Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways - PubMed (original) (raw)

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

Nataliya N Bulayeva et al. Steroids. 2004 Mar.

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-alpha (mERalpha). 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.

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Figures

Fig. 1

Optimization conditions for the fixed cell-based ELISA for MAPKs. Values are means ± S.E.; n: the number of wells from a 96-well plate. (A) Signal from control cells (treated with 0.001% acetic acid vehicle) vs. EGF-treated (50 ng/ml, 5 min) cells (n = 29) at different primary pMAP Ab concentrations. pNp/CV stands for the pMAP signal normalized to the CV signal. *: Statistically significant (P < 0.05) vs. nonspecific binding (no. 1 Ab); #: statistically significant (P < 0.05) vs. vehicle-treated cells (n = 8). Background (signal from nonspecific binding) was not subtracted from data in this graph, so that the relative amount of background signal could be viewed. (B) Western blot (inset) and plate assay (n = 16) show time-dependent pMAP kinase changes after EGF treatment. pMAP kinase Ab was used at 1:2000 and 1:400 dilutions for these methods, respectively. C: control for the inset; *: statistically significant (P < 0.05) EGF-treated cells vs. vehicle-treated controls. (C) Plate assay results (n = 30) after PD (10 μM, 20 min pretreatment) action. *: Statistically significant (P < 0.05) from control; #: statistically significant (P < 0.05) from EGF-treated cells.

Fig. 2

Signals from pMAP kinase (n = 8) and tMAP kinase (n = 8) directly correlate with cell density in both control and EGF treatment groups. Values are means ± S.E.; n: the number of wells from a 96-well plate; *: statistically significant (P < 0.05) EGF-treated cells vs. vehicle-treated controls.

Fig. 3

E2 (1 nM) effects on ERK 1/2 phosphorylation. *: Statistical significance (P < 0.05) when compared with ethanol (EtOH, 0.00001%) vehicle-treated controls. Data are represented as % of control level, which was set to 100%. (A) Western analysis (four experiments); each band was scanned to produce a densitometry value and the values presented are means±S.E. “Sum” (solid line) indicates the average between ERK 1 (44) and ERK 2 (42) phosphorylation values. The inset shows a single representative Western blot. C: vehicle-treated control. (B) Plate assay; values are means ± S.E. with 100% set as control; solid line represent E2 action in GH3/B6/F10 cell line (n = 75–90 wells/point taken from six different plates); dash line: D9 (n = 83–90 wells/point taken from three different plates). (C) Plate assay; values are means ± S.E. with 100% set as control (n = 66 wells/point taken from three different plates).

Fig. 4

Effects of different types of inhibitors on basal levels of MAP kinase phosphorylation. Cells were pretreated with 1 μM ICI 182, 870 (ICI) for 40 min, 50 μg/ml Nystatin (Nys) for 40 min, 10 μM BAPTA-AM (B-TA) for 40 min, 10 μM PP2 (PP2) for 20 min, 10 μM Ly 294002 (Ly) for 40 min, 250 nM AG 14 tyrphostin (AG 14) for 20 min, 250 nM nonactive tyrphostin analog AG 9 (AG 9) for 20 min or 0.1% DMSO vehicle (control) for 40 min, followed by plate assay. Values are means ± S.E.; n = 120–140 wells from nine different plates; *: statistical significance (P < 0.05) vs. cells treated with DMSO vehicle alone.

Fig. 5

Effects of different inhibitors on E2-induced MAP kinase activation. (A, B) Cells were pretreated with inhibitors (see Fig. 4 for times, concentrations and abbreviations) and then stimulated with E2 (1 nM) for the times indicated. Values are means ± S.E.; n = 40–90 wells from three to six different plates; *: statistical significance for E2 treatment (P < 0.05) vs. vehicle control; #: statistical significance (P < 0.05) vs. time-specific E2 alone stimulated controls. (C) Cell extracts were probed by immunoblot analysis for ERα levels at different times of ICI treatment (upper panel). Total ERK 42 levels were also monitored (lower panel) to demonstrate that a different protein did not change during this treatment time course. (D) The effect of different times of ICI pretreatment was assessed for effects on the 3 min E2-induced ERK activation. Values are means ± S.E.; n = 60 wells from three different experimentsl; *: statistical significance for E2 treatment (P < 0.05) vs. vehicle control; #: statistical significance for ICI treatments compared to 3 min E2 treatment alone (P < 0.05).

Fig. 6

DES (1 nM) effects on ERK 1/2 phosphorylation. *: Statistical significance (P < 0.05) vs. EtOH (0.00001%) vehicle-treated controls. Data are presented as % of control values, which were set to 100%. (A) Plate assay, means ± S.E.; n = 65–79 wells from six different plates. (B) Western analysis, four experiments, each band scanned to produce a densitometry value; values presented are means ± S.E. “Sum” (solid line) indicates the average between ERK 1 (44) and ERK 2 (42) phosphorylation values. Inset is a representative Western blot where C indicates vehicle-treated control.

Fig. 7

Inhibitors diminish MAP kinase activation by DES at different time points. Cells were pretreated with inhibitors and then stimulated with DES (1 nM). Values are means ± S.E.; n = 39–79 wells from three to six plates; *: statistical significance for DES treatment (P < 0.05) vs. vehicle control; #: statistical significance (P < 0.05) vs. time-specific DES-stimulated values.

Fig. 8

E2–P (1 nM) effects on MAP kinase phosphorylation. *: Statistical significance (P < 0.05) vs. EtOH (0.00001%) vehicle-treated controls. Data are presented as % of control values, which were set to 100%. E2 was administrated at 1 nM (calculated based on the amount of E2 present in the conjugate). (A) Plate assay, means ± S.E.; n = 85–90 wells from six plates. (B) Western analysis, three experiments, each band scanned to produce a densitometry value; values presented are means ± S.E. “Sum” (solid line) indicates the average of ERK 1 (44) and ERK 2 (42) phosphorylation values. The inset shows a Western blot after E2–P treatment at the same time points; C: vehicle-treated control.

Fig. 9

Inhibitors decrease MAP kinase responses to E2–P at different time points. Cells were pretreated with inhibitors and then stimulated with E2–P (1 nM based on E2 present in the conjugate). Values are means ± S.E.; n = 35–90 wells from three to six plates; *: statistical significance for E2–P treatment (P < 0.05) vs. EtOH (0.00001%) vehicle control; #: statistical significance (P < 0.05) vs. time-specific E2–P.

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Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiated signaling pathways - PubMed (original) (raw)