Involvement of estrogen receptors alpha  and beta  in the regulation of cervical permeability

George I. Gorodeski1,2 and Dipika Pal1

Departments of 1 Reproductive Biology, and 2 Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen increases the permeability of cultured human cervical epithelia (Gorodeski, GI. Am J Physiol Cell Physiol 275: C888-C899, 1998), and the effect is blocked by the estrogen receptor modulators ICI-182780 and tamoxifen. The objective of the study was to determine involvement of estrogen receptor(s) in mediating the effects on permeability. In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites, in a specific and saturable manner. Scatchard analysis revealed a single class of binding sites with a dissociation constant of 1.3 nM and binding activity of ~0.5 pmol/mg DNA. Estradiol increased the density of estrogen-binding sites in a time- and dose-related manner (half time approx  4 h, and EC50 approx  1 nM). RT-PCR assays revealed the expression of mRNA for the estrogen receptor alpha  (alpha ER) and estrogen receptor beta  (beta ER). Removal of estrogen from the culture medium decreased and treatment with estrogen increased the expression of alpha ER and beta ER mRNA. In cells not treated with estrogen, ICI-182780 and tamoxifen increased beta ER mRNA. In cells treated with estrogen, neither ICI-182780 nor tamoxifen had modulated significantly the increase in alpha ER or beta ER mRNA. The transcription inhibitor actinomycin D blocked the estrogen-induced increase in permeability, and it abrogated the estradiol-induced increase in estrogen binding sites. These results suggest that the estrogen-dependent increase in cervical permeability is mediated by an alpha ER-dependent increase in transcription.

human; cervical cells; epithelium; transepithelial transport; cervical mucus; transcription; ICI-182780; tamoxifen


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CERVICAL MUCUS IS A MIXTURE of mucins and cervical plasma that are produced continuously throughout the woman's life but change in quantity and composition during different phases of life (8). The main function of the cervical mucus is to lubricate the lower genital canal and to prevent entry of microorganisms and cells into the uterus. During reproductive years changes in cervical mucus in the preovulatory phase allow for sperm penetration into the cervix and for sperm capacitation and migration (22). Abnormal secretion of cervical mucus may lead to infertility and to states of disease such as mucorrhea and dryness dyspareunia (8).

Estrogens increase cervical secretions in the woman (8). Estrogen regulation of mucin secretion is relatively well understood (8); in contrast, less is known about estrogen regulation of cervical plasma. The cervical plasma comprises 80%-99% of the total weight of the cervical mucus and is believed to originate by transudation of fluid and solutes from the blood into the cervical canal through the paracellular pathway (8). We have developed novel methods to culture human cervical epithelial cells on filters (16) and have recently reported that estrogen increases the paracellular permeability of cultured cervical epithelia (10). The proposed mechanism for the increase in permeability is enhanced cell deformability: estrogen shifts actin steady state toward G-actin and produces a more flexible cytoskeleton (10). This renders cells more sensitive to stimuli, such as hydrostatic gradients, and subsequently results in a decrease in size (9). A decrease in cell size is accompanied by parallel, though reciprocal changes in the size of the intercellular space (27). As a result, the paracellular permeability increases, allowing greater flow of fluid and solutes across the cervical epithelium via the intercellular space, and an increase in mucus production.

The signaling mechanism by which estrogen increases cervical permeability is unknown. Estrogens can affect target cells by genomic and nongenomic mechanisms (24). Genomic mechanisms usually involve activation of the nuclear estrogen receptor, followed by transcription regulation and upregulation of protein synthesis (2, 24). We (13) and others (7, 19, 26) have previously shown that human cervical epithelial cells express nuclear receptors for estrogen; these data suggest that the effect of estrogen on cervical permeability involves the estrogen receptor. The objective in the present paper was to study the degree to which estrogen regulation of cervical permeability involves the estrogen receptor.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Collection of endocervical and ectocervical tissues. Endocervical and ectocervical tissues were obtained from uteri of premenopausal women who underwent hysterectomy for indications unrelated to the study and who had histologically normal cervix. After removal of the uterus, the cervical tissues were washed, minced and transferred to the lab in ice-cold saline.

Cell cultures. Three types of cell cultures were used: 1) human ectocervical epithelial cells (hECE), which are a model of the stratified ectocervical epithelium (16), were obtained from minces of normal human ectocervix and used in third passage (16); 2) ECE16/1 are a stable line of immortalized hECE cells with the human papilloma virus 16 (see Ref. 13; kindly provided by Dr. R. L. Eckert, Department of Physiology and Biophysics at Case Western Reserve University (CWRU) School of Medicine, Cleveland OH) and are a model of the squamous metaplastic epithelium (16); 3) CaSki cells are a stable line of transformed cervical epithelial cells that express phenotypic markers of the endocervix (16). Cells were grown and maintained in culture dish at 37°C in a 91% O2-9% CO2 humidified incubator. For electrophysiological experiments cells were plated on filters as we have described (15). Cells were routinely tested for mycoplasma. In most experiments cells were shifted to steroid-free medium (SFM) for 3-5 days (10). Before experiments, filters containing cells were washed three times and preincubated for 15 min at 37°C in a modified Ringer buffer (10). For treatments, all agents were added from stock (1,000×) solutions. For experiments on filters, agents were added to both the luminal and subluminal solutions.

Changes in paracellular permeability. Changes in paracellular permeability were determined in terms of changes in the transepithelial electrical conductance (GTE); the method, including conditions for optimal determinations of GTE across low-resistance epithelia, calibrations and controls, potential pitfalls, and the appropriate measures to prevent artifacts was previously discussed by us (14, 15) and by others (27). Briefly, changes in GTE were determined continuously across filters mounted vertically in a modified Ussing chamber (15) from successive measurements of the transepithelial electrical current (I; obtained by measuring the current necessary to clamp the offset potential to zero, and normalized to the 0.6-cm2 surface area of the filter) and of the transepithelial potential difference (PD; lumen negative) and switching between I (pulses of 200-1,400 µA/cm2) and PD at a rate of 20 Hz: GTE = I/PD.

17beta -[3H]estradiol binding assay. Scatchard analysis was performed on total cell extracts using 17beta -[3H]estradiol as the tracer and hydroxylapatite (HAP) to separate bound and free ligand (13). Before extraction and preparation of lysates, viability was determined using trypan blue exclusion and was generally >95%. Preconfluent cultures of hECE, ECE16/1, and CaSki cells were harvested, resuspended in iced phosphate buffer (5 mM sodium phosphate, pH 7.4 at 4°C, 10 mM thioglycerol, 10 mM sodium molybdate, 10% glycerol), homogenized in a Dounce homogenizer (B-pestle; Kontes, Vineland, NJ), and extracted for 60 min at 4°C by the addition of 3 vol high-salt buffer (10 mM Tris · HCl, pH 8.5 at 4°C, 1.5 mM EDTA, 10 mM thioglycerol, 10% glycerol, 10 mM sodium molybdate, 0.8 M KCl). The extracts were then centrifuged at 180,000 g for 30 min at 4°C, and the supernatant (total cell extract) was diluted with 3 vol iced phosphate buffer and used immediately. To assess total and nonspecific binding, 300 µl of extract were incubated for 4 h at 30°C with 17beta -[3H]estradiol at concentrations ranging from 1 × 10-11 to 6 × 10-8 M in the presence or absence of a 100-fold excess of diethylstilbestrol (DES). These conditions (4 h at 30°C) measure total (empty plus filled) receptor sites (13). The tubes were boiled to 4°C, and an aliquot was removed and counted to accurately determine the final 17beta -[3H]estradiol concentration. The remaining sample was treated with HAP for 30 min at 4°C to adsorb the hormone-receptor complex, the HAP was washed four times with phosphate buffer to remove residual free 17beta -[3H]estradiol, and the HAP pellet was counted in scintillation fluid (13). Specific binding was determined by subtracting nonspecific from total binding at each concentration. The results were analyzed by a Scatchard plot (13). In some experiments incubations were included at 0-4°C for 20 h to determine the fraction of filled and empty sites (13). These values agreed with the values obtained at 30°C, indicating that essentially all of the receptor sites were empty. Specific receptor binding was expressed per milligram DNA (11).

Isolation of RNA. Tissues (endocervix and ectocervix) were pulverized and then homogenized by tissue-tearor using the Qiagen kit (Qiagen, Chatsworth, CA) with lysis buffer plus beta -mercaptoethanol (beta ME) at 350 µl/30 mg. Total RNA from cultured cells was isolated with the Qiagen kit, using lysis buffer plus beta ME at 350 µl/107 cells. The final total RNA pellets were resuspended in 50 µl diethyl pyrocarbonate (DEPC)-water and quantitated by measuring optical density at 260 nm as we have described (12).

RT-PCR. A Perkin Elmer DNA thermal cycler (Cetus, Norwalk CT) was utilized for the assays using a RT-PCR kit (Boehringer Mannaheim, Indianapolis, IN). Total RNA (1.5 µg), denatured at 65°C for 5 min was reverse transcribed in a final volume of 20 µl of reaction mixture containing 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2, 1 mM dNTPs, 5 µM oligo(dT)15 (Promega, Madison, WI), 40 units RNase inhibitor, and 25 units avian myeloblastosis virus (AMV) RT (Boehringer Mannheim, Germany). For mock reaction a similar tube was added without the oligo(dT) and without the AMV RT. The reaction was allowed to proceed at 42°C for 60 min and was terminated by heating to 99°C for 2 min. The sample was diluted 1:5 with double distilled H2O. PCR was then performed in a 50-µl volume using 5 µl of the diluted sample, 5× PCR buffer (Amersham, Arlington Heights, IL), 1 µM of each primer, 0.01 mM dNTPs, 5 units Taq polymerase (Amersham), and 1.4 µM Taq antibody (Clonetech, Palo Alto, CA) in a Perkin Elmer 460 DNA thermal cycler. Samples were heated for 5 min at 94°C, and then the following conditions were applied: estrogen receptor alpha  (alpha ER): 35 cycles of 1-min denaturation step at 94°C, 1 min of annealing step at 62°C, and 2 min of extension step at 72°C; estrogen receptor beta  (beta ER): 30 cycles of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C; glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 30 cycles of 1 min at 94°C, 1 min at 60°C, 1 min at 72°C; beta -actin: 30 cycles of 1 min at 94°C, 1 min at 62°C, and 1 min at 72°C. Samples were then kept for 7 min at 72°C, cooled at 4°C (soak file), and frozen at -80°C to facilitate removal of the mineral oil. The following oligonucleotide primers were used: human estrogen receptor alpha  (17) forward (sense) 5'-CAG GGG TGA AGT GGG GTC TGC TG -3'; reverse (antisense) 5'-ATG CGG AAC CGA GAT GAT GTA GC -3'; human estrogen receptor beta  (20) forward (sense) 5'-TGC TTT GGT TTG GGT GAT TGC-3'; reverse (antisense) 5'-TTT GCT TTT ACT GTC CTC TGC-3'; human GAPDH (5) forward (sense) 5'-TGA AGG TCG GAC TCA ACG GAT TTG GT-3', reverse (antisense) 5'-GTG GTG GAC CTC ATG GCC CAC ATG-3'; human beta -actin (GenBank AI 527892) forward (sense) 5'-GTT GCT ATC CAG GCT GTG CT-3', reverse (antisense) 5'-GGC CAT CTC TTG CTC GAA GT-3'. The sequences used to amplify the primers were synthesized by the Molecular Biology Core Laboratory at CWRU School of Medicine and were prepared as 10 µM stocks. Amplified samples (20 µl) were analyzed on 1.5% agarose gel, stained with ethidium bromide, and photographed. Parallel experiments were routinely done using DNase I before RT, to exclude amplification of genomic cDNA contaminants. The DNA molecular weight markers were from Hinc II digests of Phi X174 DNA (United States Biochemical, Cleveland, OH), or 1-kb ladder (GIBCO, Grand Island, NY).

To validate that the RT-PCR can yield semiquantitative estimates of changes in mRNA, the following steps were taken. 1) The cDNAs of the alpha ER, beta ER, beta -actin, and the GAPDH were amplified in parallel tubes, and changes in alpha ER and beta ER RNA were determined relative to the changes in GAPDH RNA. 2) Experiments using DNase I before the RT-PCR were routinely run, to control for amplification of genomic cDNA. 3) In control experiments GAPDH was amplified using different doses of the GAPDH cDNA to determine the semiquantitative changes in the amplification rates, as we (12) and others (34) have described.

Densitometry. The X-ray films were analyzed with a laser densitometer Sciscan 5000 (United States Biochemical, Cleveland, OH) and normalized relative to GAPDH RNA.

Statistical analysis of the data. Data are presented as means ± SD, and significance of differences among means was estimated by ANOVA. Trends were calculated using GB-STAT V5.3 (Dynamic Microsystems, Silver Spring, MD) and analyzed with ANOVA. Best fit of regression equations (least-squares criterion) was achieved with SlideWrite Plus (Advanced Graphics Software, Carlsbad, CA), which uses the Levenberg-Marquardt Algorithm, and analyzed using ANOVA.

Chemicals and supplies. Anocell (Anocell-10) filters were obtained from Anotec (Oxon, UK). Fluorescent microspheres (FluoresBrite beads, calibration grade) were obtained from Polysciences (Warrington, PA). ICI-182780 was a gift from Dr. Alan Wakeling (31). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Estrogen increases cervical permeability. In cultured epithelia of hECE, ECE16/1, and CaSki cells that were grown in SFM baseline levels of GTE ranged from 30 to 80 mS/cm2 (Fig. 1, ~12-30 Omega  · cm2). These levels are similar to those previously reported by us (10) and indicate that human cervical epithelial cells form a relatively permeable epithelium on filters (27). Treatment with 10 nM 17beta -estradiol increased GTE two- to threefold (Fig. 1). This effect confirms our previous results (10), indicating that treatment with physiological concentrations of 17beta -estradiol increases the permeability across cultured human cervical epithelia.


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Fig. 1.   Means ± SD of transepithelial electrical conductance (GTE) across human cervical epithelial cells on filters. Cells were plated on filters and after 2 days were shifted to steroid-free medium (SFM) for an additional 2-3 days in the presence of 10 nM 17beta -estradiol (E) or the vehicle (control, C). One day before the experiment, 10 µM tamoxifen (TMX) or 10 µM ICI-182780 (ICI) was added to some filters. There were 3-4 filters in each treatment group. * P < 0.01 compared with C.

Treatment of hECE, ECE16/1, and CaSki cells grown in SFM with the estrogen-receptor modulators tamoxifen or ICI-182780 had no significant effect on GTE (Fig. 1). In contrast, tamoxifen and ICI-182780 blocked the estrogen-induced increase in GTE (Fig. 1). These results indicate that tamoxifen and ICI-182780 block the estrogen-induced increase in paracellular permeability.

Human cervical cells express receptors for estrogen. Our main objective in this paper was to understand the signaling cascade involved in the estrogen regulation of transcervical permeability. In the first experiment we determined the binding characteristics of 17beta -[3H]estradiol to lysates of hECE, ECE16/1, and CaSki cells. Cells on filters were treated with 10 nM 17beta -estradiol for 2 days, and binding of 17beta -[3H]estradiol to total extracts of the cells was assayed as we (11) and others (33) have described. Estradiol binding was saturable, and Scatchard analysis revealed a single class of binding sites with Kd of 1.6 ± 0.2 (SD) nM for hECE cells, 1.1 ± 0.3 nM for ECE16/1 cells, and 1.2 ± 0.2 nM for CaSki cells (Fig. 2A). The binding activity ranged from 1.1 ± 0.2 (SD) pmol/mg DNA for hECE cells, 0.4 ± 0.1 pmol/mg DNA for ECE16/1 cells, and 0.3 ± 0.2 pmol/mg DNA for CaSki cells (Fig. 2A). Based on determinations of DNA per cell, these levels correspond to 1 × 10-7, 0.5 × 10-7, and 0.5 × 10-7 mg DNA/cell, respectively, for hECE, ECE16/1, and CaSki cells. These levels are similar to values reported in the cervix in vivo (7, 11, 19, 26). Competition binding assays I extracts of hECE cells revealed the following competition profile: 17beta -estradiol = diethylstilbestrol >> estriol = ICI-182780 = tamoxifen >> hydrocortisone = progesterone = testosterone (Fig. 2B). These results indicate that estradiol binds in human cervical epithelial cells to high-affinity, low-capacity sites, in a specific and saturable manner.


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Fig. 2.   A: Scatchard plot of 17beta -[3H]estradiol binding to total extracts of hECE, ECE16/1, and CaSki cells; shown is 1 example of 3 experiments. Cells were treated with 10 nM 17beta -estradiol for 2 days, and binding of 17beta -[3H]estradiol to total extracts of the cells was assayed as is described in METHODS. The data were fitted into straight lines (P < 0.01 in each case), and the means of Kd and binding activities for each cell type are given in RESULTS. B/F, bound-to-free ratio. B: competition binding assays: hECE cells were treated with 10 nM 17beta -estradiol for 2 days, and binding of 25 nM 17beta -[3H]estradiol (E2) to total extracts of the cells was assayed in the absence or presence of 1 µM of 1 of the following agents: diethylstilbestrol (DES), estriol (E3), tamoxifen (TMX), ICI-182780 (ICI), hydrocortisone (HC), progesterone (P), and testosterone (T). Nonspecific binding was determined by incubating cells with 1 µM 17beta -estradiol. Means ± SD of 3-4 experiments in each group were normalized to E2 binding. The following trends were significant: (E2 = DES) >> (E3 = ICI = TMX) >> (HC = P = T) (P < 0.01).

In the second experiment we studied the expression of mRNA for alpha ER and beta ER using the RT-PCR technique. Experiments were done on extracts of minces of human endocervical and ectocervical tissues, as well as on lysates of cultured hECE, ECE16/1, and CaSki cells. Using oligonucleotide primers complementary to cloned human alpha ER (17) and beta ER (20), single cDNA fragments of 483 and 283 bp, respectively, were amplified by RT-PCR from human endocervical and ectocervical homogenates and from lysates of the cultures of cervical cells (Fig. 3). These cDNA fragments were isolated, amplified, and purified, and the products were sequenced by the dideoxy chain termination method. Sequence analysis of the cloned segments revealed homologies of 98% (sense and antisense) with the human alpha ER and beta ER (the differences were sequence errors; not shown). These results indicate that human cervical epithelial cells express mRNA for alpha ER and beta ER.


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Fig. 3.   Human cervical cells express mRNA for estrogen receptor alpha  (alpha ER) and estrogen receptor beta  (beta ER). Experiments utilized the RT-PCR technique and were done on extracts of endocervical (EndoCx) and ectocervical (EctoCx) minces or on lysates of cultured CaSki, ECE16/1, and hECE cells. Oligonucleotide primers complementary to the cloned alpha ER and beta ER were used to amplify single cDNA fragments of 483 and 283 bp, respectively. M, molecular size markers. The experiment was repeated twice.

Estrogen increases estradiol binding sites. Treatment of human cervical epithelial cells with 17beta -estradiol increased the density of binding sites for 17beta -[3H]estradiol in a time- and dose-related manner (Fig. 4, A and B). In hECE cells the effect of estradiol required at least 1-3 h of treatment with the hormone and was maximal already after 6 h (Fig. 4A). It was previously shown that 17beta -estradiol increases the permeability across cultured human cervical epithelia in a time-related manner: effects began after 1 h of treatment and reached a plateau after 6-9 h (10). Our present and previous results therefore indicate that, in cultured human cervical epithelia, the estradiol-induced increase in 17beta -estradiol binding sites precedes the increase in permeability.


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Fig. 4.   Estrogen increases estradiol binding sites in human cervical epithelial cells. A: hECE cells grown in SFM were treated with 10 nM 17beta -estradiol for periods of 1-48 h. B: hECE cells grown in SFM were treated with 17beta -estradiol at concentrations ranging from 0.1 to 100 nM for 14 h. C: hECE, ECE16/1 or CaSki cells grown in SFM were treated with 10 nM 17beta -estradiol (Estrogen) or vehicle (Control) for 16 h. At the completion of treatments, binding of 17beta -[3H]estradiol (25 nM) to total extracts of cells was assayed as in Fig. 2. Shown are means ± SD of 3-4 experiments in each group. In C, levels in the estrogen group for all 3 cell types were higher than in the control group (P < 0.03-0.01).

The increase in estradiol binding sites began already with 0.1 nM of 17beta -estradiol and reached saturation at 10 nM, with an EC50 of estradiol of ~1 nM (Fig. 4B). As is summarized in Fig. 4C, in hECE, ECE16/1, and CaSki cells treatment with 10 nM 17beta -estradiol for 6 h increased the density of 17beta -[3H]estradiol binding sites three- to fourfold. 17beta -Estradiol also increases the permeability across cultured human cervical epithelia in a dose-related manner: we have previously shown that effects begin with 0.1 nM of the hormone and reach saturation at 10 nM, with an EC50 of estradiol of ~1 nM (10). Our present and previous results therefore indicate that, in cultured human cervical epithelia, estradiol exerts similar potency for increasing the permeability and for upregulation of 17beta -estradiol binding sites.

Estrogen increases alpha ER and beta ER mRNA. We also determined the effect of estrogen on the expression of alpha ER and beta ER mRNA in ECE16/1 and CaSki cells. Cells were shifted to SFM and treated with 10 nM 17beta -estradiol or the vehicle for 2-3 days. In parallel experiments the effect of estrogen on cells maintained in regular medium was also determined, and a representative experiment is shown in Fig. 5. Incubation in SFM had no effect on beta -actin mRNA or on GAPDH mRNA, but it decreased alpha ER and beta ER mRNA. Densitometry results of three experiments in ECE16/1 cells and three experiments in CaSki cells revealed that incubation in SFM decreased alpha ER mRNA relative to GAPDH mRNA threefold and beta ER/GAPDH mRNA fivefold (Table 1). Treatment of cells that were maintained in regular culture medium with 10 nM 17beta -estradiol increased alpha ER/GAPDH mRNA 5-fold and beta ER/GAPDH mRNA 12-fold (Fig. 5 and Table 1). Treatment of cells grown in SFM with 10 nM 17beta -estradiol increased alpha ER/GAPDH mRNA 17-fold and beta ER/GAPDH mRNA 78-fold (Fig. 5 and Table 1).


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Fig. 5.   Effects of SFM and estrogen on the expression of mRNA for alpha ER, beta ER, beta -actin, and GAPDH in ECE16/1 cells. Cells were grown either in regular culture medium (RM) or in SFM and treated with 10 nM 17beta -estradiol (+E) or the vehicle for 2-3 days. Experiments were repeated 3 times. In mock reactions [lacking the oligo(dT) and the avian myeloblastosis virus (see METHODS)], no detectable bands were found (not shown).


                              
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Table 1.   Effects of estradiol, steroid-free medium, ICI-182780, and tamoxifen on changes in the ratios of alpha ER/GAPDH mRNA and beta ER/GAPDH mRNA in ECE16/1 and CaSki cells

Effects of ICI-182780 and tamoxifen on alpha ER and beta ER mRNA levels. To determine the effect of ICI-182780 and tamoxifen on mRNA levels of alpha ER and beta ER, ECE16/1 and CaSki cells were shifted to SFM and treated with 10 µM ICI-182780 or 10 µM tamoxifen for 24 h. The results of an experiment with ECE16/1 cells are shown in Fig. 6; similar results were obtained with CaSki cells (not shown). The results were analyzed by densitometry and are summarized in Table 1. ICI-182780 had no significant effect on alpha ER/GAPDH mRNA, but it increased beta ER/GAPDH mRNA by 47-fold. Tamoxifen increased alpha ER/GAPDH mRNA 3-fold and beta ER/GAPDH mRNA 53-fold (Fig. 6 and Table 1).


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Fig. 6.   Effects of ICI-182780 or tamoxifen on the expression of alpha ER, beta ER, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in ECE16/1 cells. Cells grown in SFM were treated with 10 nM 17beta -estradiol (+E) or vehicle (control, C) for 2-3 days. In some plates cells were also treated with 10 µM ICI-182780 or 10 µM tamoxifen one day before experiments. Experiments were repeated 3 times.

To determine the effect of ICI-182780 and tamoxifen on the expression of alpha ER and beta ER mRNA in estrogen-treated cells, ECE16/1 and CaSki cells were shifted to SFM and treated with 10 nM 17beta -estradiol (or the vehicle) for 2 days. One day before experiments, 10 µM ICI-182780 or 10 µM tamoxifen (or the vehicle) were added. In cells treated with estrogen, neither ICI-182780 nor tamoxifen modulated significantly the effects of estrogen on the expression of alpha ER/GAPDH mRNA or beta ER/GAPDH mRNA (Fig. 6 and Table 1). These results suggest that ICI-182780 and tamoxifen have little effect on the expression of alpha ER mRNA but that both agents increase the expression of beta ER mRNA.

Effects of actinomycin D on permeability and on estradiol binding sites. Estrogens modulate cell functions via several known mechanisms, including transcription regulation (2, 24). The objective of the next experiment was to study the effects of the transcription inhibitor actinomycin D on the estrogen-induced increase in GTE and on the estrogen-induced increase in estradiol binding sites. Cells grown in SFM were treated with 10 nM 17beta -estradiol (or the vehicle) for 2 days, followed by 10 µM actinomycin D for an additional 2.5 h before experiments. This period of time is sufficiently long to enable a measurable effect of estradiol on permeability (10) and on estradiol binding sites (Fig. 4A). In addition, treatment with actinomycin D for 2.5 h is not excessively toxic to cervical cells (14).

Actinomycin D had little effect on baseline conductance, but it blocked the increase in GTE that was induced by estrogen (Fig. 7). Actinomycin D had no significant effect on baseline specific binding of 17beta -[3H]estradiol, but it abrogated the estradiol-induced increase in binding of 17beta -[3H]estradiol (Fig. 8). This was in contrast to a lack of a significant effect of tamoxifen on the estradiol-induced increase in binding of 17beta -[3H]estradiol (Fig. 8). Collectively, these results suggest that an estrogen-receptor-dependent increase in transcription is necessary for the estrogen-dependent increase in cervical permeability.


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Fig. 7.   Effects of actinomycin D (Act-D) on changes in GTE in estrogen-treated CaSki cells. Cells grown in SFM were treated with 10 nM 17beta -estradiol (E) or vehicle (control, C) for 2 days, followed by 10 µM actinomycin D for an additional 2.5 h. Data are means ± SD of 3-4 experiments. * P < 0.01.



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Fig. 8.   Effects of actinomycin D and tamoxifen on changes in 17beta -[3H]estradiol binding in estrogen-treated CaSki cells. Cells were grown in SFM and then treated with 1 or more of the following agents: 10 nM 17beta -estradiol (E) for 2 days; 10 µM tamoxifen, added 1 day before experiments; or 10 µM actinomycin D, added 2.5 h before experiments. Data are means ± SD of 3-4 experiments. * P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study confirms our previous results (10) that estrogen increases the permeability across cultured human cervical epithelia. Our results suggest that the effect is mediated by estrogen receptor(s) and that it involves upregulation of transcription.

Estradiol had no acute effect on permeability (10), and the hormone did not change acutely estradiol binding sites (present study). These results effectively rule out direct activation by estrogen of ion transport mechanism(s) such as Na+-K+-ATPase (6), K+ channels (25), Cl- channels (18), Ca2+ channels (23, 32), and proton transport (30) as mediators of the estrogen increase in permeability. However, the results do not rule out that ion transport mechanisms are activated by estrogen-dependent transcriptional regulation. In contrast, the present results suggest that the effect of estrogen on permeability is mediated by the nuclear estrogen receptor(s). This conclusion is supported by the following experimental findings. 1) In cultured human cervical epithelial cells estradiol binds to high-affinity, low-capacity sites that are specific to estrogens and do not bind other steroid hormones. 2) Estradiol increased both estradiol binding (present study) and permeability (10). 3) The increase in estradiol-binding sites preceded the increase in permeability, suggesting that upregulation of estrogen receptor(s) mediates the increase in permeability. 4) Both effects were dose related: the permeability (10) and the density of estradiol binding sites (present results) could be modulated by concentrations of estradiol that are in the physiological range for women (29). The EC50 of estradiol for both responses was ~1 nM, which corresponds to a Kd of 1 nM for the classical estrogen receptor in the human uterus (24). 5) Actinomycin D blocked both the increase in permeability and the increase in binding of 17beta -[3H]estradiol, suggesting that upregulation of transcription is necessary for the estrogen-dependent increase in cervical permeability. 6) The estrogen receptor modulators ICI-182780 and tamoxifen blocked the estradiol-induced increase in permeability. 7) The effects of ICI-182780 and tamoxifen were not acute and required the presence of these agents for at least 3 h (not shown). These results rule out direct modulation by ICI-182780 and tamoxifen of permeability-related mechanisms and suggest that ICI-182780 and tamoxifen block the estradiol-induced increase in permeability by interacting with the estrogen receptor(s). 8) The RT-PCR assays revealed that human cervical tissues, as well as cultured human cervical epithelial cells, express mRNA for both the alpha ER and beta ER. Incubation of cells in steroid-free medium downregulated alpha ER and beta ER mRNA, indicating that the low amount of estrogens present in the regular culture medium, and possibly phenol red (3), are sufficient to increase levels of alpha ER and beta ER mRNA. 9) Treatment with estradiol upregulated mRNA of both receptors, similar to the effect of estrogen in other estrogen-responsive cells (2, 24).

The present results raise an additional number of issues. First, estradiol increased mRNA levels of both alpha ER and beta ER. The increase in beta ER mRNA was not necessary for stimulating an increase in permeability, because ICI-182780 and tamoxifen blocked the estrogen-induced increase in permeability but stimulated an increase in beta ER mRNA. It was previously suggested that alpha ER is the dominant species in the uterus and that beta ER modulates alpha ER effects (21, 28). The present results support this statement also in the cervix, suggesting that the alpha ER is the key isoform in the cervix for stimulating an increase in permeability. The role of beta ER in the cervix is at present unclear.

Second, ICI-182780 and tamoxifen had no significant effect on alpha ER mRNA levels, but both agents increased beta ER mRNA levels. These results suggest that, in cervical cells, the alpha ER and beta ER isoforms are products of distinct genes.

Third, tamoxifen had no significant effect on binding density of 17beta -[3H]estradiol, but it increased levels of beta ER mRNA. This finding can be explained by a relatively low expression of beta ER mRNA in cervical cells compared with alpha ER mRNA. Because mRNA levels of alpha ER did not significantly change following treatment with tamoxifen, changes in the expression of the less-abundant beta ER isoform should not affect significantly the total level of the estrogen receptors (i.e., binding density of 17beta -[3H]estradiol).

Fourth, ICI-182780 and tamoxifen had no significant effect on levels of alpha ER mRNA, but they blocked the estradiol-induced increase in permeability. Based on this important finding, we propose that the effect of estrogen on permeability involves two transcription-regulated signaling steps: 1) an increase in estrogen receptor, where the active isoform regarding changes in permeability is the alpha ER. 2) An estrogen-receptor-dependent activation of a secondary mechanism (possibly nitric oxide synthase, see below). This hypothesis can explain the effects of ICI-182780 and tamoxifen on binding to the estrogen receptor(s) and on the permeability. Accordingly, ICI-182780 and tamoxifen bind to the estrogen receptor(s), but their effects on signaling relative to changes in permeability depend on the site of interaction with the estradiol-activated estrogen receptor(s): interaction of ICI-182780 or tamoxifen with the estradiol-activated estrogen receptor that is bound with DNA at the site of the estrogen-receptor promoter will either have no effect (e.g., on alpha ER) or will stimulate estrogenic activity (e.g., upregulation of beta ER). If, on the other hand, ICI-182780 or tamoxifen interacts with the estradiol-activated estrogen receptor that is bound with DNA at the site of the promoter of the secondary signaling mechanism, ICI-182780 or tamoxifen will act as estrogen antagonists. This will block the increase in permeability.

The nature of the secondary signaling mechanism in human cervical cells is at present unknown. In human endothelial cells estrogen increases permeability by modulating nitric oxide (5). Human cervical cells produce nitric oxide and express different isoforms of the nitric oxide synthase (Gorodeski, unpublished results). Furthermore, nitric oxide synthase genes express estrogen response elements that can interact with the activated estrogen receptor (4). It is possible that in human cervical cells the effect of estrogen on permeability is mediated by estrogen-receptor-dependent modulation of nitric oxide synthase gene(s), and this subject is currently being investigated in our lab.

The present results may contribute to our understanding of cervical mucus production in women. Estrogen increases secretion of the fluid component of cervical mucus in women. Estrogen deficiency, such as after menopause, decreases fluid secretion, while estrogen replacement to postmenopausal women restores lubrication (8). Our previous studies suggest that the effect of estrogen is the result of increased permeability of the lateral intercellular space (10), and the proposed mechanism is transformation of the cytoskeleton into a more flexible structure (10). This renders cervical epithelial cells more sensitive to decreases in cell size in response to stimuli that operate in vivo, and consequently to increases in the size of the intercellular space. The end result is an increase in the permeability of the lateral intercellular space, which allows greater flow of plasma from the blood into the cervical canal. The present results suggest that two transcription-regulated signaling steps mediate the effect of estrogen on the cytoskeleton: the alpha ER, and an alpha ER-dependent secondary signaling system.

The present results may also have pharmacological significance for women. For instance, women treated with tamoxifen often complain of vaginal dryness. Tamoxifen blocks the estrogen-induced increase in permeability of cultured cervical cells, including in hECE cells. hECE cells retain a squamous stratified nonkeratinizing phenotype (13), similar to that of the vaginal epithelium (8). A decrease in permeability of ectocervical or vaginal epithelia increases the resistance for flow of fluid from the blood into the lumen of the lower genital tract and diminishes lubrication. Women experiencing this condition complain of vaginal dryness. Our results provide, for the first time, an explanation at the cellular level for the clinical side effect of tamoxifen.

The present results may also provide a basis for future research into nonestrogenic modulation of fluid transport in the woman's reproductive tract. Until recently, relatively little was known about mechanisms of regulation of cervical and vaginal permeability, and hence of fluid regulation in the woman's genital tract. Traditionally, most cases of diminished cervical and/or vaginal fluid secretion were attributed to low estrogen (8). This explanation has met with difficulty, because treatment with estrogen may not improve mucus production in all women. Our experiments revealed a complex signaling of estrogen-dependent modulation of cervical permeability. It is possible that some cases of defective fluid secretion are the results of dysregulation at sites distal to the estrogen receptor. Because pharmacological agents can modulate these sites, it may be possible to target steps downstream to the estrogen receptor to regulate the permeability of cervical and vaginal epithelia, and modulate fluid secretion.


    ACKNOWLEDGEMENTS

This study was supported in part by National Institutes of Health Grants HD-00977, HD-29924, and AG-15955 to G. I. Gorodeski.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. I. Gorodeski, Univ. MacDonald Women's Hospital, Univ. Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: gig{at}po.cwru.edu).

Received 25 March 1999; accepted in final form 19 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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