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Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells.

Zivadinovic D, Watson CS - Breast Cancer Res. (2004)

Bottom Line: The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors).These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness.Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA. ddzivadi@utmb.edu

ABSTRACT

Introduction: We examined the participation of a membrane form of estrogen receptor (mER)-alpha in the activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells.

Methods: We immunopanned and subsequently separated MCF-7 cells (using fluorescence-activated cell sorting) into mER-alpha-enriched (mERhigh) and mER-alpha-depleted (mERlow) populations. We then measured the expression levels of mER-alpha on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-alpha and intracellular ER-alpha. Western analysis was used to determine colocalized estrogen receptor (ER)-alpha and caveolins in membrane subfractions. The levels of activated ERK1 and ERK2 were determined using a fixed cell-based enzyme-linked immunosorbent assay developed in our laboratory.

Results: Immunocytochemical studies revealed punctate ER-alpha antibody staining of the surface of nonpermeabilized mERhigh cells, whereas the majority of mERlow cells exhibited little or no staining. Western analysis demonstrated that mERhigh cells expressed caveolin-1 and caveolin-2, and that ER-alpha was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mERhigh versus mERlow cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mERhigh cells but not in mERlow cells. Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

Conclusion: Our results suggest that the levels of mER-alpha play a role in the temporal coordination of phosphorylation/dephosphorylation events for the ERKs in breast cancer cells, and that these signaling differences can be correlated to previously demonstrated differences in E2-induced cell proliferation outcomes in these cell types.

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Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E2), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mERhigh) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.
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Figure 11: Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E2), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mERhigh) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.

Mentions: To characterize the pharmacologic properties of ERK activation, we used mERhigh cells to study the effectiveness of the potent antiestrogen ICI182,780 (1 μmol/l) and the inactive E2 stereoisomer 17α-estradiol (10 nmol/l). We used concentrations of these compounds shown by others to be effective in inhibiting the transcriptional activity of E2. We had also previously shown that a 10 nmol/l 17α-estradiol concentration could elicit another type of nongenomic estrogenic effect in our GH3/B6/F10 pituitary tumor cell model – rapid prolactin release [15]. ICI182,780 alone induced an activation pattern very similar to that seen with E2 but with an earlier first peak (Fig. 11a). A 30 min ICI182,780 preincubation before a subsequent 1 pmol/l E2 challenge did not significantly change the E2 activation pattern, although the first peak again appeared at 6 min (the ICI182,780 pattern) and there was a much more pronounced inactivation at 10 and 20 min (compare Fig. 11b with Figs 7a and 11a). However, simultaneous application of ICI182,780 (1 μmol/l) and E2 (1 pmol/l) blunted the response and altered the kinetics of ERK phosphorylation, shifting the now single weak activation to later times (20–60 min; Fig. 11c). The E2 stereoisomer (17α-estradiol) provoked a slightly delayed and blunted response also, but with some other interesting features (Fig. 11d). A significant dephosphorylation occurred before the major activation peak, a return to baseline phosphorylation levels followed the 20 min activation peak, and no second activation peak occurred at 60 min.


Membrane estrogen receptor-alpha levels predict estrogen-induced ERK1/2 activation in MCF-7 cells.

Zivadinovic D, Watson CS - Breast Cancer Res. (2004)

Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E2), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mERhigh) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1064105&req=5

Figure 11: Effects of ICI182,780 (ICI), ICI + 17β-estradiol (E2), and 17α-estradiol on extracellular signal-regulated kinase (ERK)1/2 activation in membrane estrogen receptor-α-enriched (mERhigh) MCF-7 cells. (a) Time course of ERK1/2 activation by 1 μmol/l ICI. (b) Pretreatment with ICI (1 μmol/l) for 30 min followed by 1 pmol/l E2 stimulation in the continued presence of ICI. (c) ICI (1 μmol/l) and E2 (1 pmol/l) applied simultaneously. (d) Time course of 17α-estradiol (10 nmol/l) activation of ERK1/2. All experiments were repeated at least three times with 24 well replicates/experiment; the averaged values ± standard error are presented. The asterisks indicate significant differences from vehicle controls.
Mentions: To characterize the pharmacologic properties of ERK activation, we used mERhigh cells to study the effectiveness of the potent antiestrogen ICI182,780 (1 μmol/l) and the inactive E2 stereoisomer 17α-estradiol (10 nmol/l). We used concentrations of these compounds shown by others to be effective in inhibiting the transcriptional activity of E2. We had also previously shown that a 10 nmol/l 17α-estradiol concentration could elicit another type of nongenomic estrogenic effect in our GH3/B6/F10 pituitary tumor cell model – rapid prolactin release [15]. ICI182,780 alone induced an activation pattern very similar to that seen with E2 but with an earlier first peak (Fig. 11a). A 30 min ICI182,780 preincubation before a subsequent 1 pmol/l E2 challenge did not significantly change the E2 activation pattern, although the first peak again appeared at 6 min (the ICI182,780 pattern) and there was a much more pronounced inactivation at 10 and 20 min (compare Fig. 11b with Figs 7a and 11a). However, simultaneous application of ICI182,780 (1 μmol/l) and E2 (1 pmol/l) blunted the response and altered the kinetics of ERK phosphorylation, shifting the now single weak activation to later times (20–60 min; Fig. 11c). The E2 stereoisomer (17α-estradiol) provoked a slightly delayed and blunted response also, but with some other interesting features (Fig. 11d). A significant dephosphorylation occurred before the major activation peak, a return to baseline phosphorylation levels followed the 20 min activation peak, and no second activation peak occurred at 60 min.

Bottom Line: The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors).These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness.Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA. ddzivadi@utmb.edu

ABSTRACT

Introduction: We examined the participation of a membrane form of estrogen receptor (mER)-alpha in the activation of mitogen-activated protein kinases (extracellular signal-regulated kinase [ERK]1 and ERK2) related to cell growth responses in MCF-7 cells.

Methods: We immunopanned and subsequently separated MCF-7 cells (using fluorescence-activated cell sorting) into mER-alpha-enriched (mERhigh) and mER-alpha-depleted (mERlow) populations. We then measured the expression levels of mER-alpha on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-alpha and intracellular ER-alpha. Western analysis was used to determine colocalized estrogen receptor (ER)-alpha and caveolins in membrane subfractions. The levels of activated ERK1 and ERK2 were determined using a fixed cell-based enzyme-linked immunosorbent assay developed in our laboratory.

Results: Immunocytochemical studies revealed punctate ER-alpha antibody staining of the surface of nonpermeabilized mERhigh cells, whereas the majority of mERlow cells exhibited little or no staining. Western analysis demonstrated that mERhigh cells expressed caveolin-1 and caveolin-2, and that ER-alpha was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-alpha detected a significant difference in mER-alpha levels between mERhigh and mERlow cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-alpha (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17beta-estradiol (E2), as well as different patterns of E2 dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mERhigh versus mERlow cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mERhigh cells but not in mERlow cells. Both 1A and 2B protein phosphatases participated in dephosphorylation of ERKs, as demonstrated by efficient reversal of ERK1/2 inactivation with okadaic acid and cyclosporin A.

Conclusion: Our results suggest that the levels of mER-alpha play a role in the temporal coordination of phosphorylation/dephosphorylation events for the ERKs in breast cancer cells, and that these signaling differences can be correlated to previously demonstrated differences in E2-induced cell proliferation outcomes in these cell types.

Show MeSH
Related in: MedlinePlus