrations (>1 μm), however, genistein has antiestrogenic actions, namely, it down-regulates osteogenesis, which is opposite to E2-induced effects. Antiestrogenic effects of genistein have been reported in many cell types and animal models, but the mechanism responsible for this is still not known (
). We show here that the antiestrogenic effects are not due to a decrease of estrogenic activity of genistein. Instead, genistein at micromolar concentrations dose-dependently increased estrogenic transcriptional activity, and the levels were even higher than those induced by E2. These results are in line with reports using different cell lines or assays (
). Moreover, antiestrogenic effects of genistein could not be restored or blocked by E2 or by the antiestrogen compound ICI164,382. Together, our results implicate that antiestrogenic effects of genistein are elicited via pathways other than the ER pathway.
Different from E2, genistein binds to and transactivates PPARγ, leading to adipogenesis. Moreover, activation of PPARγ may also be due to an inhibition of the MAPK pathway. It is well known that the A/B domain of PPARγ contains a consensus MAPK site (
Z.-C. Dang, V. Audinot, S. E. Papapoulos, J. A. Boutin, and C. W. G. M. Löwik, unpublished observations.
It is therefore possible that an inhibition of p42/44 MAPKs contributes to an activation of PPARγ. By using a pure PPARγ ligand, ciglitazone, we showed that activation of PPARγ down-regulates osteogenesis in KS483 cells. These results are consistent with observations in MC3T3-E1 cells and in U33 cells (
). An increase in adipogenesis and a decrease of osteogenesis by genistein at concentrations of 25 μm or higher indicate that PPARγ actions dominate at higher genistein concentrations.
Genistein concurrently activates two different transcriptional factors, ERs and PPARγ. These two transcriptional factors have opposite effects on osteogenesis or adipogenesis. We showed that activation of PPARγ by genistein at the micromolar concentrations down-regulates its estrogenic transcriptional activity, while activation of ERα or ERβ down-regulates PPARγ transcriptional activity. It is plausible that genistein at certain concentrations activates ERs and PPARγ to a different extent. The balance between activated ERs and PPARγ determines the biological effects of genistein, i.e.osteogenesis and adipogenesis, which are fully concentration-dependent.
Our findings provide the molecular basis of the mechanism of action of genistein and may have wide implications. Diverse effects of genistein in different tissues have been explained by the high binding affinity for ERβ because ERβ can act as a dominant negative regulator of estrogenic activity. These dominant negative effects were only observed below the micromolar concentrations of genistein (
). We show that the balance between activated ERs and PPARγ determines the biological effects of genistein, which might explain its diverse biological effects in different organs. Therefore, the biological effects of genistein in certain tissues strongly depend on the concentration of genistein present and the levels of ERs and PPARγ within that particular tissue. There is accumulating evidence that health benefits occur only when phytoestrogens are consumed in sufficient quantities (
). It has been reported that plasma concentration of genistein is relatively low and generally less than 40 nm in humans consuming diets without soy, whereas it can reach 4 μm in the plasma of Japanese who consume high amount of soy products (
). Our findings might explain why genistein functions only at a certain level. For example, genistein at the micromolar concentration range inhibits growth of ER-positive breast cancer cells like MCF7 and T47 D as well as ER-negative breast cancer cells like MDA-MD-231 cells (
), it is plausible that only when PPARγ is activated, genistein at certain levels could inhibit the growth of cancer cells.
We are grateful to Drs. E. Kalkhoven, M. G. Parker, J. Auwerx, G. Kuiper, K. van der Lee, M. van Bilsen, K. W. Kinzler and B. Vogelstein for supplying constructs. We thank colleagues from the Endocrinology department for the technical support and Numico Research B. V. for financial support.