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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.
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.