Bisphenol A induces a rapid activation of Erk1/2 through GPR30 in human breast cancer cells
Introduction
2,2-Bis(4-hydroxyphenyl) propane, or bisphenol A (BPA), has been used worldwide in industry for the production of polycarbonate plastics and epoxy resins, owing to its unique physical and chemical properties (Ikezuki et al., 2002). Once it has contaminated the environment, BPA is expected to be degraded rapidly in air, whereas it is more persistent in water and soil (Tsai, 2006). Humans are exposed to BPA mainly at the time of consumption of water and foods through the materials used for containers, packages and others (Vandenberg et al., 2007, Brotons et al., 1995, Lambert et al., 1998). Indeed, BPA has been detected in multiple human tissue compartments including serum, follicular and amniotic fluids (Ikezuki et al., 2002, Tsutsumi, 2005), urine (Ouchi and Watanabe, 2002), breast milk (Ye et al., 2006), saliva (Sasaki et al., 2005) and adipose tissue (Fernandez et al., 2007). Moreover, in fetal serum and full-term amniotic fluid, traces of BPA have been found due to its passage through the placenta (Ikezuki et al., 2002).
Exposure to BPA has been associated with recurrent miscarriage (Sugiura-Ogasawara et al., 2005). Serum levels of BPA were greater in women with the polycystic ovarian syndrome than in healthy controls (Takeuchi and Tsutsumi, 2002). Furthermore, BPA levels were positively correlated to the levels of testosterone and androstenedione, suggesting an interaction between androgen and the metabolism of BPA (Takeuchi et al., 2004). Of particular relevance, epidemiological studies have highlighted a correlation between the level of BPA in the environment and the incidence of cancer (Maffini et al., 2006, Keri et al., 2007). Furthermore, in experimental animal studies, BPA treatment has been shown to accelerate growth and puberty, to alter the ovarian cycle in females (Mlynarcíková et al., 2005, Rasier et al., 2006), to interfere with embryonic development (Takai et al., 2000), and to induce aneuploidy (Hunt et al., 2003). BPA has harmful effects on the multipotent neural progenitor (Kim et al., 2007) and increases depression-like behavior in rats (Fujimoto et al., 2006). These lines of evidence suggest that BPA can act as an endocrine disruptor and a mitogenic substance inducing cell proliferation, and, consequently, affect significantly human and animal health.
The mechanism by which BPA exerts its biological actions has been proposed. BPA binds to both estrogen receptors (ERs) α and β (ERα and ERβ), which have been reported as the foremost molecular mediators of the in vitro and in vivo effects exerted by BPA (Welshons et al., 2006, Wetherill et al., 2007, Khurana et al., 2000, Singleton et al., 2006, Bredhult et al., 2007). Because ERs belong to the nuclear receptor super-family (Ascenzi et al., 2006), research has been mainly focused on the ability of BPA to modulate transcriptional activity through these nuclear receptors (Welshons et al., 2006, Khurana et al., 2000, Singleton et al., 2006, Bredhult et al., 2007, Bergeron et al., 1999, Matthews et al., 2001). Recently, it was found that the ERs localized at the plasma membrane initiated a 17β-estradiol (E2)-induced rapid signaling (Ascenzi et al., 2006). Indeed, ERα-dependent rapid membrane-starting actions are crucial for the E2-induced response of cancer promotion in several target cells (Ascenzi et al., 2006). Some evidence suggests that BPA also binds to G-protein-coupled receptor 30 (GPR30), a seven-transmembrane receptor structurally unrelated to the nuclear ERs, and mediates rapid actions of estrogens (Thomas and Dong, 2006, Filardo et al., 2002, Revankar et al., 2005). Therefore, it is reasonable to hypothesize that BPA binds to GPR30 to mediate non-genomic estrogenic actions and alters these rapid signals.
The aims of the present study are to elucidate whether the mechanism by which BPA induces the activation of Erk1/2 is distinguishable from that behind ERα-mediated signaling, and to evaluate the signaling pathway by which BPA-induced non-genomic actions of estrogen affect the expression of genes. The human breast cancer cell lines MCF-7, MDA-MB-231 and SKBR3 were used as experimental models to discriminate between ER-dependent, putative ER-independent, and/or GPR30-associated effects.
Section snippets
Reagents
17β-Estradiol (E2), AG1478, and PD98059 were purchased from Sigma–Aldrich (St. Louis, MO), ICI 182,780 from Tocris Cookson (Ellisville, MO), and cycloheximide (CHX) from Santa Cruz Biotechnology (Santa Cruz, CA). All compounds were solubilized in dimethyl sulfoxide (DMSO). The final concentration of the solvent in the culture medium did not exceed 0.1%, which did not affect the yield of cells. Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT). The
Phosphorylation of Erk1/2 is stimulated by BPA
Estrogens and xenoestrogens were shown to generate a rapid signal via second messengers, such as Ca2+, cAMP, nitric oxide, and G-proteins, which in turn activate various downstream kinases (Bulayeva and Watson, 2004, Watson et al., 2007). To evaluate whether BPA is involved in the rapid cellular response exerting the estrogenic activity, we investigated its ability to induce phosphorylation of Erk1/2 in MCF-7 cells (Fig. 1). Interestingly, BPA stimulated phosphorylation of Erk1/2 with a
Discussion
It has long been acknowledged that estrogenic chemicals interact with human ERs (Dodds and Lawson, 1938, Krishnan et al., 1993, Olea et al., 1996), and act as an antagonist against the human androgen receptor (AR) (Sohoni and Sumpter, 1998, Xu et al., 2005). However, BPA’s ability to bind ERs and AR and its hormonal activity are extremely weak, approximately 1000–10,000 times less than that for natural hormones, making the intrinsic significance of the low-dose effects intangible and obscure (
Conclusion
The present study demonstrates that BPA induces a rapid activation of Erk1/2 in both ERα/β-positive and negative breast cancer cells and that this effect is not blocked with an ER antagonist, ICI 182,780, suggesting the involvement of an ER-independent pathway. BPA-induced signaling is likely associated with GPR30 in human breast cancer cells, because the expression of GPR30 is necessary for BPA-induced activation of Erk1/2 and transcriptional regulation of c-fos. In addition, the expression of
Acknowledgments
This research was supported by a Grant-in-aid for Basic Areas from the Ministry of Education, Science, Sports and Culture of Japan, by a grant for preventing public pollution from the Ministry of the Environment of Japan, and by a grant of R&D for small and medium enterprises from the Ministry of Economy, Trade and Industry of Japan.
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