Elsevier

Steroids

Volume 69, Issue 3, March 2004, Pages 181-192
Steroids

Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways

https://doi.org/10.1016/j.steroids.2003.12.003Get rights and content

Abstract

Estradiol (E2) and other steroids have recently been shown to initiate various intracellular signaling cascades from the plasma membrane, including those stimulating mitogen-activated protein kinases (MAPKs), and particularly extracellular-regulated kinases (ERKs). In this study we demonstrated the ability of E2 to activate ERKs in the GH3/B6/F10 pituitary tumor cell line, originally selected for its enhanced expression of membrane estrogen receptor-α (mERα). We compared E2 to its cell-impermeable analog (E2 conjugated to peroxidase, E2–P), and to the synthetic estrogen diethylstilbestrol (DES). Time-dependent ERK activation was quantified with a novel fixed cell-based immunoassay developed to efficiently determine activation by multiple compounds over multiple parameters. Both E2 and DES produced bimodal responses, but with distinctly different time courses of enzyme phosphorylation (activation) and inactivation; E2–P induced a monophasic ERK activation. E2 also phosphorylated ERKs in concentration-dependent manner with two concentration optima (10−14 and 10−8 M). Inhibitors were employed to determine pathway (ER, EGFR, membrane organization, PI3 kinase, Src kinase, Ca2+) involvement and timing of pathway activations; all affected ERK activation as early as 3–6 min, suggesting simultaneous, not sequential, activation. Therefore, E2 and other estrogenic compounds can produce rapid ERK phosphorylations via nongenomic pathways, using more than one pathway for signal generation.

Introduction

Recent studies have demonstrated that E2 can regulate cell proliferation, apoptosis, vasodilatation, regulated hormone release, cell differentiation, and other specific cell functions by rapid (from seconds to a few minutes) stimulation of signaling pathways in different cell types [1], [2]. This occurs in addition to classical genomic effects mediated by estrogen receptors (ERs) [3]. A transcriptionally inactive form of ER (classical nuclear receptor with only the ligand-binding domain remaining) can activate signaling cascades leading to cell survival and DNA synthesis [4], [5]. Other examples of signaling pathways initiated by E2 include increased levels of intracellular Ca2+, modulation of potassium channels, cAMP production, nitric oxide formation, interaction with G protein-coupled receptors and G proteins, and stimulation of mitogen-activated protein (MAP) kinases [6], [7], [8], [9], [10], [11], [12], [13].

One type of MAP kinase, the extracellular-regulated kinases (ERK 1 and 2), can be activated by dual phosphorylation on Thr183 and Tyr185. The ERK 1/2 cascade has been shown to be involved in cell differentiation, proliferation, and increased cell motility and migration—all responses that can be initiated by estrogens as well. These responses can also be activated by many other mitogens, such as EGF, PDGF, thromboxane A2, angiotensin II, and insulin [13]. Classical ERK 1/2 pathway activations start from receptor tyrosine kinases (RTKs) after ligand binding and auto-phosphorylation. The adaptor protein Shc can then recognize the phosphorylated site on the receptor, bind to it, and become phosphorylated itself. Another entry point into this pathway is phosphorylation of Shc by Src family kinases. The receptor–Shc complex then further binds with the adapter protein GRB and the nucleotide exchange factor SOS to catalyze the conversion of GDP to GTP bound on the small G protein Ras. Ras, in turn, interacts with the kinases Raf and MEK that subsequently affect ERKs. One consequence is that ERKs translocate to the nucleus and alter the phosphorylation status of transcription factors. Additionally, other pathways (involving G proteins, Src, [Ca2+]i elevation, and PI3K/Akt) can lead to ERK activation [14], [15]. Therefore, there are many different elements of the signaling web that can lead to ERK activation, and resultant cell functions. Some of these pathways (PI3K and Src activation, Ca2+ level changes, channel openings) are stimulated by E2 [7], [16], [17]. ERK phosphatases are inducible by stimuli that activate ERK pathways, providing negative feedback control for ERK activity [18].

Additionally, E2 can share the signaling pathways of EGF. E2 was recently shown to activate a metalloproteinase which in turn releases a soluble form of EGF (heparin or sHB-EGF) by cleavage from its membrane-anchored tether [11], [19]. Consequently, sHB-EGF is able to bind to EGF receptor and activate MAP kinases through the classical RTK pathway.

To produce rapid non-genomic effects, ERs are thought to be located in or near the plasma membrane, where they can access the machineries of signal generation. Multiple antibodies (Abs) directed against different epitopes of nuclear ERα recognize antigen on the plasma membrane of live or fixed prolactin-producing pituitary cells [20], [21], as well as in other cell types [22], [23], [24]. Multiple studies now suggest that mERs can associate with membrane rafts and caveolae [25], [26]. These specialized structures contain high concentrations of cholesterol and sphingolipids, and provide a location for the interaction of different types of receptors, integrins, G proteins and other signaling molecules [27]. Localization of mER in these structures may allow the sharing of signals that ERs transduce, thus causing integration and simultaneous triggering of multiple signaling cascades.

To address the ERK-linked signaling consequences of estrogens in lactogenic pituitary tumor cells selected for mERα expression [28], [29], we assessed the effects of various specific signaling pathway inhibitors to determine the involvement of signaling partners and the sequencing of the multiple signaling events. To accomplish this more effectively, we developed a quantitative plate immunoassay for ERK phosphorylation status. This allowed us to efficiently compare the ability of different estrogenic compounds (E2, E2–P and DES) to activate ERKs over an extensive time course.

Section snippets

Experimental

Phenol red-free DMEM and horse serum were purchased from Gibco-BRL (Grand Island, NY). Defined, supplemented, fetal calf and goat sera were obtained from Hyclone (Logan, UT). From Vector Laboratories (Burlingame, CA), we purchased biotinylated universal anti-mouse/rabbit IgG, Vectastain ABC-AP (avidin:biotinylated enzyme complex with alkaline phosphatase) detection system, levamisol (endogenous alkaline phosphatase subtype inhibitor), and para-nitrophenol–phosphate (pNpp, the substrate for our

Cell culture

GH3/B6/F10 and GH3/B6/D9, clonal rat prolactinoma cell lines, were selected for high and low expression (respectively) of mERα [31]. Cells were routinely cultured in DMEM medium containing 12.5% horse serum, 2.5% defined-supplemented calf serum and 1.5% fetal calf serum. For individual experiments, cells were deprived of steroids for 48 h after plating by substituting DMEM medium containing only 1% serum that had been charcoal-stripped four times.

Western blots

We used approximately 120,000 cells/25 cm2 culture flask (Corning Incorporated, Corning, NY) for MAPK assay. Cells were treated with different hormones and blocking agents for the times indicated, usually over a 60 min time course. Cells were then rinsed twice with ice-cold PBS and solubilized in 0.5 ml of lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 1 mM PMSF, 1 mM DTT) at 4 °C. After

Fixed cell-based 96-well ELISA

Cells were plated at approximately 10,000 cells/well in a 96-well poly-d-lysine coated plate (Corning Incorporated) and then exposed to medium (containing 1% serum stripped of steroids) for 48 h. The cells were then treated with hormones and other reagents for 3–60 min, followed by fixation with 2% paraformaldehyde/0.2% picric acid at 4 °C for 48 h. After fixation, the cells were washed twice with PBS and incubated with blocking buffer (2% BSA, 0.1% Triton X-100 in PBS) for 1 h at RT. Primary Ab for

Crystal violet (CV) cell quantification assay

This procedure for normalization of the pNp signal to cell number [28] involved washing alkaline phosphatase reaction reagents from the plate twice with ddH2O, then drying the plates at RT for at least 12 h. Crystal violet solution (0.1% in water, filtered) was then added to each well (50 μl/well) and incubated for 30 min at RT. The cells were then washed at least 3× with ddH2O and dried at RT. Dye was released from the cells with 50 μl/well acetic acid (10% in water) at RT for 30 min. The A590 was

Statistics

Data were compared for significance of differences using Sigma Stat 2 (Jandel Scientific, San Rafael, CA) and the one-way ANOVA test.

Fixed cell-based ELISA (plate assay)

Western blotting analyses of the activation/phosphorylation of signaling molecules are laborious and difficult to quantitate. Quantification of bands via densitometry requires subjective judgments about which part of the bands to scan, and what to designate as a background signal. These considerations predispose the data to variations that can only be overcome by numerous repetitions. Small, though important regulatory changes are not likely to be measured as significant with such systems.

Discussion

Our studies demonstrated rapid activation of a kinase signaling endpoint (ERKs) by E2 and different estrogenic mimetics. Using a spectrum of compounds that specifically inhibit signaling pathways known to lead to ERK activation, we demonstrated the utilization of all of these mechanisms in pituitary tumor cells bearing membrane ERα. Temporal patterns of activation between different types of estrogens were evident from our studies, and demonstrate why it is important to carefully document

Acknowledgments

This work was supported by NIEHS grant no. 010987. We are grateful for the skilled editing and scientific comments of Dr. David Konkel.

References (52)

  • E Gaetjens et al.

    Synthesis of fluorescein labelled steroid hormone–albumin conjugates for the fluorescent histochemical detection of hormone receptors

    J. Steroid Biochem.

    (1980)
  • M Ushio-Fukai et al.

    Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling

    J. Biol. Chem.

    (2001)
  • H Endoh et al.

    Rapid activation of MAP kinase by estrogen in a bone cell line

    Biochem. Biophys. Res. Commun.

    (1997)
  • Watson CS. Signaling themes shared between peptide and steroid hormones at the plasma membrane. In: Science’s signal...
  • K.J Ho et al.

    Nonnuclear actions of estrogen

    Arterioscler. Thromb. Vasc. Biol.

    (2002)
  • T.H Hamilton

    Control by estrogen of genetic transcription and translation

    Science

    (1968)
  • M.V Castoria G et al.

    Non-transcriptional action of oestradiol and progestin triggers DNA synthesis

    EMBO J.

    (1999)
  • A Nadal et al.

    Rapid insulinotropic effect of 17-β-estradiol via a plasma membrane receptor

    FASEB J.

    (1998)
  • S.M Aronica et al.

    Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription

    Proc. Natl. Acad. Sci. U.S.A.

    (1994)
  • V Prevot et al.

    Estradiol coupling to endothelial nitric oxide stimulates gonadotropin-releasing hormone release from rat median eminence via a membrane receptor

    Endocrine

    (1999)
  • E.J Filardo et al.

    Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF

    Mol. Endocrinol.

    (2000)
  • M.M Belcheva et al.

    Diversity of G protein-coupled receptor signaling pathways to ERK/MAP kinase

    Neurosignals

    (2002)
  • A Migliaccio et al.

    Tyrosine kinase/P21 (RAS)/MAP-kinase pathway activation by estradiol–receptor complex in MCF-7 cells

    EMBO J.

    (1996)
  • T Simoncini et al.

    Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase

    Nature

    (2000)
  • A.M Norfleet et al.

    Estrogen receptor-α detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry

    Endocrine

    (1999)
  • T.C Pappas et al.

    Membrane estrogen receptors identified by multiple antibody and impeded ligand labeling

    FASEB J.

    (1995)
  • Cited by (101)

    • Xenoestrogen interference with nongenomic signaling actions of physiological estrogens in endocrine cancer cells

      2019, Steroids
      Citation Excerpt :

      Also note that GPR30, which has a disputed location (plasma membrane vs. intracellular), is shown in both locations by our analysis. We next examined the phospho-ERK (pERK) response in prostate cancer cells (Fig. 4), as we have previously examined in multiple cell types [19,26,28,30,34,42,43,45,47,61,62]. We compared ERK responses to E2 (100 pM) vs. DES (1µM) in the same early-stage vs. late-stage prostate cancer cell lines.

    View all citing articles on Scopus
    View full text