Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses

INTRODUCTION: 17β-estradiol (E(2)) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mER(low)) and high (mER(high)) expression of the membrane form of estrogen receptor (mER)-α, and thus addressed the receptor subform involved in cAMP signaling. METHODS: MCF-7 cells were immunopanned and subsequently separated by fluorescence activated cell sorting into mER(high )(mER-α-enriched) and mER(low )(mER-α-depleted) populations. Unique (compared with previously reported) incubation conditions at 4°C were found to be optimal for demonstrating E(2)-induced cAMP production. Time-dependent and dose-dependent effects of E(2 )on cAMP production were determined for both cell subpopulations. The effects of forskolin, 8-CPT cAMP, protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ 22,536) on E(2)-induced cell proliferation were assessed using the crystal violet assay. RESULTS: We demonstrated a rapid and transient cAMP increase after 1 pmol/l E(2 )stimulation in mER(high )cells; at 4°C these responses were much more reliable and robust than at 37°C (the condition most often used). The loss of cAMP at 37°C was not due to export. 3-Isobutyl-1-methylxanthine (IBMX; 1 mmol/l) only partially preserved cAMP, suggesting that multiple phosphodiesterases modulate its level. The accumulated cAMP was consistently much higher in mER(high )cells than in mER(low )cells, implicating mER-α levels in the process. ICI172,780 blocked the E(2)-induced response and 17α-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E(2 )dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E(2 )concentrations in mER(high )cells. Proliferation of mER(low )cells was stimulated over the whole range of E(2)concentrations, whereas the number of mER(high )cells was greatly decreased at concentrations above 1 nmol/l, suggesting that estrogen over-stimulation can lead to cell death, as has previously been reported, and that mER-α participates. E(2)-mediated activation of adenylyl cyclase and downstream participation of protein kinase A were shown to be involved in these responses. CONCLUSION: Rapid mER-α-mediated nongenomic signaling cascades generate cAMP and downstream signaling events, which contribute to the regulation of breast cancer cell number

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

INTRODUCTION: We examined the participation of a membrane form of estrogen receptor (mER)-α 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-α-enriched (mER(high)) and mER-α-depleted (mER(low)) populations. We then measured the expression levels of mER-α on the surface of these separated cell populations by immunocytochemical analysis and by a quantitative 96-well plate immunoassay that distinguished between mER-α and intracellular ER-α. Western analysis was used to determine colocalized estrogen receptor (ER)-α 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-α antibody staining of the surface of nonpermeabilized mER(high )cells, whereas the majority of mER(low )cells exhibited little or no staining. Western analysis demonstrated that mER(high )cells expressed caveolin-1 and caveolin-2, and that ER-α was contained in the same gradient-separated membrane fractions. The quantitative immunoassay for ER-α detected a significant difference in mER-α levels between mER(high )and mER(low )cells when cells were grown at a sufficiently low cell density, but equivalent levels of total ER-α (membrane plus intracellular receptors). These two separated cell subpopulations also exhibited different kinetics of ERK1/2 activation with 1 pmol/l 17β-estradiol (E(2)), as well as different patterns of E(2 )dose-dependent responsiveness. The maximal kinase activation was achieved after 10 min versus 6 min in mER(high )versus mER(low )cells, respectively. After a decline in the level of phosphorylated ERKs, a reactivation was seen at 60 min in mER(high )cells but not in mER(low )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-α 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 E(2)-induced cell proliferation outcomes in these cell types

Fluorescence immunocytochemical detection of membrane estrogen receptor (mER)-α in nonpermeabilized MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "Membrane estrogen receptor-α levels predict estrogen-induced ERK1/2 activation in MCF-7 cells"</p><p>Breast Cancer Research 2004;7(1):R130-R144.</p><p>Published online 26 Nov 2004</p><p>PMCID:PMC1064105.</p><p>Copyright © 2004 Zivadinovic and Watson, licensee BioMed Central Ltd.</p> 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. mER-α-enriched (mER) MCF-7 cells; the arrows indicate some of the punctate staining. mER-α-depleted (mER) MCF-7 cells have no staining. The bar in panel b represents 10 μm

Digital deconvolution of membrane estrogen receptor (mER)-α fluorescent image in mER-α-enriched (mER) MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "Membrane estrogen receptor-α levels predict estrogen-induced ERK1/2 activation in MCF-7 cells"</p><p>Breast Cancer Research 2004;7(1):R130-R144.</p><p>Published online 26 Nov 2004</p><p>PMCID:PMC1064105.</p><p>Copyright © 2004 Zivadinovic and Watson, licensee BioMed Central Ltd.</p> Fifteen different focal planes starting from the top of the cells and moving toward the bottom are displayed. The sequential slices are from left to right in each row of images. These cells are nonpermeabilized and treated as described in the previous figure legend. The corresponding transmitted light image is displayed in the upper left-hand corner. FITC filter images were captured, pseudo-colored, and processed by digital deconvolution

Quantification of membrane estrogen receptor (mER) and total estrogen receptor (tER) in mER-α-enriched (mER) and mER-α-depleted (mER) MCF-7 cells

<p><b>Copyright information:</b></p><p>Taken from "Membrane estrogen receptor-α levels predict estrogen-induced ERK1/2 activation in MCF-7 cells"</p><p>Breast Cancer Research 2004;7(1):R130-R144.</p><p>Published online 26 Nov 2004</p><p>PMCID:PMC1064105.</p><p>Copyright © 2004 Zivadinovic and Watson, licensee BioMed Central Ltd.</p> Different numbers of cells were fixed in the same manner as described in Fig. 1 and the level of mER was assessed with 8 μg/ml C-542 antibody. Closed and open circles represent mERand mERcells, respectively. The data were approximated with an exponential decay curve and compared by evaluating the difference between the sums of squares of the residuals from each curve with the sum of squares of the residuals for the combined curve using the F-test. The curves are significantly different (= 0.0003). The diamond represents MDA-MB-231 cells and the cross-hatched horizontal bar the level of binding of a mouse IgGisotype control antibody. Level of total ER (intracellular estrogen receptor [iER] plus mER) for mER(closed circles) and mER(open circles) under the same binding conditions except for permeabilization of the cells. Where error bars for standard error are not visible, they are smaller than the size of the symbols. CV, crystal violet; pNp, paranitrophenol

Kinetics of cAMP decrease in MCF-7 cells enriched for membrane estrogen receptor-α (mER) cells

<p><b>Copyright information:</b></p><p>Taken from "Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses"</p><p>Breast Cancer Research 2004;7(1):R101-R112.</p><p>Published online 24 Nov 2004</p><p>PMCID:PMC1064104.</p><p>Copyright © 2004 Zivadinovic et al., licensee BioMed Central Ltd.</p> Cells were treated with 1 pmol/l 17β-estradiol (E) for different time intervals at 37°C in the presence of 1 mmol/l 3-isobutyl-1-methylxanthine (IBMX). The intracellular cAMP (circles) and that in the medium (squares) from the same cells were assessed. cAMP was produced by directly stimulating adenylyl cyclase with 10 μmol/l forskolin for 15 min at 37°C. The decrease in cAMP in the cytosol was tested at 37°C in the absence (open circles) and presence of 1 mmol/l IBMX (closed circles). The entire regression lines were compared by evaluating the differences between the sums of squares of the residuals of individual lines with the sum of squares of the residuals of the combined line using an F-test. The data are presented as means ± standard error. The regression lines were significantly different (= 0.0001)