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(The FASEB Journal. 2002;16:1917-1927.)
© 2002 FASEB

Regulation of the membrane estrogen receptor-{alpha}: role of cell density, serum, cell passage number, and estradiol

CELESTE H. CAMPBELL, NATALIYA BULAYEVA, DAVID B. BROWN, BAHIRU GAMETCHU* and CHERYL S. WATSON1

Human Biological Chemistry and Genetics Department, University of Texas, Medical Branch, Galveston Texas, USA; and
* Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

1Correspondence: Department of Human Biological Chemistry and Genetics, University of Texas, Medical Branch, Galveston, TX 77555-0645, USA. E-mail: cswatson{at}utmb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We used modified immunocytochemical conditions to quantify a membrane form of estrogen receptor-{alpha} (mER{alpha}) in a rat pituitary tumor cell line, GH3/B6/F10. We studied the regulation of mER{alpha} vs. levels of intracellular ER{alpha} (iER{alpha}) using our 96-well plate immunoassay. The anti-ER{alpha} antibody C542 was used to label the ER{alpha} (via conjugated alkaline phosphatase) in fixed permeabilized (for iER{alpha}) vs. nonpermeabilized cells (for mER{alpha}). Expression of mER{alpha} was highest at low cell densities (<1000 cells/well) and decreased significantly at densities where cellular processes touched, whereas the more abundant iER{alpha} increased with increasing cell density over the same range. Serum starvation for 48 h caused increases in mER{alpha}, whereas iER{alpha} levels showed no significant changes. A large decline in mER{alpha} and iER{alpha} levels with cell passage number was observed. Minutes after nM 17ß-estradiol (E2) treatment, a portion of the cells rounded up and detached from the culture plate, whereas nM cholesterol had no such effect. Although E2 treatment did not change mER{alpha} levels, the antigen was reorganized from a fine particulate to aggregation into asymmetric large granules of staining. That common culturing conditions favor down-regulation of mER{alpha} may explain the relatively few reports of this protein in other experimental systems.—Campbell, C. H., Bulayeva, N., Brown, D. B., Gametchu, B., Watson, C. S. Regulation of the membrane estrogen receptor-{alpha}: role of cell density, serum, cell passage number, and estradiol.


Key Words: estrogens • nongenomic effects of steroids • rapid • nuclear receptors


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEVERAL MEMBERS OF the steroid receptor family have isoforms located intracellularly and in the membrane (reviewed in ref 1 ). Studies of the membrane form of the glucocorticoid (2) , estrogen (3 4 5 6) , and progesterone (7) receptors have shown that the membrane versions can be related to the intracellular form of the receptor. In other systems, however, data have been interpreted to suggest that membrane steroid receptors are unique proteins unrelated to nuclear receptors (1) . Whereas the membrane and intracellular forms may or may not be related, they play different roles in the cell (1 , 8) . Nuclear steroid receptors are involved in synthetic genomic functions that occur over extended periods whereas the proximal actions of membrane steroid receptors have been associated with rapid, nongenomic signaling functions (1 2 3 4 , 7 , 9) .

The membrane estrogen receptor-{alpha} (mER{alpha}) has been identified in multiple systems (10 11 12) and has been correlated with rapid responses to estrogen, including calcium release (13) , prolactin release (9 , 12 , 14 15 16) , MAP kinase activation (6 , 17 18 19) , and cell proliferation (6 , 20) . In some of these studies, immunocytochemical evidence suggests that mER{alpha} and the intracellular estrogen receptor-{alpha} (iER{alpha}) are closely related (4 , 9 , 17) and originate from the same genetic coding sequence (4 5 6) . Our studies have focused on mER{alpha} in the rat pituitary tumor cell line GH3/B6 (3 , 4 , 12 , 15) . Using live- and fixed-cell immunocytochemical techniques, we identified a subclone, GH3/B6/F10 (F10) with high mER{alpha} expression levels (12) . We recently developed a plate assay combining our immunocytochemical methodology with the soluble alkaline phosphatase product para-nitrophenol (pNp) (21) . This assay allows quantification of the amount of mER{alpha} present in this subclone and direct comparison with the amount of iER{alpha} in permeabilized cells (4) .

In earlier studies we demonstrated that the mER{alpha} is a dynamic protein whose expression varies greatly under different experimental conditions (12 , 15) . In this paper, we use our quantitative assay to examine the regulation of the mER{alpha} by such common culture variant factors as cell density, serum starvation, and cell passage number. For a comparison with the mER{alpha}, we examined regulation of the iER{alpha} with respect to these same parameters, using almost identical methodologies. Cellular rapid responses, mER{alpha} levels, and expression patterns, as perturbed by brief treatment with 17ß-estradiol (E2) treatment, were also assessed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Ham’s F-10 medium and phenol red-free DMEM were purchased from GIBCO-BRL (Grand Island, NY). All sera were obtained from Hyclone (Logan, UT). Cytoseal 280 and a 25% stock solution of glutaraldehyde were from Electron Microscopy Services (Ft. Washington, PA). Biotinylated universal anti-mouse/rabbit IgG, VECTASTAIN ABC-AP (avidin:biotinylated enzyme complex with alkaline phosphatase as the detection system), levamisole, Vector Red, and para-nitrophenylphosphate (pNpp) were from Vector Laboratories (Burlingame, CA). The anti-estrogen receptor-{alpha} antibody C542 (raised against residues 582–595 of human ER{alpha}) (22) was provided by Drs. Dean Edwards and Nancy Weigel; this antibody is now commercially available from StressGen (Collegeville, PA). All other reagents were purchased from Sigma (St. Louis, MO). Please note that since the non-ionic detergent Nonidet P-40 is no longer produced, IGEPAL CA-630 is recommended as an acceptable substitute.

Cell culture
GH3/B6/F10 cells were subcloned from GH3/B6 cells (obtained from Dr. Bernard Dufy; Universitie de Bordeaux II, Bordeaux, France) by limited dilution and found to be enriched for mER{alpha} as determined by live cell labeling with the ER{alpha} R3 antibody (12) . Cells used in these studies were from passages 3 through 25. The GH3/B6/F10 cells were routinely maintained in Ham’s F-10 medium supplemented with 12.5% heat-inactivated equine serum, 2.5% defined-supplemented calf-serum, and 1.5% FCS at 37°C in 5% CO2. We have often observed that cells grown in sera-supplemented medium have lower levels of mER{alpha} compared with serum-starved cells. Therefore, in some experiments the cells were transferred to a defined medium consisting of DMEM containing 5 µg/mL insulin, 5 µg/mL transferrin, 5 ng/mL sodium selenite, 0.1% BSA, 20 mM sodium pyruvate, and 25 mM HEPES.

96-well plate assay
Cells were trypsinized and plated at a density of 1000 cells per well for most of these studies; for density experiments, cells were seeded at densities ranging from 100 to 6000 cells per well. For growth on poly-D-lysine-coated plates, 100 µL of 10 µg/mL poly-D-lysine was incubated in each well for 10 min; excess poly-D-lysine was removed and the wells were rinsed twice with 200 µL PBS before cells were plated.

Two days after plating, the sera-supplemented Ham’s F-10 medium was removed and cells were serum-starved for 48 h in the defined medium. For experiments in which the effects of serum starvation were examined, sera-supplemented medium was removed and the cells were cultured in defined medium for 24, 48, 72, and 96 h. The defined medium was removed by turning each plate upside down and flicking it side to side, then up and down to remove all remaining liquid; this method was used for all reagent removal steps. E2 was solubilized in 0.001% ethanol in the defined medium. For the E2 exposure experiments, vehicle or E2 was removed after 1, 3, 15, 30, or 60 min and the cells were washed once with PBS. For cell attachment experiments, cholesterol was prepared similarly to E2 and added to the cells at varying concentrations for 3 or 60 min. Membrane antigens were preserved by fixing cells with 50 µL/well of 2% para-formaldehyde, 0.1% glutaraldehyde (P/G) in PBS, pH 7.4, for 30 min at room temperature. Intracellular antigens were exposed by permeabilizing the cells with the addition of 0.5% Nonidet P-40 and 0.15 M sucrose to the P/G fixative; cells were thus fixed and permeabilized for 30 min. Fixation was followed by two PBS washes (5 min each) delivered to the plate using a Combi Cell Harvester (Skatron Instruments Inc., Leucadia, CA). Free reactive aldehyde groups were reduced using 50 µL/well 13 mM NaBH4, 70 mM NaHPO4 in ddH20 for 15 min at room temperature, followed by two PBS washes (5 min each). Nonspecific antibody binding was blocked with 350 µL/well 0.1% coldwater fish skin gelatin in PBS, at room temperature for 45 min. The primary antibody (C542) was diluted to 1 µg/mL in 0.1% gelatin in PBS and incubated with the cells overnight at 4°C; this primary antibody concentration was chosen because it had been found to be optimal for immunocytochemical use (15) and for saturation of the epitope in the plate assay (21) . The unbound primary antibody solution was then removed and a brief PBS rinse was followed by three PBS washes (5 min each). Cells were incubated for 1 h at room temperature with 50 µL/well of 5 µg biotinylated secondary antibody per milliliter of 0.1% gelatin in PBS. After three PBS washes (5 min each), cells were incubated with 100 µL Vectastain ABC-AP. Nonpermeabilized cells were washed five times with PBS (5 min each) whereas permeabilized cells required four washes with 0.1% Nonidet P-40 in PBS (5 min each), followed by a single 5 min wash in PBS. The Vectastain substrate solution was prepared immediately before use by mixing 500 µL of 100 mM pNpp with 10 mL of 100 mM sodium bicarbonate, then adding 40 µL of 125 mM levamisole to inhibit generation of pNp by endogenous alkaline phosphatase present in our cells (21) ; the final concentrations were 5 mM of pNpp and 0.5 mM of levamisole. Vectastain substrate solution (100 µL) was then added to each well, and the plates were incubated in the dark in a SpectraMax Plus Microplate Spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA) at 37°C. Hydrolysis of pNpp was monitored at 405 nm in the dark for up to 30 min at 37°C, with the caveat that the instrument could not quantitate absorbance (A405) readings of > 2. We used readings taken at 15 min, previously determined to be well within the linear range of the assay (21) .

Determination of cell number, density-based growth patterns, and normalization of receptor levels to cell number
To allow normalization of the pNp A405 readings to cell number in individual wells, the crystal violet (CV) assay (23 24 25) was performed on the same plates to measure cellular protein levels. Comparison of GH3/B6/F10 cell counts via hemocytometer to CV absorbances confirmed a linear relationship between CV absorbances and cell number (C. H. Campbell and C. S. Watson, unpublished observation). The pNp solution was removed and two 5 min washes were performed in ddH2O. Cells were incubated for 30 min at room temperature in 50 µL 0.1% crystal violet (filtered within 2 wk of use to remove crystallized dye). After three brief washes with ddH2O to remove unbound dye, absorbed dye was released with 50 µL 0.2% Triton X-100 during a 30 min incubation at room temperature. The absorbance was then measured at 562 nm (A562). When cells were grown on poly-D-lysine-coated plates, only a portion of the crystal violet dye was solubilized with Triton X-100 (C. H. Campbell, B. Jakubas, and C. S. Watson, unpublished observation), so the dye was released with a 15 min incubation with 10% acetic acid and absorbance was measured at 590 nm (A590) (25) . The pNp A405 readings were divided by the CV A 562 or CV A 590 readings, yielding a value representative of the number of receptors per cell. The relative levels of mER{alpha} and total ER{alpha} in the F10 subclone were determined with this method, and values for the iER{alpha} were obtained by subtracting the mER{alpha} (unpermeabilized) values from the total ER{alpha} (permeabilized) values. The percentages of cells scored in each growth pattern (Fig. 1 c) were determined by counting 16 wells (2 fields each). All controls were performed on separate plates because E2 caused the cells in non-E2-treated wells to detach from the culture plate. The extent of E2’s effect on cell attachment was determined by calculating the percent difference from control. Data points > 2 SD from the average were excluded. Data were compared for significance of differences using SigmaStat 2 (Jandel Scientific, San Rafael, CA). A one-way ANOVA test was followed, where appropriate, by a Tukey test.



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  		Figure 1. Expression of the mER{alpha} and iER{alpha} is dependent on cell density. a) A group of cells with mER{alpha} staining along the edge of the clump. The bar represents 10 µm. b) Cells were plated at the indicated densities and total ER-{alpha} (•), mER{alpha} ({blacktriangledown}), and iER{alpha} ({circ}) were measured at each density. Expression of mER{alpha} decreased significantly at densities of >= 1500 cells/well, P < 0.001. Expression of iER{alpha} at 3000 cells per well was significantly different from expression levels on cells plated at 250-1000 cells per well and >= 4000 cells/well, both P < 0.001, as determined by the ANOVA and Tukey test. Error bars are ± SE. c) Two fields of cells in each well were counted and scored according to their growth pattern. Cells that grew as singlets or doublets, cells that touched other cells, and those that grew in multicell clusters were the three patterns scored. Percentages > 40 are boxed to show where the majority of cell morphologies fall.

Determination of cell viability and plating capacity after E2 treatment
To check the viability and growth potential of E2-detached cells, we plated the cells in 25 cm2 flasks and applied 1 nM E2 for 5, 10, 15, and 30 min. After E2 exposures as indicated, we collected the medium from each flask and pelleted the detached cells (5 min at 500 g). Cells from each group were then resuspended in F10 medium and cell number was measured with a hemocytometer. Viability of the cells was determined by trypan blue exclusion assay, then each group of cells was plated in a separate well of a 6-well plate. After 5 h of incubation, the medium was collected from each well and the number of cells floating (not attached) was calculated with another hemocytometer reading. The percentage of plated cells able to attach to new plate was calculated by comparative hemocytometer readings.

Immunocytochemistry
Coverslips were placed into T-75 flasks and cells were plated in the flasks. After 48 h, the sera-supplemented medium was removed and cells were incubated in defined medium for 72 h. After 72 h, the defined medium was removed and cells were exposed to either vehicle (0.001% ethanol in defined medium) or 10 nM E2 (in vehicle) for 3 or 60 min. At the end of this incubation period, the medium was removed and cells that had come unattached were recovered by cytocentrifugation at 800 rpm for 5 min (low acceleration setting) in a Cytospin 3 cytocentrifuge (Shandon, Pittsburgh, PA).

Cells still adhering to the coverslips, and those recovered via cytocentrifugation, were fixed for 30 min at room temperature with the P/G fixative. After two PBS washes (5 min each), free reactive aldehyde groups were reduced using 13 mM NaBH4, 70 mM NaHPO4 in ddH2O for 15 min at room temperature, followed by two PBS washes (5 min each). Nonspecific antibody binding was blocked with 0.1% coldwater fish skin gelatin in PBS at room temperature for 45 min. C542 was diluted to 1 µg/mL in 0.1% gelatin in PBS and incubated with the cells overnight at 4°C.

After the unbound primary antibody was removed and samples were rinsed twice with PBS, they were incubated with 5 µg biotinylated secondary antibody per milliliter of 0.1% gelatin in PBS. Three PBS washes (5 min each) were followed by incubation in Vectastain ABC-AP for 1 h. Cells were then washed three times with PBS (5 min each). The Vector Red alkaline phosphatase substrate was prepared immediately before use according to the manufacturer’s instructions. To each 10 mL of substrate solution, 40 µL of 125 mM levamisole was added to inhibit endogenous alkaline phosphatase; the final levamisole concentration was 0.5 mM. The Vector Red substrate solution was then added to each coverslip; after a 5 min incubation, the substrate solution was removed and cells were rinsed with ddH2O. Samples were then dehydrated with two changes (30 s each) of 70% ethanol, followed by two changes (30 s each) of 100% ethanol. Coverslips were cleared in three xylene washes (3 min each) and mounted with Cytoseal 280. Slides were examined using a Leitz Wetzlar Orthoplan microscope with a rhodamine filter. Fluorescence images were captured at 200x magnification, with an exposure setting of 1/2, using an Optronics Engineering DEI750 digital image capture system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell density affects expression of mER{alpha} and iER{alpha}
We observed that cells growing in clumps were predominantly labeled for mER{alpha} on cells growing at the outer edge of the clumps (Fig. 1a ). Because this might indicate higher expression on cells, which have less contact with adjacent cells, we examined the effect of cell density on mER{alpha} expression and found that expression of mER{alpha} and iER{alpha} was dependent on cell density (Fig. 1b ). When cells were plated sparsely (<1500 cells/well), mER{alpha} expression was at its highest. At plating densities of >= 1500 cells per well, there was a significant decline (P<0.001) in mER{alpha} expression that occurred when singlet or doublet cells branched out to touch other cells via long, thin processes; this decrease in mER{alpha} levels remained as the cells became confluent. At low cell densities, iER{alpha} was present at levels much lower than mER{alpha}, but as plating densities were increased to 3000 cells per well, iER{alpha} levels became almost sevenfold greater than mER{alpha} levels. Cells plated at very high densities (>=4000 cells/well) exhibited a significant decrease in expression of iER{alpha} as well (P<0.001). Despite the dramatic changes in mER{alpha} and iER{alpha} expression, total ER{alpha} expressed did not change significantly at densities of 250-3000 cells per well.

Cells were scored on the following categories of growth patterns: singlets or doublets (two cells touching extensively, apparently having just divided), cells touching via processes, and cells clustered in multicell groups. When cells were least dense, growing mostly as singlets or doublets, they expressed the highest level of mER{alpha}; when single or doublet cells touched or formed clusters, mER{alpha} expression decreased significantly (P<0.001; Fig. 1b, c ). Immunocytochemistry confirmed that cells growing singly or in small groups (several cells) expressed a great amount of mER{alpha} over the entire cell surface. The outer edges of cell clusters expressed mER{alpha} more abundantly (Fig. 1a ).

Length of serum starvation affects the amount of mER{alpha} expressed
Previous immunocytochemical measurements of mER{alpha} via live cell labeling showed an increase in mER{alpha} levels cultured for some time in defined medium (12 , 15) . We addressed this question quantitatively in F10 cells using our plate assay. Levels of the mER{alpha} and iER{alpha} were compared when cells were grown in serum-replete medium vs. serum-starved conditions for 24, 48, 72, or 96 h. The only significant increase in mER{alpha} was observed after cells were serum-starved for 48 h (P<0.001; Fig. 2 a). A comparison of iER{alpha} expression in cells grown in serum-replete medium vs. those serum-starved showed no significant difference in the amount of iER{alpha} detected at each point (Fig. 2b ). Compared to cells serum-starved for 48 h, however, cells serum-starved for 96 h showed a significant decrease in mER{alpha} and iER{alpha} levels (P<0.001); these decreases may be due to a lack of necessary nutrients or growth factors after 96 h in serum-deprived medium. Though decreases were observed in receptor levels in cells grown in serum-replete medium, those decreases might also be influenced by the much higher cell densities achieved with longer periods in culture.



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  		Figure 2. Serum starvation enhances the amount of mER{alpha} protein expressed. a) Comparison of cells grown in serum-replete vs. serum-starved media reveals that at 48 h, the mER{alpha} signal in serum-starved cells is significantly greater than the signal in cells grown in serum-replete media, P < 0.001, as determined by ANOVA and Tukey tests. Error bars are ± SE. b) Serum starvation does not significantly enhance iER{alpha} expression. Error bars are ± SE.

Increases in cell passage can alter expression of the mER{alpha}
We noticed that F10 cells at high passage numbers appeared to express little to no mER{alpha}. The pNp assay was used to confirm these early qualitative observations. Cells closest in passage number to the initial separation and isolation of the F10 subclone tended to express more mER{alpha} than cells that were subcultured for longer periods (Fig. 3 a). There were some exceptions, perhaps attributable to other, as yet unknown parameters. Comparison of data for mER{alpha}, iER{alpha}, and total ER{alpha} showed that cells assayed for ER{alpha} earlier than passage 11 expressed significantly more of both receptor subtypes than cells at later passages (passage 15 or greater, P<0.001; Fig. 3b ).



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  		Figure 3. The expression of ER-{alpha} decreases with passage number. a) Data from 69 experiments show that as cells are propagated longer in culture, expression of mER{alpha} decreases. b) Expression of both forms of the ER-{alpha} is dependent on passage number. Early (before passage 11) and late passage cells (passage 15 and above) were compared for total ER-{alpha}, iER{alpha}, and mER{alpha} levels. *Significant differences between early and late passage ER-{alpha} levels, P < 0.001, as determined by ANOVA and Tukey tests. Error bars are ± SE.

E2 treatment rapidly alters cell adherence
We previously detected mER{alpha} in the absence of E2 (using defined media), so we sought to examine the effect of acute E2 exposure on mER{alpha} expression in F10 cells. We collected crystal violet data in each plate assay experiment to normalize pNp receptor values to cell number. Comparison of the crystal violet data between E2-treated and untreated cells revealed that E2 treatment affected the number of cells remaining attached to the plate at the end of each experiment. We knew this could not be due to the mechanics of the assay itself, as we have already shown that untreated cells remain well attached throughout the assay (21) . A significant decrease in cell number was observed when cells were treated with 1 nM E2 for 1 h compared with vehicle controls (P<0.001; Fig. 4 a). Exposure times as short as 1–3 min consistently resulted in a 10–13% decrease in cell number. Hour-long exposure to E2 concentrations ranging from 10-7 to 10-15 M induced detachment of 20–30% of the cells from the plates (Fig. 4b ). Further examination revealed that untreated wells located up to several rows away from the E2-treated wells experienced at least a 10% loss of cells, and wells in rows adjacent to E2-treated cells suffered greater losses of up to 35% (Fig. 4c ). Samples from experiments previously believed to have demonstrated no E2 effect were then compared with controls from non-hormone-treated plates from the same preparation. In all cases, these comparisons revealed that when any row of wells on a plate was treated with E2, almost all of the wells experienced some loss of cells. Therefore, we found it necessary to use separate, non-E2-treated plates for the vehicle control conditions as opposed to including vehicle controls on the same plate as the E2 exposed samples. E2 specificity of this occurrence was tested by exposing cells to cholesterol at concentrations from 10-7 to 10-15 M for 3 or 60 min. There was no resultant decrease in cell adhesion (data not shown).



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  		Figure 4. E2 treatment induces cells to detach from the culture plate. a) Exposures to 1 nM E2 of 1–60 min. *Significant difference from control, P < 0.05, as determined by ANOVA and Tukey tests. Error bars are ± SE. b) 1 h. E2 treatments at concentrations from 10-8 to 10-15 cause significant detachment of cells from the culture plate. *Significant difference from control, P < 0.05, as determined by ANOVA and Tukey tests. Error bars are ± SE. c) E2 causes cells in adjacent untreated wells to lift off the plate. *Significant difference from control, P < 0.05, as determined by ANOVA and Tukey tests. Error bars are ± SE. All vehicle controls were performed on a separate plate. The mean CV A562 control value for these experiments was 0.1146 ± 0.0048 (SE).

Cytocentrifugation of medium removed from E2-exposed cells confirmed that cells were lost with E2 treatment; cells recovered via cytocentrifugation are referred to as "detached cells." After 3 min of treatment with 10 nM E2, the detached cells isolated were either single cells or small clusters (Fig. 5 h). After 60 min of 10 nM E2 treatment, however, the majority of the detached cells were grouped in large clusters (Fig. 5j ). Examination of the corresponding (60 min) E2-exposed cells that were still attached revealed many stained cellular "footprints" and left-behind membrane fragments where cells had previously been attached (26) (Fig. 5f ). These cell "tracks" were not observed among cells remaining adherent after a 3 min exposure to E2 (Fig. 5d ).



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  		Figure 5. The effects of E2 and attachment status on cell shape and mER{alpha} staining pattern. Cells were grown on coverslips and treated with E2 or vehicle for 3 or 60 min. Controls performed included exclusion of primary antibody (a) and exclusion of primary and secondary antibodies (b). Remaining left panels show cells treated with vehicle that remained attached after 3 (c) and 60 (e) min and those cells that detached after 3 (g) and 60 (i) min. The panels on the right show cells treated with E2 that remained attached after 3 (d) and 60 (f) min and those that detached after 3 (h) and 60 (j) min. a) Bar represents 10 µm; all panels are at the same magnification.

E2 treatment induces aggregation of the mER{alpha} in a time-dependent manner
The mER{alpha} staining pattern was altered on cells treated with E2, and the degree of alteration was dependent on the length of E2 exposure. After 3 min of E2 treatment, mER{alpha} on attached cells had shifted to one side of the cell surface and the label appeared as large punctate granules interspersed with very fine dots (Fig. 5d ). After 60 min of E2 treatment, the entire surface of the attached cells was labeled evenly with large granules, which appeared in greater number around the periphery of the cells (Fig. 5f ); the staining is especially apparent on blebs extending from the cell surface.

Immunocytochemical staining of detached cells that had been exposed to E2 for 3 and 60 min revealed that the mER{alpha} antigen was present as concentrated aggregates of staining. After 3 min, staining was observed as large granules along the entire periphery of the cell, with granules occasionally confined to one edge of the cell (data not shown) and as fine punctate labeling across the cell surface (Fig. 5h ). After 60 min of hormone treatment, large granules were observed to be evenly distributed across the surface of all cells (Fig. 5j ).

E2 alters cell shape and morphology
Since E2 treatment caused cells to detach from the culture dish, we monitored coincident changes in morphology between adherent and detached cells. The cytoplasm of the non-E2-treated cells was spread out and the cells were found to have long cytoplasmic extensions (Fig. 5 , panels c, e, g, i). E2-treated cells, however, were very round with few cytoplasmic extensions (Fig. 5 , panels d, f, h, j). We observed the cytoplasm of these cells to be a thin rim surrounding the nucleus, with some smaller, peripheral bleb-like extensions of cytoplasm. E2-treated cells that remained attached to the coverslip tended to be flatter than the control cells, causing the nucleus to protrude markedly from the rest of the cell. All of the morphological changes described were present after E2 treatment for 3 and 60 min, but were more pronounced at 60 min.

Attachment status affects mER{alpha} distribution pattern
Comparison of cells still attached after vehicle treatment vs. those that became detached revealed differences in the organization, but not the appearance, of the mER{alpha} label. The majority of mER{alpha} signal on vehicle-treated, attached cells was dispersed as fine dots, distributed irregularly and asymmetrically on the cell surface, especially along the edge of a clump (Fig. 5c, e ). Detached cells of the same treatment group displayed regular staining as fine dots distributed evenly across the cell surface that were most apparent about the cell’s periphery (Fig. 5g, i ). A similar shift from the asymmetric staining seen in attached cells, to even distribution on detached cells, occurred in the E2-treated groups. The detached cells appeared to have lost their orientation, allowing the antigen to be redistributed evenly across the now unpolarized cell surface.

E2-detached cells are viable and can be replated
Most cells that detached from the flask after 5, 10, 15, and 30 min E2 treatment were viable according to their exclusion of trypan blue dye (94%). Most of the detached cells (75–80%) were able to attach to a new substrate after 5 h in fresh medium (see Table 1 ).


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  		Table 1. Viability and subsequent attachment of E2-detached cellsa

E2 treatment does not alter the amount of mER{alpha} expressed
Although immunocytochemical studies revealed that the mER{alpha} staining pattern changed with E2 exposure, it was difficult to assess quantitative changes visually. Because E2 treatment induced cells to detach from the culture plate, poly-D-lysine was used to anchor the cells to the 96-well plates so that we could assay the mER{alpha} content of all treated cells. Cells grown on this substrate did not lift off the plate when treated with 10 nM E2 for either 3 or 60 min (Fig. 6 a). Assay of the mER{alpha} status in these same cells showed that treatment with 10 nM E2 did not alter the amount of mER{alpha} expressed per cell (Fig. 6b ).



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  		Figure 6. Effect of E2 on cell attachment and mER{alpha} expression on cells grown in wells coated with poly-D-lysine. a) Cells grown on poly-D-lysine do not detach with 10 nM E2 treatment. Each bar is the average of 4 experiments, 16 wells per experiment. Error bars are ± SE. Mean CV A562 control values were 0.1107 ± 0.0197 (SE) at 3 min and 0.1214 ± 0.0088 at 60 min. b) Treatment with 10 nM E2 does not alter mER{alpha} expression levels detected with the C542 antibody in anchored cells. All vehicle controls were performed on a separate plate. Each bar is the average of seven experiments, 16 wells per experiment. Error bars are ± SE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Characterization of factors that regulate the mER{alpha} may lead to a new understanding of its role in biological processes. The data presented here demonstrate how cell–cell interactions, serum, and hormonal factors affect ER{alpha} (nuclear and membrane) expression. Past studies have focused on measurements of the intracellular forms of the estrogen receptors and have showed that dynamic expression of the ERs plays a role in development, hormonal cycling in females, behavior, and progression of endocrine cancers. For example, expression of estrogen receptors in specific regions of the brain peaks 7 days after birth in neonatal rats. Soon after weaning, staining patterns for estrogen receptors change to reflect those found in adult rat brains (27) . Levels of estrogen receptors in the pituitary glands of female rats fluctuate with the different stages of the estrous cycle (28) . As breast cancers become more invasive, their growth and metastasis typically become hormone independent; only 40% of these non-E2-responsive cancers remain estrogen receptor positive (29) . Though these data demonstrate that receptor dynamics influence biological functions, the role of the mER{alpha} in these processes is not understood. An important role for the mER{alpha} is suggested by its apparent high turnover rate. Antisense treatment blocks mER{alpha} expression entirely whereas iER{alpha} expression is barely affected during the same time frame (4) . Since mER and iER appear to come from the same gene sequence, differential expression may be due to differences in alternate splicing or translational control, as seen with the glucocorticoid receptor (30 , 31) , or may be the result of post-translational modifications or interacting comodulator proteins (reviewed in ref 32 ). Improved methods of separately quantitating the mER{alpha} and the iER{alpha} will allow us to elucidate the separate or coordinate roles of these steroid receptors in biological processes.

Significant changes in mER{alpha} expression with cell density may indicate that the mER{alpha} plays a role in cell proliferation, particularly when the amount of mER{alpha} plunges to almost undetectable levels as cells begin to make contact with each other. At higher cell densities, the iER{alpha} becomes the predominant form of ER{alpha} and perhaps is better suited to perform the signaling required for gene expression and cell maintenance. This idea is further supported by immunocytochemical data for each receptor subform. Expression of the mER{alpha} varies according to the cells’ position in a cluster. Cells located on the outer edges of a clump of cells are least restricted by cell–cell contacts, express the most mER{alpha}, and are in a position to proliferate and expand the cluster, whereas cells at the center of a clump of cells express less mER{alpha} and rarely, if ever, replicate. In contrast, all of the cells within a cluster express similar amounts of iER{alpha} (21) . Our work shows the importance of determining mER{alpha} and iER{alpha} signals separately instead of combining both signals as total ER{alpha}. If only total ER{alpha} expression were examined at the lowest cell densities (250–3000 cells/well), the dependence of any particular form of ER{alpha} expression on cell density would be missed. Other investigators have correlated the expression of greater levels of estrogen receptors with increasing cell density (33 , 34) , but the focus of those steroid binding studies was on the iER. In those studies of high-affinity binding sites for estrogens, ER{alpha} and ERß were probably detected, as Sertoli and endometrial epithelial cells have since been shown to express ERß in many species (35 36 37) . Although ERß has been found in the GH3 pituitary cell line (38) , we have shown that the GH3/B6/F10 subclone does not express this form of the estrogen receptor (4 , 21) . Our study is the first systematic characterization of cell density vs. the expression of ER{alpha} at densities low enough to encompass changes in cell growth patterns representing the change from individual cells to those that touch one another by cytoplasmic processes and to those sharing significant borders in larger clusters.

When first characterizing mER{alpha} expression in GH3/B6 cells, we reported that cells selected via immunopanning for high mER{alpha} expression showed a decrease in the percentage of cells expressing mER{alpha} by ~80% after 10 wk in culture (16) . Our data for the F10 subclone relating the loss of mER{alpha} with increasing cell passage numbers corroborated these initial findings with cells selected by a different technique and revealed that over the same period, iER{alpha} expression was lowered. The decrease in expression of ER{alpha} subforms may be related, as both originate from the same coding sequence (4 5 6) . Other investigators have shown that ER transcripts decrease and eventually disappear in cultured cells that are repeatedly passaged (39) .

Increased expression of ERs has been reported after in vitro serum starvation (40) and in vivo estrogen deprivation (41) , and we found that serum starvation maximized the amount of mER{alpha} detectable via immunoassay in the GH3/B6/F10 cells. This increase in mER{alpha} levels after serum starvation may be due to removal of the chronic down-regulating influence on mER{alpha} by serum components. Removal of serum, which contains not only steroids but a myriad of other regulators, for 48 to 72 h also optimizes mER{alpha}-mediated responses (12 , 15 , 16 , 42) . This increased mER{alpha} expression may cause the cells to enter a mode of heightened sensitivity to E2, as suggested by our studies showing that serum starvation allows detection of rapid responses to very low E2 concentrations (4) .

Although other rapid effects of E2 on GH3/B6 cells have been documented (9 , 12 , 14 15 16 , 42 , 43) , we were surprised to observe the rapid change of cell shape and adherence properties in response to E2. We previously showed that when F10 cells were serum-starved, their morphology changed from a rounded shape to a flattened, elongated shape (4 , 12) , perhaps due to the removal of steroids and/or other growth factors contained in the serum. With E2 treatment, cells appeared to retract their cytoplasmic extensions and adopt a rounder shape, in some cases leading to detachment from the coverslip; cells that remained on the coverslip were still flattened out. However, reorganization of the cell surface was not limited to changes in cell shape and adherence. Immunocytochemistry showed that before E2 treatment, the mER{alpha} was expressed at moderate levels across the cell surface. E2 exposure caused aggregation of the mER{alpha} into large punctate granules. Others have reported similar mER aggregation with various detection systems that depended on ligand labeling (reviewed in ref 44 ) and, more recently, antibody recognition (45) ; we previously observed similar aggregation of mER{alpha} in live cells treated with the anti-ER{alpha} antibody R4 (46) , an antibody that also elicits an mER{alpha}-triggered cellular response (15) . Aggregation of mER{alpha} could be a consequence of partitioning into membrane rafts that become caveolae, as these organelles sequester a variety of signaling molecules and have been shown to harbor membrane estrogen receptors (47 , 48) .

Loss of cells in the control wells (untreated or vehicle-treated) of a plate containing E2-treated cells was a surprising demonstration of the great sensitivity of ER-mediated responses in serum-starved cells. Even femtomolar concentrations of E2 caused the GH3/B6/F10 cells to lift off the plate in nearby vehicle-treated wells. Though this occurrence alerted us to the changes in morphology and adhesion triggered by E2, it also left some experiments without control samples for comparison until we examined duplicate plates that had been used for other experiments not involving E2. E2 may be volatilized and recondensed into other wells at 37°C, as has been shown for other estrogenic compounds (49 , 50) , warranting administration of vehicle and steroid test compounds in separate plates. Further characterization of the effects of E2 on membrane and intracellular ER{alpha} expression in the F10 cells required that cells be prevented from lifting off the plates by coating the wells with an artificial matrix of poly-D-lysine before the cells were plated. Other investigators have reported that after chronic E2 treatment, whole cell or intracellular ER expression increases (28) , decreases, or remains the same (51 , 52) ; some of those studies measured {alpha} and ß forms of the ER due to the detection methods used. Our studies found no net change in mER{alpha} levels after acute E2 exposure. Factors in media (such as those provided by serum) may be required for regulation of mER{alpha} protein levels; alternatively, a longer time frame may be required for this type of regulation, such as was seen in our studies demonstrating elevation of mER{alpha} after serum removal. Although it has been postulated that the mER{alpha} is internalized in response to E2, our data do not demonstrate acute relocalization of the mER{alpha} away from the cell surface. However, our use of the C542 carboxyl-terminal antibody for ER{alpha} detection could not rule out the possibility that the receptor could be cleaved during activation (53) , potentially leaving only the carboxyl-terminal fragment in the membrane as a result.

The changes in cell shape, cell adherence, and mER{alpha} distribution induced by E2 treatment are probably related to each other and to distinct biological functions. Others have reported changes in cell morphology after exposure to nanomolar amounts of E2. These alterations include microtubule shortening and the rapid emergence and disappearance of microvilli (54) , an increase in the number of processes (55) , and expansion and retraction at the cell’s periphery accompanied by ruffling of the leading edge (56) . Heinrich et al. reported that GH3 cells adopted a rounder shape and detached in response to E2 via an estrogen receptor signaling pathway that caused decreased N-cadherin protein on the cell surface (57) . The ER antagonist ICI 182,780 blocked these E2-induced changes and increased N-cadherin protein on the cell surface, causing the cells to spread out and adhere tightly to each other. Activation of the mER{alpha} may be involved in this response. Several studies have linked activation of mER{alpha} to cell proliferation (6 , 19) and suggested roles for estrogen receptors in cell migration (26) . Certainly the cells released by E2 treatment in our studies remained viable and able to reattach to a new substrate. Cells in the G2 or M phases of the cell cycle often "round up" when preparing to divide and then become flatter when going through the G1 and S phases of the cell cycle (58) . Our progressive partial detachment response (only 10–15% of cells at 3 min, but 25% of cells after 1 h) may indicate that cells at different stages of the cell cycle were differentially responsive to this effect of estrogens. When cells are migrating, there is usually intermittent detachment from the substrate [retraction of cellular extensions in one direction, leaving bits of cytoplasm and membrane behind (26) , whereas the cell expands its pseudopodia in a different direction (59) ]. The surface changes we observed in F10 cells may indicate that activation of the mER{alpha} by E2 is causing the cells to divide and/or migrate.

In summary, the development of a quantitative plate assay for membrane and intracellular ER{alpha} (21) has allowed us to explore the regulatory control of these receptor subforms using techniques, which are sufficiently similar to allow direct comparisons. Use of this assay confirmed and further explained some aspects of the extremely dynamic expression of the mER{alpha}. The inability of others to detect the mER{alpha} in the past may have been due to this dynamism of its expression. Careful attention to the regulatory changes in mER{alpha} described here may improve the success of membrane steroid receptor detection, allowing further elucidation of the role of mER{alpha} in E2-mediated functions and potential new approaches to their modulation. Further study of why the expression of both ER{alpha} subtypes are turned off with extended culturing may lead to a better understanding of the mechanisms contributing to the loss of specific steroid receptor expression and the accompanying loss of hormonal control over the proliferation of some aggressive cancers.


   ACKNOWLEDGMENTS
 
Supported by NICHD 32481. C.H.C. was supported by the Zelda Zinn Casper Fellowship Fund, the NIEHS Toxicology Training Grant 5232ES07254, and the UTMB Centennial Center for Environmental Toxicology. The authors wish to thank Dr. Darrell Carney for use of his plate reader and microscope and Dr. David Konkel for critical review of the manuscript. Some data contained in this manuscript were presented as a poster at the Endocrine Society Annual Meeting in Denver, June 2001.

Received for publication February 26, 2002. Accepted for publication August 9, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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