Advertisement

Peroxisome Proliferator-activated Receptor γ (PPARγ) as a Molecular Target for the Soy Phytoestrogen Genistein*

Open AccessPublished:November 05, 2002DOI:https://doi.org/10.1074/jbc.M209483200
      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.
      ER
      estrogen receptor
      PPARγ
      peroxisome proliferator-activated receptor-γ
      E2
      17β-estradiol, MEM, minimum essential medium
      ALP
      alkaline phosphate
      MAPK
      mitogen-activated protein kinase
      In recent years, soy phytoestrogens have attracted wide attention due to their potential beneficial effects on some common medical disorders (
      • Barnes S.
      ,
      • Adlercreutz H.
      • Mazur W.
      ,
      • Anderson J.J.
      ). Genistein, the principal soy phytoestrogen, has an array of biological actions and is widely available in herbal tablets (
      • Anderson J.J.
      ,
      • Setchell K.D.
      ,
      • Bouker K.B.
      • Hilakivi-Clarke L.
      ). It binds to estrogen receptors (ERs),1 ERα and ERβ, and has ER-mediated effects (
      • Kuiper G.G.
      • Lemmen J.G.
      • Carlsson B.
      • Corton J.C.
      • Safe S.H.
      • van der Saag P.T.
      • van der B.B.
      • Gustafsson J.A.
      ,
      • Tham D.M.
      • Gardner C.D.
      • Haskell W.L.
      ). In addition, it has antiestrogenic effects, but the underlying mechanism is still unknown (
      • Barnes S.
      ,
      • Adlercreutz H.
      • Mazur W.
      ,
      • Setchell K.D.
      ). Non-ER mediated genistein actions such as an inhibition of protein tyrosine kinase, DNA topoisomerases I and II and ribosomal S6 kinase have also been reported (
      • Akiyama T.
      • Ishida I.
      • Nakagawa S.
      • Ogawara H.
      • Watanabe S.I.
      • Itoh N.
      • Shibuya M.
      • Fukami Y.
      ,
      • Okura A.
      • Arakawa H.
      • Oka H.
      • Yoshinari T.
      • Monden Y.
      ,
      • Markovits J.
      • Linassier C.
      • Fosse P.
      • Couprie J.
      • Pierre J.
      • Jacquemin-Sablon A.
      • Saucier J.M., Le
      • Pecq J.B.
      • Larsen A.K.
      ). These actions are most likely mediated through transcriptional processes rather than via direct effects on enzyme activity (
      • Kim H.
      • Peterson T.G.
      • Barnes S.
      ,
      • Barnes S.
      • Kim H.
      • Darley-Usmar V.
      • Patel R., Xu, J.
      • Boersma B.
      • Luo M.
      ).
      Peroxisome proliferator-activated receptor-γ (PPARγ), one of the subtypes of PPARs, is a ligand-dependent transcription factor of the nuclear hormone receptor superfamily (
      • Willson T.M.
      • Wahli W.
      ). PPARγ is most highly expressed in adipose tissue and is involved in critical physiological functions such as adipogenesis and glucose and cholesterol metabolism (
      • Rosen E.D.
      • Spiegelman B.M.
      ). It is a target for therapeutic intervention in cardiovascular diseases, various cancers, and diabetes (
      • Kliewer S.A., Xu, H.E.
      • Lambert M.H.
      • Willson T.M.
      ).
      PPARγ is the essential transcriptional factor for adipogenesis (
      • Lazar M.A.
      ,
      • Rosen E.D.
      • Hsu C.H.
      • Wang X.
      • Sakai S.
      • Freeman M.W.
      • Gonzalez F.J.
      • Spiegelman B.M.
      ,
      • Ren D.
      • Collingwood T.N.
      • Rebar E.J.
      • Wolffe A.P.
      • Camp H.S.
      ). Adipocytes and the bone-forming cells, the osteoblasts, arise from the same bone marrow mesenchymal precursor cells (
      • Nuttall M.E.
      • Gimble J.M.
      ,
      • Bianco P.
      • Gehron R.P.
      ). The osteoprogenitor KS483 cells, which are cloned from mouse calvaria (
      • Yamashita T.
      • Asano K.
      • Takahashi N.
      • Akatsu T.
      • Udagawa N.
      • Sasaki T.
      • Martin T.J.
      • Suda T.
      ,
      • Yamashita T.
      • Ishii H.
      • Shimoda K.
      • Sampath T.K.
      • Katagiri T.
      • Wada M.
      • Osawa T.
      • Suda T.
      ), 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 (
      • Dang Z.C.
      • van Bezooijen R.L.
      • Karperien M.
      • Papapoulos S.
      • Löwik C.
      ). 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.

      DISCUSSION

      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.
      Figure thumbnail gr8
      Figure 8Molecular mechanisms of action of genistein. Genistein concurrently activates two different types of transcriptional factors, ERs and PPARγ, which have opposite effects on osteogenesis or adipogenesis. These transcriptional factors influence each other and the balance between activated ERs and PPARγ determines the biological effects of genistein on osteogenesis and adipogenesis.
      At low concentrations (≤1 μm), genistein has ER-dependent effects on osteogenesis and adipogenesis; the effects are similar to those of E2 (
      • Dang Z.C.
      • van Bezooijen R.L.
      • Karperien M.
      • Papapoulos S.
      • Löwik C.
      ). 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 (
      • Barnes S.
      ,
      • Adlercreutz H.
      • Mazur W.
      ,
      • Setchell K.D.
      ,
      • Calabrese E.J.
      ). 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 (
      • Kuiper G.G.
      • Lemmen J.G.
      • Carlsson B.
      • Corton J.C.
      • Safe S.H.
      • van der Saag P.T.
      • van der B.B.
      • Gustafsson J.A.
      ,
      • Morito K.
      • Hirose T.
      • Kinjo J.
      • Hirakawa T.
      • Okawa M.
      • Nohara T.
      • Ogawa S.
      • Inoue S.
      • Muramatsu M.
      • Masamune Y.
      ,
      • De Boever P.
      • Demare W.
      • Vanderperren E.
      • Cooreman K.
      • Bossier P.
      • Verstraete W.
      ). 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 (
      • Hu E.
      • Kim J.B.
      • Sarraf P.
      • Spiegelman B.M.
      ,
      • Adams M.
      • Montague C.T.
      • Prins J.B.
      • Holder J.C.
      • Smith S.A.
      • Sanders L.
      • Digby J.E.
      • Sewter C.P.
      • Lazar M.A.
      • Chatterjee V.K.
      • O'Rahilly S.
      ,
      • Camp H.S.
      • Tafuri S.R.
      ). Inhibition of PPARγ phosphorylation by the specific MAPK inhibitor PD98059 stimulates adipogenesis (
      • Chan G.K.
      • Deckelbaum R.A.
      • Bolivar I.
      • Goltzman D.
      • Karaplis A.C.
      ). Genistein inhibits p42/44 MAPKs in KS483 cells.
      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 (
      • Jackson S.M.
      • Demer L.L.
      ,
      • Lecka-Czernik B.
      • Moerman E.J.
      • Grant D.F.
      • Lehmann J.M.
      • Manolagas S.C.
      • Jilka R.L.
      ). It has been shown that PPARγ2 plays a dominant role in the determination of the fate of mesenchymal progenitor cells (
      • Lecka-Czernik B.
      • Gubrij I.
      • Moerman E.J.
      • Kajkenova O.
      • Lipschitz D.A.
      • Manolagas S.C.
      • Jilka R.L.
      ). 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 (
      • Pettersson K.
      • Delaunay F.
      • Gustafsson J.A.
      ). However, the distinct genistein effects in different tissues are often observed at the micromolar concentrations (
      • Barnes S.
      ,
      • Adlercreutz H.
      • Mazur W.
      ,
      • Setchell K.D.
      ,
      • Barnes S.
      • Boersma B.
      • Patel R.
      • Kirk M.
      • Darley-Usmar V.M.
      • Kim H.
      • Xu J.
      ). 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 (
      • Barnes S.
      ,
      • Adlercreutz H.
      • Mazur W.
      ,
      • Setchell K.D.
      ). 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 (
      • Barnes S.
      ,
      • Adlercreutz H.
      • Mazur W.
      ,
      • Setchell K.D.
      ). 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 (
      • Shao Z.M.
      • Shen Z.Z.
      • Fontana J.A.
      • Barsky S.H.
      ). Since it is now well established that ligand activation of PPARγ inhibits cell growth and induces apoptosis in these cancer cells (
      • Sasaki T.
      • Fujimoto Y.
      • Tsuchida A.
      • Kawasaki Y.
      • Kuwada Y.
      • Chayama K.
      ,
      • Rumi M.A.
      • Sato H.
      • Ishihara S.
      • Ortega C.
      • Kadowaki Y.
      • Kinoshita Y.
      ,
      • Shimada T.
      • Terano A.
      ), it is plausible that only when PPARγ is activated, genistein at certain levels could inhibit the growth of cancer cells.

      Acknowledgments

      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.

      REFERENCES

        • Barnes S.
        Proc. Soc. Exp. Biol. Med. 1998; 217: 386-392
        • Adlercreutz H.
        • Mazur W.
        Ann. Med. 1997; 29: 95-120
        • Anderson J.J.
        J. Clin. Endocrinol. Metab. 2001; 86: 39-40
        • Setchell K.D.
        Am. J. Clin. Nutr. 1998; 68: 1333S-1346S
        • Bouker K.B.
        • Hilakivi-Clarke L.
        Environ. Health Perspect. 2000; 108: 701-708
        • Kuiper G.G.
        • Lemmen J.G.
        • Carlsson B.
        • Corton J.C.
        • Safe S.H.
        • van der Saag P.T.
        • van der B.B.
        • Gustafsson J.A.
        Endocrinology. 1998; 139: 4252-4263
        • Tham D.M.
        • Gardner C.D.
        • Haskell W.L.
        J. Clin. Endocrinol. Metab. 1998; 83: 2223-2235
        • Akiyama T.
        • Ishida I.
        • Nakagawa S.
        • Ogawara H.
        • Watanabe S.I.
        • Itoh N.
        • Shibuya M.
        • Fukami Y.
        J. Biol. Chem. 1987; 262: 5592-5595
        • Okura A.
        • Arakawa H.
        • Oka H.
        • Yoshinari T.
        • Monden Y.
        Biochem. Biophys. Res. Commun. 1988; 157: 183-189
        • Markovits J.
        • Linassier C.
        • Fosse P.
        • Couprie J.
        • Pierre J.
        • Jacquemin-Sablon A.
        • Saucier J.M., Le
        • Pecq J.B.
        • Larsen A.K.
        Cancer Res. 1989; 49: 5111-5117
        • Kim H.
        • Peterson T.G.
        • Barnes S.
        Am. J. Clin. Nutr. 1998; 68: 1418S-1425S
        • Barnes S.
        • Kim H.
        • Darley-Usmar V.
        • Patel R., Xu, J.
        • Boersma B.
        • Luo M.
        J. Nutr. 2000; 130: 656S-657S
        • Willson T.M.
        • Wahli W.
        Curr. Opin. Chem. Biol. 1997; 1: 235-241
        • Rosen E.D.
        • Spiegelman B.M.
        J. Biol. Chem. 2001; 276: 37731-37734
        • Kliewer S.A., Xu, H.E.
        • Lambert M.H.
        • Willson T.M.
        Recent Prog. Horm. Res. 2001; 56: 239-263
        • Lazar M.A.
        Genes Dev. 2002; 16: 1-5
        • Rosen E.D.
        • Hsu C.H.
        • Wang X.
        • Sakai S.
        • Freeman M.W.
        • Gonzalez F.J.
        • Spiegelman B.M.
        Genes Dev. 2002; 16: 22-26
        • Ren D.
        • Collingwood T.N.
        • Rebar E.J.
        • Wolffe A.P.
        • Camp H.S.
        Genes Dev. 2002; 16: 27-32
        • Nuttall M.E.
        • Gimble J.M.
        Bone. 2000; 27: 177-184
        • Bianco P.
        • Gehron R.P.
        J. Clin. Invest. 2000; 105: 1663-1668
        • Yamashita T.
        • Asano K.
        • Takahashi N.
        • Akatsu T.
        • Udagawa N.
        • Sasaki T.
        • Martin T.J.
        • Suda T.
        J. Cell. Physiol. 1990; 145: 587-595
        • Yamashita T.
        • Ishii H.
        • Shimoda K.
        • Sampath T.K.
        • Katagiri T.
        • Wada M.
        • Osawa T.
        • Suda T.
        Bone. 1996; 19: 429-436
        • Dang Z.C.
        • van Bezooijen R.L.
        • Karperien M.
        • Papapoulos S.
        • Löwik C.
        J. Bone Miner. Res. 2002; 17: 394-405
        • Ferry G.
        • Bruneau V.
        • Beauverger P.
        • Goussard M.
        • Rodriguez M.
        • Lamamy V.
        • Dromaint S.
        • Canet E.
        • Galizzi J.P.
        • Boutin J.A.
        Eur. J. Pharmacol. 2001; 417: 77-89
        • He T.C.
        • Chan T.A.
        • Vogelstein B.
        • Kinzler K.W.
        Cell. 1999; 99: 335-345
        • Calabrese E.J.
        Crit. Rev. Toxicol. 2001; 31: 503-515
        • Morito K.
        • Hirose T.
        • Kinjo J.
        • Hirakawa T.
        • Okawa M.
        • Nohara T.
        • Ogawa S.
        • Inoue S.
        • Muramatsu M.
        • Masamune Y.
        Biol. Pharm. Bull. 2001; 24: 351-356
        • De Boever P.
        • Demare W.
        • Vanderperren E.
        • Cooreman K.
        • Bossier P.
        • Verstraete W.
        Environ. Health Perspect. 2001; 109: 691-697
        • Hu E.
        • Kim J.B.
        • Sarraf P.
        • Spiegelman B.M.
        Science. 1996; 274: 2100-2103
        • Adams M.
        • Montague C.T.
        • Prins J.B.
        • Holder J.C.
        • Smith S.A.
        • Sanders L.
        • Digby J.E.
        • Sewter C.P.
        • Lazar M.A.
        • Chatterjee V.K.
        • O'Rahilly S.
        J. Clin. Invest. 1997; 100: 3149-3153
        • Camp H.S.
        • Tafuri S.R.
        J. Biol. Chem. 1997; 272: 10811-10816
        • Chan G.K.
        • Deckelbaum R.A.
        • Bolivar I.
        • Goltzman D.
        • Karaplis A.C.
        Endocrinology. 2001; 142: 4900-4909
        • Jackson S.M.
        • Demer L.L.
        FEBS Lett. 2000; 471: 119-124
        • Lecka-Czernik B.
        • Moerman E.J.
        • Grant D.F.
        • Lehmann J.M.
        • Manolagas S.C.
        • Jilka R.L.
        Endocrinology. 2002; 143: 2376-2384
        • Lecka-Czernik B.
        • Gubrij I.
        • Moerman E.J.
        • Kajkenova O.
        • Lipschitz D.A.
        • Manolagas S.C.
        • Jilka R.L.
        J. Cell. Biochem. 1999; 74: 357-371
        • Pettersson K.
        • Delaunay F.
        • Gustafsson J.A.
        Oncogene. 2000; 19: 4970-4978
        • Barnes S.
        • Boersma B.
        • Patel R.
        • Kirk M.
        • Darley-Usmar V.M.
        • Kim H.
        • Xu J.
        Biofactors. 2000; 12: 209-215
        • Shao Z.M.
        • Shen Z.Z.
        • Fontana J.A.
        • Barsky S.H.
        Anticancer Res. 2000; 20: 2409-2416
        • Sasaki T.
        • Fujimoto Y.
        • Tsuchida A.
        • Kawasaki Y.
        • Kuwada Y.
        • Chayama K.
        Pathobiology. 2001; 69: 258-265
        • Rumi M.A.
        • Sato H.
        • Ishihara S.
        • Ortega C.
        • Kadowaki Y.
        • Kinoshita Y.
        J. Lab. Clin. Med. 2002; 140: 17-26
        • Shimada T.
        • Terano A.
        J. Lab. Clin. Med. 2002; 140: 4-5