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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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Fluorescence immunocytochemical detection of membrane estrogen receptor (mER)-α in nonpermeabilized MCF-7 cells. Cells were fixed with 2% pararaformaldehyde/0.1% glutaraldehyde, probed with C-542 carboxyl-terminal estrogen receptor-α antibody, and visualized with a biotinylated secondary antibody–avidin conjugated alkaline phosphatase fluorescent Vector red product. Fluorescence images were photographed using the FITC filter and 100 × magnification. (a) mER-α-enriched (mERhigh) MCF-7 cells; the arrows indicate some of the punctate staining. (b) mER-α-depleted (mERlow) MCF-7 cells have no staining. The bar in panel b represents 10 μm.
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Figure 1: Fluorescence immunocytochemical detection of membrane estrogen receptor (mER)-α in nonpermeabilized MCF-7 cells. Cells were fixed with 2% pararaformaldehyde/0.1% glutaraldehyde, probed with C-542 carboxyl-terminal estrogen receptor-α antibody, and visualized with a biotinylated secondary antibody–avidin conjugated alkaline phosphatase fluorescent Vector red product. Fluorescence images were photographed using the FITC filter and 100 × magnification. (a) mER-α-enriched (mERhigh) MCF-7 cells; the arrows indicate some of the punctate staining. (b) mER-α-depleted (mERlow) MCF-7 cells have no staining. The bar in panel b represents 10 μm.

Mentions: Immunopanning and subsequent FACS successfully separated MCF-7 cells into two populations according to the expression of mER-α observed in immunocytochemistry experiments. Punctate staining can be seen on the surface of unpermeabilized mERhigh cells (Fig. 1a), whereas the majority of mERlow cells did not exhibit this staining (Fig. 1b). Whenever occasional staining was present on cells in the mERlow population, its appearance was similar to that seen on mERhigh cells (data not shown). Secondary antibody staining alone was at levels similar to that shown for the mERlow cells in Fig. 1b (not shown). When permeabilized, both subpopulations of cells exhibited plentiful cytoplasmic and nuclear staining (not shown) at similar levels. Digital deconvolution (in 15 separate cell planes) performed on a grouping of three unpermeabilized mERhigh cells clearly demonstrated punctuate staining all along the periphery of these cells (Fig. 2).


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

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

Fluorescence immunocytochemical detection of membrane estrogen receptor (mER)-α in nonpermeabilized MCF-7 cells. Cells were fixed with 2% pararaformaldehyde/0.1% glutaraldehyde, probed with C-542 carboxyl-terminal estrogen receptor-α antibody, and visualized with a biotinylated secondary antibody–avidin conjugated alkaline phosphatase fluorescent Vector red product. Fluorescence images were photographed using the FITC filter and 100 × magnification. (a) mER-α-enriched (mERhigh) MCF-7 cells; the arrows indicate some of the punctate staining. (b) mER-α-depleted (mERlow) MCF-7 cells have no staining. The bar in panel b represents 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064105&req=5

Figure 1: Fluorescence immunocytochemical detection of membrane estrogen receptor (mER)-α in nonpermeabilized MCF-7 cells. Cells were fixed with 2% pararaformaldehyde/0.1% glutaraldehyde, probed with C-542 carboxyl-terminal estrogen receptor-α antibody, and visualized with a biotinylated secondary antibody–avidin conjugated alkaline phosphatase fluorescent Vector red product. Fluorescence images were photographed using the FITC filter and 100 × magnification. (a) mER-α-enriched (mERhigh) MCF-7 cells; the arrows indicate some of the punctate staining. (b) mER-α-depleted (mERlow) MCF-7 cells have no staining. The bar in panel b represents 10 μm.
Mentions: Immunopanning and subsequent FACS successfully separated MCF-7 cells into two populations according to the expression of mER-α observed in immunocytochemistry experiments. Punctate staining can be seen on the surface of unpermeabilized mERhigh cells (Fig. 1a), whereas the majority of mERlow cells did not exhibit this staining (Fig. 1b). Whenever occasional staining was present on cells in the mERlow population, its appearance was similar to that seen on mERhigh cells (data not shown). Secondary antibody staining alone was at levels similar to that shown for the mERlow cells in Fig. 1b (not shown). When permeabilized, both subpopulations of cells exhibited plentiful cytoplasmic and nuclear staining (not shown) at similar levels. Digital deconvolution (in 15 separate cell planes) performed on a grouping of three unpermeabilized mERhigh cells clearly demonstrated punctuate staining all along the periphery of these cells (Fig. 2).

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