Departments of 1 Reproductive Biology, and 2 Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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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 4 h, and EC50
1 nM). RT-PCR assays revealed the expression of mRNA for the
estrogen receptor
(
ER) and estrogen receptor
(
ER).
Removal of estrogen from the culture medium decreased and treatment
with estrogen increased the expression of
ER and
ER mRNA. In
cells not treated with estrogen, ICI-182780 and tamoxifen increased
ER mRNA. In cells treated with estrogen, neither ICI-182780 nor
tamoxifen had modulated significantly the increase in
ER or
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
ER-dependent increase in transcription.
human; cervical cells; epithelium; transepithelial transport; cervical mucus; transcription; ICI-182780; tamoxifen
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INTRODUCTION |
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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.
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METHODS |
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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.
17-[3H]estradiol binding
assay.
Scatchard analysis was performed on total cell extracts using
17
-[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 17
-[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
17
-[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
17
-[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).
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RESULTS |
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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 · 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 17
-estradiol increased
GTE two- to threefold (Fig. 1). This effect
confirms our previous results (10), indicating that treatment with
physiological concentrations of 17
-estradiol increases the
permeability across cultured human cervical epithelia.
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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
17-[3H]estradiol to lysates of hECE,
ECE16/1, and CaSki cells. Cells on filters were treated with 10 nM
17
-estradiol for 2 days, and binding of
17
-[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:
17
-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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In the second experiment we studied the expression of mRNA for ER
and
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
ER (17) and
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
ER
and
ER (the differences were sequence errors; not shown). These
results indicate that human cervical epithelial cells express mRNA for
ER and
ER.
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Estrogen increases estradiol binding sites. Treatment of human
cervical epithelial cells with 17-estradiol increased the density of
binding sites for 17
-[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 17
-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 17
-estradiol binding
sites precedes the increase in permeability.
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The increase in estradiol binding sites began already with 0.1 nM of
17-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 17
-estradiol for 6 h increased the density of
17
-[3H]estradiol binding sites three- to
fourfold. 17
-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 17
-estradiol
binding sites.
Estrogen increases ER and
ER mRNA. We also determined the
effect of estrogen on the expression of
ER and
ER mRNA in ECE16/1 and CaSki cells. Cells were shifted to SFM and treated with 10 nM
17
-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
-actin mRNA or on GAPDH mRNA, but it decreased
ER and
ER mRNA.
Densitometry results of three experiments in ECE16/1 cells and three
experiments in CaSki cells revealed that incubation in SFM decreased
ER mRNA relative to GAPDH mRNA threefold and
ER/GAPDH mRNA
fivefold (Table 1). Treatment of cells that
were maintained in regular culture medium with 10 nM 17
-estradiol
increased
ER/GAPDH mRNA 5-fold and
ER/GAPDH mRNA 12-fold (Fig. 5
and Table 1). Treatment of cells grown in SFM with 10 nM
17
-estradiol increased
ER/GAPDH mRNA 17-fold and
ER/GAPDH mRNA
78-fold (Fig. 5 and Table 1).
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Effects of ICI-182780 and tamoxifen on ER and
ER mRNA levels.
To determine the effect of ICI-182780 and tamoxifen on mRNA levels
of
ER and
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
ER/GAPDH mRNA, but it increased
ER/GAPDH mRNA by 47-fold.
Tamoxifen increased
ER/GAPDH mRNA 3-fold and
ER/GAPDH mRNA
53-fold (Fig. 6 and Table 1).
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To determine the effect of ICI-182780 and tamoxifen on the expression
of ER and
ER mRNA in estrogen-treated cells, ECE16/1 and CaSki
cells were shifted to SFM and treated with 10 nM 17
-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
ER/GAPDH mRNA or
ER/GAPDH mRNA (Fig. 6 and Table 1). These results suggest that
ICI-182780 and tamoxifen have little effect on the expression of
ER
mRNA but that both agents increase the expression of
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
17-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
17-[3H]estradiol, but it abrogated the
estradiol-induced increase in binding of
17
-[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 17
-[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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DISCUSSION |
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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 17
-[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
ER and
ER. Incubation of cells in steroid-free medium
downregulated
ER and
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
ER and
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 ER and
ER. The increase
in
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
ER mRNA. It was previously suggested that
ER is the dominant species in the uterus and that
ER modulates
ER effects (21, 28).
The present results support this statement also in the cervix,
suggesting that the
ER is the key isoform in the cervix for
stimulating an increase in permeability. The role of
ER in the
cervix is at present unclear.
Second, ICI-182780 and tamoxifen had no significant effect on ER
mRNA levels, but both agents increased
ER mRNA levels. These results
suggest that, in cervical cells, the
ER and
ER isoforms are
products of distinct genes.
Third, tamoxifen had no significant effect on binding density of
17-[3H]estradiol, but it increased levels of
ER mRNA. This finding can be explained by a relatively low
expression of
ER mRNA in cervical cells compared with
ER mRNA.
Because mRNA levels of
ER did not significantly change following
treatment with tamoxifen, changes in the expression of the
less-abundant
ER isoform should not affect significantly the total
level of the estrogen receptors (i.e., binding density of
17
-[3H]estradiol).
Fourth, ICI-182780 and tamoxifen had no significant effect on levels of
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
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
ER)
or will stimulate estrogenic activity (e.g., upregulation of
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 ER, and an
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.
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ACKNOWLEDGEMENTS |
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This study was supported in part by National Institutes of Health Grants HD-00977, HD-29924, and AG-15955 to G. I. Gorodeski.
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FOOTNOTES |
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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.
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