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

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.

Estradiol activates mast cells via a non-genomic estrogen receptor-α and calcium influx

“…Rather our findings suggest that E 2 's effects on mast cell mediator release are through a membrane-associated (non-genomic) form of ER-α. This type of ER stimulation has been shown to induce rapid cellular responses through G-protein activation, (Bulayeva et al, 2004;Collins and Webb, 1999;Doolan et al, 2000;Improta-Brears et al, 1999;Stefano et al, 1999;Watson et al, 1999) which can include various signaling pathways involving Ca 2+ fluxes, and modulation of mitogen-activated protein kinases and adenyl cyclase (Bulayeva et al, 2004;Collins and Webb, 1999;Doolan and Harvey, 2003;Nadal et al, 2000;Song et al, 2004;Zivadinovic et al, 2005). Indeed, we found that intracellular Ca 2+ was increased within the first 2.5 min after estrogenic stimulation of RBL.…”

Xenoestrogens are potent activators of nongenomic estrogenic responses

“…This could reflect membrane steroid receptors in two different lipid compartments in the plasma membrane (such as rafts vs. invaginated caveolae or rafts vs. non-raft membrane [37;50;51]), but this has not been directly demonstrated. These potent responses (at the pM to nM concentrations range, [40]) contrast dramatically to the μM-mM effective concentrations required for genomic responses, and also used for studies on ERK activation by others [25]. In other studies we also showed that inhibitors of signaling pathways that lead to ERK activation inhibit both E 2 -and xenoestrogen-induced kinase phosphorylation [22].…”

“…First, we directly examined the ability of E 2 and xenoestrogens to raise intracellular Ca ++ levels, as Ca ++ signaling is likely to be involved in other downstream events, including both ERK activation [40] and regulation of secretion of peptide hormones like PRL [43]. E 2 , E 2 -P (E 2 conjugated to peroxidase to impede its entry into cells), and all xenoestrogens caused increased Ca ++ spikes at low concentrations [24;41].…”

“…Our clonal cell line GH3/B6/F10 was selected for its natural (not transfection-driven) expression of high levels of a membrane form of the estrogen receptor-α (mERα). Expression of mERα was correlated with very sensitive responses to E 2 , including those for ERK activation [40], Ca ++ entry [41], and rapid PRL release [41]. We first observed changes in mERα levels detected in the membrane when cells were treated with low concentrations of xenoestrogens just before fixation for immunocytochemistry.…”

“…To estimate ERK phosphorylation quantitatively, we used a cell-based ELISA, which we previously developed and described [40]. Briefly, cells (10 4 cells/well) were plated in 96-well plates (Corning Incorporated, Corning, NY) and withdrawn from serum hormones by incubation in medium containing 1% charcoal-stripped serum for 48 hr before experiments began.…”