Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium

Abstract

Estrogens and growth factors such as epidermal growth factor (EGF) act as mitogens promoting cellular proliferation in the breast and in the reproductive tract. Although it was considered originally that these agents manifested their mitogenic actions through separate pathways, there is a growing body of evidence suggesting that the EGF and estrogen-mediated signaling pathways are intertwined. Indeed, it has been demonstrated recently that 17beta-estradiol (E2) can induce a rapid activation of mitogen-activated protein kinase (MAPK) in mammalian cells, an event that is independent of both transcription and protein synthesis. In this study, we have used a pharmacological approach to dissect this novel pathway in MCF-7 breast cancer cells and have determined that in the presence of endogenous estrogen receptor, activation of MAPK by E2 is preceded by a rapid increase in cytosolic calcium. The involvement of intracellular calcium in this process was supported by the finding that the presence of EGTA and Ca2+-free medium did not affect the activation of MAPK by E2 and, additionally, that this response was blocked by the addition of the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. Cumulatively, these data indicate that the estrogen receptor, in addition to functioning as a transcription factor, is also involved, through a nongenomic mechanism, in the regulation of both intracellular calcium homeostasis and MAPK-signaling pathways. Although nongenomic actions of estrogens have been suggested by numerous studies in the past, the ability to link estradiol and the estrogen receptor to a well defined signaling pathway strongly supports a physiological role for this activity.

Figure 1

Activation of MAPK in response to estrogen in MCF-7 cells. Western blot analysis of total lysates from MCF-7 and HeLa cells, stained with anti-active MAPK antibody (Upper) and with an antibody recognizing Erk1 and Erk2 (Lower). MCF-7 cells were mock-treated (C) or treated with either EGF for 5 min and 18 hr (lanes 2 and 3, respectively) or with estradiol (E2, lanes 4 and 5). HeLa cells were mock-treated (C) and treated for 5 min with E2 and EGF.

Figure 2

Nuclear translocation of MAPK in response to E2. Indirect immunofluorescence staining of MCF-7 cells using primary antibodies anti-Erk1/2 (Upper Left) and anti-active-MAPK antibodies (Upper Right and Lower). Cells either were left untreated (Upper) or treated with EGF (Lower Left) and estrogen (Lower Right) for 30 min before incubation with FITC secondary antibody.

Figure 3

E2-induced MAPK activation is not mediated by cRaf-1. Immunoblot stained with anti-c-Raf1 antibody. MCF-7 cells were treated with either EGF or E2 as in Fig. 1, with the exception that cRAF-1 activation also was examined after treatment with E2 + the pure antiestrogen ICI 182,780 or ICI 82,780 alone. Extracts were analyzed on 7.5% SDS/PAGE. A change in mobility of the band corresponding to phosphorylated c-Raf 1 is clearly detectable after treatment with EGF. (Lower) Normalization for Erk1/2.

Figure 4

MAPK activation after treatments of MCF-7 cells with 8-bromo cAMP, calcium ionophore A23187, and TG. E2, E2/ICI, and EGF treatments were carried out in Ca²⁺/EGTA medium as indicated (Upper). The same blots then were stripped and probed with Erk1/2 as a control (Lower).

Figure 5

Cytosolic calcium oscillations in MCF-7 cells. (A) Free intracellular calcium measurements in response to treatment with ATP, bradykinin (BK), TG are shown in A1. (A2 and A3) The calcium measurements after estrogen treatment (E2) in regular medium and in Ca²⁺-free/EGTA medium, respectively. (B) Measurements of intracellular calcium at a single-cell level by fluorescence confocal microscopy. MCF-7 cells were stimulated as indicated in the text with estradiol (E2, B1), ICI (B2), and a combination of estradiol and ICI (E2/ICI, B3). The data shown are representative of a single experiment, and a minimum of four experiments were carried out for each treatment. Values are expressed as relative fluorescence units (RFI), with each color representing the calcium response in a single cell. In response to E2, 14 of 20 cells had an RFI >30% of the baseline; in the presence of ICI, it was 14 of 25, and in the presence of E2/ICI, it was 6 of 21 cells.

Figure 6

Intracellular calcium requirement for E2-induced MAPK activation. Shown is Western blot analysis of MCF-7 cells in response to E2 in the presence and absence of the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate. The blot was stained first with anti-active MAPK (Upper) and then subsequently stripped and restained with anti-Erk1/2 antibody.