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Departments of Reproductive Biology (R.Z., X.L., G.I.G.), Physiology and Biophysics (G.I.G.), and Oncology (G.I.G.), Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Address all correspondence and requests for reprints to: George I. Gorodeski, M.D., Ph.D., University MacDonald Women’s Hospital, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: gig{at}cwru.edu.
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Introduction |
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Previous studies have shown that short-term treatment with estrogen increases transcervical permeability by decreasing the RLIS (1). The effect of estrogen involves modulation of the cytoskeleton, and the signaling pathway involves activation of the estrogen receptor-
(2) and up-regulation of nitric oxide and cGMP (3); cGMP-dependent activation of protein kinase stimulates ADP-ribosylation of monomeric G-actin and fragmentation of the cytoskeleton (4). Cells become more deformable and tend to decrease their size more readily in response to stimuli that modulate the cytoskeleton, e.g. the transepithelial hydrostatic gradient. Decreases in cell size cause reciprocal increase in the size of the intercellular space and increase the paracellular permeability (5). In vivo this pathway could play a role during the preovulatory phase of the menstrual cycle, when acute increases in plasma estradiol increase cervical permeability and promote secretion of cervical mucus.
More recently we discovered that estrogen could also modulate the transcervical RTJ. Those studies used normal human cervical epithelial cells and required longer treatments with estrogen (6). The objective of the present study was to understand the involvement of tight junctions in the control of transcervical permeability, and the mechanism by which estrogen regulates the RTJ. In other types of cells, tight junctions control transepithelial and transendothelial transport, and dysfunction of tight junctions can lead to water and solutes imbalance (7). Tight junctions also play an important role in development (8) and aging (9), and tight-junction dysfunction could be associated with inflammation (10), bacterial and viral infections (11), vascular disease (12), organ failure (13), and cancer invasiveness (14). Understanding how estrogen modulates cervical RTJ could improve our understanding of transcervical permeability and provide a greater understanding of the biology of the cervix.
Tight junctions are part of the apical junctional complex, which includes the desmosomes, adherens junctions, and intercellular tight junctions. The main function of desmosomes and adherens junctions is to mechanically link adjacent cells (15). The intercellular tight junctions have three known functions: occlusion of the intercellular space; adhesion of adjacent cells; and functional division of the plasma membrane into apical and basolateral domains by sorting integral proteins into these domains and restricting the lateral diffusion of proteins and lipids past the tight junctions (reviewed in Ref.16). On freeze-fracture electron microscopy, the tight junctions appear as continuous anastomosing intramembranous strands on the P face, with complementary grooves on the E face (17). These structures are thought to represent linear polymers of transmembrane proteins with extracellular stretches that associate laterally with tightjunction strands in the apposing membrane of adjacent cells to seal the intercellular space.
Tight junctions are composed of cytoplasmic and transmembrane proteins. Cytoplasmic tight-junction proteins such as the zonula-occludens proteins (ZOs) couple the transmembrane tight-junctional proteins to the cortical actin cytoskeleton (Reviewed in Ref.18). Of the three groups of transmembrane tight-junction proteins, junctional adhesion molecules, claudins, and occludin, only the latter two are expressed in epithelial cells. The claudins and occludin are four-transmembrane domain proteins exhibiting two short hydrophobic extracellular loops that possibly form homotypic or heterotypic interactions to connect adjacent cells (16). Human occludin is a 65-kDa protein with a predicted 65-amino-acid N terminus, two extracellular loops of 56 and 48 amino acids separated by an 11-amino-acid cytosolic segment; four transmembrane stretches of 11–24 amino acids; and a C terminus tail of 256 amino acids that interacts with the cytoplasmic tight-junction proteins (19).
Occludin is important for cell adhesivity (20) and gating of the tight junctions (21). In some types of cells, occludin is not sufficient to form an effective barrier; for instance, embryonic stem cells lacking occludin maintain a partial permeability barrier (22), and transfection experiments in mouse fibroblasts have shown that occludin alone forms only partial junctional fibrils (23). However, in most types of epithelial cells, occludin is distributed throughout tight-junctional fibrils (16, 24), and it is a necessary component of the junctional mechanism (16). Depletion of occludin or transfections of cells with truncated or mutated forms of occludin resulted in aberrant formation of tight junctions, with marked decrease in the number of tight-junction strands and abrogation of the paracellular resistance (25). Transfection of occludin into fibroblasts, which lack endogenous expression of the protein, induced cell-cell adhesiveness (24). Overexpression of occludin in Madin-Darby canine kidney cells resulted in hyperaggregation of globular, hydrophobic, dense particles in regions of the tight junctions (as viewed by electron microscopy) and increased the paracellular resistance (26). Collectively, these data underscore the importance of occludin for conferring the RTJ and suggest that occludin extracellular loops compose in part the intercellular tight-junction strands.
The objective of the present study was to understand the degree to which estrogen modulates the RTJ across human cervical epithelia and the mechanisms involved. A secondary objective was to understand the effects of estrogen on the expression of the apical junctional complex proteins in human cervical epithelial cells. Our results indicate that estrogen stimulates modulation of occludin and that the changes in occludin expression could be the molecular mechanism by which estrogen abrogates transcervical RTJ.
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Materials and Methods |
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The experiments used two types of cells: human ectocervical epithelial cells (hECEs) and CaSki cells. Primary to tertiary cultures of hECEs, cells were generated from minces of the ectocervix as described (27). Discarded ectocervical tissues were collected by the Cooperative Human Tissue Network at the University Hospitals of Cleveland and Case Western Reserve University according to the institutional review board protocol 03-90-TG. Cells were grown and maintained in culture dishes at 37 C in DMEM/Ham’s F12 (3:1) supplemented with nonessential amino acids, adenine (1.8/10–4 M), penicillin (100 U/ml), streptomycin (100 mg/ml), gentamicin (50 ng/ml), L-glutamine (2 mM), insulin (5 µg/ml), hydrocortisone (1/10–6 M), transferrin (5 µg/ml), triiodothyronine (2/10–9 M), epidermal growth factor (10 ng/ml), and 8% fetal calf serum (Sigma, St. Louis, MO) in a 91% O2/9% CO2 humidified incubator (27). Cells were routinely tested for mycoplasma.
For experiments with estrogen, cells on filters were shifted to steroid-free medium (1). This medium is composed of phenol-red-deficient (DMEM)/Ham’s F12 or RPMI 1640 (Sigma) containing 8% heat-inactivated fetal bovine serum that was previously treated with charcoal to remove steroids. Preparation of charcoal-treated serum was described (1); briefly, dextran-coated charcoal (Sigma) was dissolved at 8% in 0.15 M NaCl, autoclaved, mixed by stirring, spun, and the pellet resuspended as 1 g/1.25 ml in H2O. Fetal bovine serum (Hyclone, Logan, UT) was mixed with the activated charcoal-dextran at 20:1 (vol/vol) and incubated for 45 min at 55 C. At the completion of incubation, the mixture was spun twice at 800 x g for 20 min, and the supernatant (serum) was decanted and collected. CaSki cells are a stable line of transformed cervical epithelial cells that were previously characterized by us (27) and are useful for high-output mechanistic studies. CaSki cells were grown and subcultured in a culture dish in RPMI 1640 supplemented with 8% fetal calf serum, 0.2% NaHCO3, nonessential amino acids, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 mg/ml), and gentamicin (50 µg/ml) at 37 C in a 91% O2/9% CO2 humidified incubator and routinely tested for mycoplasma (27). Experiments were done on cells plated on Anocell-10 filters (Anocell, Oxon, UK, obtained through Sigma), which are ceramic-base filters, pore size of 0.02 µm width and 50 µm depth. Filters were coated on their upper (luminal) surface with 3–5 µg/cm2 collagen type IV and incubated at 37 C overnight. The remaining collagen solution was aspirated and the filter was dried at 37 C. Before plating, both sides of the filters were rinsed three times with Hanks’ balanced salt solution. Cells were plated on the upper surface of the filter at 3/105 cells/cm2. By plating at this high density, the cultures become confluent within 12 h after plating. For experiments, cells were shifted to steroid-free medium for 24 h and treated with 17ß-estradiol or the vehicle (estrogen-deprived cells) for 2 additional days (27). All treatments involved adding drugs to both the luminal and subluminal solutions.
Measurements of transepithelial electrical resistance (RTE)
Before experiments, filters containing cells were washed three times and preincubated for 15 min at 37 C in a modified Ringer buffer composed of (in micromoles) NaCl (120), KCl (5), NaHCO3 (10, before saturating with 95% O2/5% CO2), CaCl2 (1.2), MgSO4 (1), glucose (5), HEPES (10) (pH 7.4), and 0.1% BSA in volumes of 4.7–5.2 ml in the luminal and subluminal compartments. Changes in paracellular permeability were determined as changes in the RTE across filters mounted vertically in a modified Ussing chamber from successive measurements of the transepithelial potential difference (
PD, lumen negative) and 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) as RTE = PD/I (28). The experimental design of the electrophysiological measurements, including calibrations and controls, the significance of the PD and I, and the conditions for optimal determinations of RTE across low-resistance epithelia, e.g. hECE, have been described and discussed (28).
Determinations of the dilution potential (Vdil) were performed in the Ussing chamber as described (28). Transepithelial Vdils were determined by measuring the effect of lowering NaCl in the luminal solution on changes in voltage generated across the epithelial culture. This was done by replacing the Ringer’s buffer in the luminal compartment (130 mM NaCl) with low (10 mM) NaCl solution. The latter buffer was similar to the Ringer’s solution except that it lacked the 120 mM NaCl and was supplemented with 240 mM sucrose to compensate for osmolarity. The methods of electrophysiological data evaluation were previously described (28). Vdil was the measured potential difference (voltageSL – voltageL) after lowering NaCl in the luminal solution, corrected for the potential-electrodes asymmetry, where the subscripts SL and L are the subluminal and luminal solutions. The Henderson diffusion equation for monocations and monoanions was used to interpret the transepithelial dilution potential in terms of ionic permeabilities. With the assumption that Na+ and Cl– are the major permeant ionic species, the relative mobilities of Na+ and Cl– in the intercellular space, uCl and uNa, can be determined as ß = uCl/uNa = (K + |Vdil|)/(K – |Vdil|), where K 
(R·T/F)·ln(NaSL/NaL) = 68.5 mV at the given [Na+SL] = 130 mM, and [Na+L] = 10 mM (29).
For simultaneous measurements of changes in RTE and Vdil, filters containing cells were mounted in the Ussing chamber in modified Ringer’s solution. After 10 min stabilization, the luminal solution was replaced with modified Ringer’s solution containing low NaCl (10 mM) plus 240 mM sucrose (to compensate for osmolarity). Drugs were added and determinations of changes in RTE and Vdil in real time were made and analyzed as described (28).
Western blot analysis
The postnuclear supernatant of cells was solubilized in lysis buffer [50 mM Tris-HCl (pH 6.8), 1% [(3-cholamidopropyl) dimethyl-ammonio]-1-propane-sulfonate, and 5 mM EDTA (pH 8.0)] containing 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml benzamidine, 10 µg/ml bacitracin, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. Aliquots normalized to 15 µg protein [about 45 µl, determined by Bio-Rad protein assay solution (Hercules, CA)] were loaded on 10% polyacrylamide sodium dodecyl sulfate gel, and vertical electrophoresis was conducted at 50 mA for 1.5 h. Gels were transferred onto Immobilon membrane (Millipore, Bedford, MA) at 200 V for 1.5 h, and membranes were blocked in 5% milk and exposed to the primary antibody at 4 C overnight. Membranes were washed three times in PBS and fluorescent stained for 1 min using an enhanced chemiluminescence kit of peroxidase-conjugated secondary antibody from Amersham (Piscataway, NJ).
Immunostaining
Cells were cultured on either Millicell-CM filters (Millipore) or Transwell filters (Costar Corp., Cambridge, MA) to confluence and fixed with cold methanol for 15 min. After blocking with blocking buffer (3% BSA, 0.1% Triton X-100 in PBS) for 30 min at room temperature, the cells were incubated with primary antibody overnight at 4 C. After washing in PBS, the cells were incubated with donkey antirabbit IgG Alexa Fluor 488 or goat antimouse IgG Alexa Fluor 594 secondary antibodies (Molecular Probes, Eugene, OR) for 1 h at room temperature. The filters were mounted in Vestashield with 4',6'-diamino-2-phenylindole (Vector H-1200, Vector Laboratories, Burlingame, CA). Immunolocalization was observed by using a confocal laser-scanning microscope (TCS-NT, Leica, Bensheim, Germany). Images were processed using Adobe Photoshop software package (San Jose, CA).
Immunoprecipitation
After labeling with [35S]methionine, cells were lysed in lysis buffer [1% Triton X-100, 0.5% Nonidet P-40, 10 mM dithiothreitol, 5 mg/ml aprotinin, 5 mg/ml leupeptin, 100 mg/ml bacitracin, 100 mg/ml benzamidine, 2 mM Na-orthovanadate, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 40 mM sodium pyrophosphate, 50 mM KH2PO4, 10 mM sodium molybdate, and 20 mM Tris-HCl (pH 7.4)] at 4 C for 20 min, spun at 10,000 x g for 15 min, and lysates were precleared with protein A/G-agarose for 90 min at 4 C. The antioccludin antibody was linked covalently to protein A/G-agarose matrix, and the lysate-antibody was subjected to immunoprecipitation for 2 h. Immune complexes were washed three times with radioimmunoprecipitation assay buffer [20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10 mM dithiothreitol, 1% Triton X-100 (pH 8.0)] and separated on a 10% linear gradient sodium dodecyl sulfate-acrylamide Laemmli gels. Polyacrylamide gels were fixed with 50% methanol and 10% acetic acid for 1 h and incubated in Amplify (Amersham) for 45 min before being dried under vacuum. Dried gels were exposed to Hyperfilm-MP (Amersham) at –80 C and analyzed by densitometry.
Antibodies
The following mouse monoclonal antihuman antibodies were from Zymed Laboratory Inc. (San Francisco, CA) and were used according to the manufacturer’s instructions: antioccludin (catalog no. 33-1500), anticlaudin-4 (catalog no. 32-9400), anti-ZO-1 (catalog no. 33-9100), anti-E-cadherin (catalog no. 33-4000, used for the immunofluorescence experiments), and anti-ß-actin. Some experiments also used mouse mononclonal antihuman E-cadherin clone 36 (BD Transduction Laboratories, San Jose, CA) at 1:1000 dilution (250 µg/µl), with similar results. The antitubulin antibody was from the Developmental Studies Hybridoma Bank at the University of Iowa (Iowa City, IA), used at 1:500 dilution for Western blot. It is a hybridoma supernatant (clone E7), with the antigen of ß-tubulin-galactosidase/ftz fusion protein.
Occludin turnover assays
For occludin synthesis, cells were rinsed once with methionine-free medium and incubated in methionine-free medium for 30 min before metabolic labeling. Filters were labeled for 1 h with 1.5 µCi [35S]methionine in methionine-free medium, and cells were extracted for immunoprecipitation after 6 h. For occludin degradation, cells cultured on filters were shifted to methionine-free medium and labeled with 0.8 µCi [35S]methionine for 14 h, and protein turnover was monitored by replacing labeling media with fresh medium. Cells were extracted for immunoprecipitation either immediately at the end of the labeling period (t = 0) or after 18 h. Cell lysates were immunoprecipitated with the antioccludin antibody as described above. Data were normalized with expression of the ubiquitous ß-actin.
RT-PCR
Total RNA was extracted from cells homogenates using RNeasy minikit (Qiagen, Valencia, CA). A Perkin-Elmer DNA thermal cycler (Cetus, Norwalk CT) was used for the assays using Invitrogen SuperScript II RnaseH– kit (Carlsbad, CA), following the manufacturer’s instructions. 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. Negative control was distilled water, and mock reaction contained a tube with total RNA in a mixture lacking the Oligo dt and without the AMV reverse transcriptase. Oligonucleotide primers were synthesized by Primer3 Input (primer3@genome.wi.mit.edu). Primers to detect human occludin mRNA were: forward (sense) 5'-TTT GTG GGA CAA GGA ACA CA-3', reverse (antisense): 5'-GCA GGT GCT CTT TTT GAA GG-3'. PCR conditions for occludin were: 94 C for 3 min, 30 cycles of 1 min denaturation at 94 C, 30 sec of annealing at 60 C, 1 min of extension at 70 C, and 10 min at 70 C (expected cDNA length 700 bp). Primers to detect human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (used as control) were: 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'. PCR conditions for GAPDH were 30 cycles of 1 min at 94 C, 1 min at 60 C, 1 min at 72 C and ß-actin, 30 cycles of 1 min at 94 C, 1 min at 62 C, and 1 min at 72 C (expected cDNA length 932 bp). 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.
Real-time RT-PCR was done using i-Cycler (Bio-Rad) following the manufacturer’s instructions and using the above primers. PCR conditions were: 95 C for 2 min, 50 cycles of 30 sec denaturation at 95 C, 30 sec of annealing at 60 C, 1 min of extension at 70 C, 80 cycles of 30 sec reaction at 55 C including data collection and real-time analysis, and cooling to 4 C. Data analysis was done using the PCR Amp-Cycle Graph, standard curve report and base line subtracted curve fit data (Bio-Rad i-Cycler) with the data analysis window set at 95.00% of a cycle, centered at the end of the cycle. The obtained standard curve correlation coefficient was 0.085 (P < 0.01) with PCR efficiency of 103.72%.
Densitometry was done using an Arcus II scanner (AGFA, New York, NY), and Un-Scan-It gel automated digital software (version 5.1, Silk Scientific, Orem, OR).
Statistical analysis of the data
Data are presented as means (± SD), and significance of differences among means was estimated by Student’s t test. Trends were calculated using GB-STAT (version 5.3, Dynamic Microsystems Inc., Silver Spring, MD) and analyzed with ANOVA.
Chemicals and supplies
All chemicals, unless specified otherwise, were obtained from Sigma. Plasmin, leupeptin, and aprotinin were from Boehringer (Petersburg, VA); matrix-metalloproteinase (MMP)-2 from Chemicon (Temecula, CA). Assays with MMP-2 involved preaddition of 0.5 mM 4-aminophenylmercuric acetate (Chemicon) to the assay to activate the latent enzyme (30).
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Results |
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Baseline levels of RTE across cultures of cervical cells grown in steroid-free medium ranged 25 ± 4 
·cm2 (Figs. 1A
, 2A, and 3A). These data confirm our previous observations (27) and indicate that human cervical epithelial cells form relatively permeable epithelia on filters. To determine the effect of estrogen on paracellular permeability, cells were treated with 100 nM 17ß-estradiol for periods of 0–96 h. The concentration of 100 nM 17ß-estradiol, which is in the higher limit of the physiological range for the woman, was chosen because it induces submaximal increase in RLIS in human cervical epithelial cells (1). Treatment with estradiol decreased RTE in a time-dependent manner; effects began already 12 h after treatment and reached a plateau after 48 h (Figs. 1A and 2A). The decreases in RTE at 48 h of treatment with 17ß-estradiol were concentration dependent in the range of 1–100 nM (Fig. 2A).
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To determine the degree to which the effect of estradiol involved abrogation of the RTJ, changes in the dilution potential were measured, and effects of estradiol on the ratio of mobilities of Cl– and Na+ (uCl/uNa) were calculated. The rationale was that cation selectivity is a property of the tight junctions, and changes in uCl/uNa reflect modulation of RTJ (28). Levels of the ratio uCl/uNa ranged 1.335 ± 0.005 and 1.351 ± 0.005, respectively, for hECE and CaSki cells incubated in steroid-free medium (Figs. 1B
, 2B, and 3B). Treatment with estrogen induced an increase in the ratio uCl/uNa to about 1.38, which began 24–36 h after treatment and reached presaturation at 48–72 h (Fig. 1B). Because an increase in the ratio of uCl/uNa indicates a decrease in tight-junctional resistance (28), the data in Fig. 1 suggest that the estrogen-induced decrease in RTE after 24 h of treatment is mediated by a decrease in RTJ. The effects of estradiol on RTJ were also concentration dependent. Decreases in RTJ were observed in cells treated with 1 nM 17ß-estradiol for 48 h, and increases in the ratio of uCl/uNa were greater with higher doses of the hormone (Fig. 2B).
Tamoxifen and progesterone modulate estrogen effects on RTE and RTJ
In vivo, the effects of estrogen on lubrication of the lower genital tract can be controlled by the estrogen-receptor modulators tamoxifen and progesterone. To better understand the mechanisms of tamoxifen and progesterone actions, experiments were conducted to determine the degree to which tamoxifen and progesterone modulated estrogen effects on the RTE and RTJ. Tamoxifen (10 µM) or progesterone alone (1 µM) had no significant effect on RTE or on the ratio of uCl/uNa (Fig. 3, A and B). Cotreatment of 10 nM 17ß-estradiol plus 10 µM tamoxifen for 48 h blocked the estrogen-induced decrease in RTE (Fig. 3A) and the estrogen-induced increase in uCl/uNa (Fig. 3B). Cotreatment with 10 nM 17ß-estradiol plus 1 µM progesterone for 48 h attenuated the estrogen-induced decrease in RTE (Fig. 3A), but it had no effect on the estrogen-induced increase in uCl/uNa (Fig. 3B). These results indicate that tamoxifen blocks estrogen effect on the RTJ; in contrast, progesterone had a lesser effect on RTE, and it did not modulate the estrogen decrease in RTJ.
Estrogen has no significant effect on the expression of E-cadherin, ZO-1, or claudin-4
A possible explanation for the effect of estrogen on the RTJ is cell contraction secondary to modulation of the cytoskeleton. Estrogen shifts actin steady state toward the monomeric G-actin, which would stimulate formation of a more flexible cytoskeleton (4) and could lead to the pulling away of cells at the zonula adherence. To determine whether the effect of estrogen involved changes in the morphology of cells, hECE cells on filters were assayed by immunofluorescence with anti-E-cadherin antibody and analyzed by laser confocal microscopy. E-cadherin is a structural membrane protein that mediates intercellular adhesion and is a useful marker of plasma membrane morphology. Treatment with 100 nM 17ß-estradiol for 48 h had no significant effect on the cellular distribution of E-cadherin (Fig. 4, A and B). Similar effects were obtained in CaSki cells (not shown).
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, A and B). Estrogen also had no significant effect on the constitutively expressed protein tubulin (Fig. 5A). In contrast to estrogen, treatment with proteinase K induced degradation of Ecadherin already by 60 min of incubation with the enzyme (Fig. 5B).
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, C and D) or claudin-4 (Fig. 4, E and F). Incubation of lysates of cells grown in steroid-free medium with anti-ZO-1 or anti-claudin-4 antibodies showed immunoreactivities at 220 and 22 kDa, respectively, and expression of the proteins was not affected by treatment with estrogen (Fig. 5, A and B). Treatment with proteinase K, on the other hand, induced degradation of claudin-4 by 60 min of incubation with the enzyme (Fig. 5B). These results rule out remodeling of E-cadherin, ZO-1, or claudin-4 as the mechanism of estrogen decrease in RTJ. Effects of estrogen on occludin
To determine whether estrogen affects the cellular distribution of occludin, cultures on filters were processed for immunofluorescence with the antioccludin antibody and analyzed by laser confocal microscopy. Treatment with 100 nM 17ß-estradiol for 48 h had no significant effect on the cellular distribution of occludin in hECE cells (Fig. 4, G and H). In contrast, treatment with estrogen affected occludin protein expression in Western blots. Immunoblots of lysates of hECE cells grown in steroid-free medium revealed specific immunoreactivity to two specific occludin forms: a 65-kDa form that was previously reported by other investigators (19) and a smaller form of 50 kDa (Figs. 6 and 7). In cells grown in steroid-free medium, the ratio of the 50/65-kDa bands ranged from 0 to 9% and tended to be lower in hECE cells than in CaSki cells (Figs. 6 and 7 and Table 1). Treatment of cells with 100 nM 17ß-estradiol for 48 h decreased the expression of the 65-kDa form and increased the expression of the 50-kDa form, thus increasing the ratio of 50/65 kDa in hECE cells from 5 to 105% and from 9 to 85% in CaSki cells (Figs. 6 and 7 and Table 1). In a control experiment, treatment with estrogen had no significant effect on the density of ß-actin protein (Fig. 6).
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and Table 1). Treatment with estradiol also increased the ratio of the 50/65-kDa forms in a dose-related manner from 7% at 1 nM estradiol to 115% at 100 nM estradiol (Fig. 7 and Table 1).
To determine whether the effect of estrogen on the expression of occludin is reversible, cells treated with 100 nM 17ß-estradiol for 48 h were shifted to steroid-free medium for 72 h. As shown in Fig. 6, under that condition the immunoreactivity was predominantly to the occludin 65-kDa form, indicating that the effect of estrogen on occludin is reversible on omission of the hormone from the culture medium. Collectively, the results in Figs. 5–7 show that treatment with 17ß-estradiol at physiological concentrations of the hormone in the woman induces a dose-dependent biphasic effect on the occludin 65-kDa form and a dose-dependent increase in the expression of the occludin 50-kDa form.
Tamoxifen, but not progesterone, modulates estrogen effects on occludin
Cotreatment with 1 µM progesterone had no significant effect on the estrogen-induced changes in the densities of the 65- and 50-kDa forms or on the ratio of 50/65 kDa (Fig. 7B). In contrast, cotreatment with 10 µM tamoxifen attenuated the estradiol (1 nM)-induced increase in the 65-kDa form and the estradiol (10 nM, 100 nM)-induced decrease in the 65-kDa form, and it blocked the estrogen-induced increase in the 50-kDa form (Fig. 7C). These results indicate that tamoxifen, but not progesterone, blocks estrogen modulation of occludin in human cervical epithelial cells.
Mechanisms of estrogen effects
No direct effect on occludin transcription.
To determine whether estrogen modulates occludin transcription, we studied effects of estrogen treatment on occludin mRNA levels. Using oligonucleotide primers complementary to cloned human occludin (NM_002538), a single cDNA fragment of 700 bp was amplified by RT-PCR from hECE and CaSki cells (Fig. 8A). The 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 occludin (the differences were sequence errors, not shown).
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). The standard RT-PCR assays were complemented by real-time PCR, which quantitates specifically and in real time the initial amount of the template and provides semiquantitative data unaffected by post-PCR processing of PCR products. Results of that assay in CaSki cells confirmed that treatment with different concentrations of 17ß-estradiol for 48 h had no significant effect on the expression of occludin mRNA relative to GAPDH mRNA (Fig. 8B). Collectively, these results indicate that treatment with estrogen does not affect the steady-state density of occludin mRNA and suggest that estrogen does not directly affect occludin transcription.
Modulation of occludin turnover.
To determine the degree to which estrogen modulates occludin translation, we assayed occludin synthesis and degradation by [35S]methioninelabeling pulse-chase assays as described in Materials and Methods. Occludin synthesis was determined in cells pulse chased with [35S]methionine for 6 h. Treatment with 10 nM 17ß-estradiol for 48 h resulted in greater immunoprecipitation of [35S]methionine-labeled occludin in hECE and CaSki cells, compared with cells not treated with the hormone (Fig. 9, A and B). The short-term pulse chase with [35S]methionine also revealed the immunoprecipitation of [35S]methionine-labeled 50-kDa occludin form in cells treated with estradiol (Fig. 9, A and B). These results suggest that estradiol accelerates occludin synthesis and stimulates the de novo expression of the 50-kDa occludin form. A similar strategy was used to study the degree to which estrogen modulates occludin degradation by using [35S]methionine-labeling pulse-chase for 14 h. Pretreatment with 10 nM 17ß-estradiol for 48 h resulted in lesser immunoprecipitation of the [35S]methionine-labeled occludin 65-kDa form and greater immunoprecipitation of the [35S]methionine-labeled occludin 50-kDa form, compared with cells not treated with the hormone (Fig. 9, A and B). These data suggest that treatment with estradiol up-regulates occludin synthesis and modulation into a 50-kDa form.
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Treatment with any of the three proteases decreased the RTE within minutes (Fig. 10A) by abrogation of the RTJ (Fig. 10B). The effects of proteinase K could be blocked by coincubation with leupeptin, the effects of plasmin by coincubation with aprotinin, and MMP-2 by coincubation with BMS-275291 plus batimastate (Fig. 10, A and B). The most likely explanation for the effects of the proteases is degradation of tight-junction proteins at extracellular sites.
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, each of the three proteases stimulated a rather unique pattern of occludin degradation that could be blocked by one of the inhibitors. Proteinase K induced expression de novo of occludin forms of 55 and 45 kDa; plasmin of 55 and 48 kDa; and MMP-2 of 60-, 58-, 53-, and 45-kDa forms (Fig. 11). In contrast to the effects of the three proteinases, treatment with estrogen generated only a single occludin 50-kDa form (Figs. 6, 7A, 9, and 11). These data suggest that the occludin 50-kDa form is an estrogen-specific-induced occludin isoform.
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Discussion |
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The present results suggest that estrogen regulates the RTJ by modulating expression of occludin. Previous studies showed that occludin could be regulated at the levels of transcription, synthesis and degradation, assembly into tight-junctional domains, scaffolding and coupling to the perijunctional actin cytoskeleton, and phosphorylation/dephosphorylation (16, 34, 35, 36). A number of studies reported the regulation of RTJ by steroid hormones, but their results did not provide in-depth understanding of the mechanisms involved. For instance, closure of tight junctions of the mammary epithelium is required for the onset of lactogenesis (37), and the effect depends on cortisol (38), the estrogen receptor (ER)-ß (39), and progesterone withdrawal (40, 41). Another group reported diminished endometrial cell-cell attachment and tight-junction closure at the time window of blastocyst implantation (42). Most other studies in the field (43, 44, 45, 46, 47) looked into effects of pathological conditions on the RTJ and occludin.
The present study reports, for the first time, regulation of occludin by estrogen under conditions that can be defined physiologically. The effect of estrogen involved the nuclear ER mechanism because tamoxifen blocked the effect. Human cervical epithelial cells express the ER and ERß, and densities of both receptors depend on the presence of estrogen: removal of estrogen from the culture medium decreases, and treatment with estrogen increases the expression of both the ER and ERß (2). Tamoxifen alone increases ERß, but it has no effect on the estrogen-induced increase in ER or ERß (2). Tamoxifen also attenuates estrogen decrease in the RLIS (1) and RTJ (present study), and it blocks estrogen-induced changes in occludin (present study). Because the effects of estrogen on RLIS and RTJ use different effector mechanisms, G-actin and occludin, respectively, it is likely that they also use different secondary signaling cascades downstream of the ER. The present data suggest that tamoxifen blocks estrogen effect at the proximal end of the signaling pathway, namely the ER. In contrast, progesterone appears to act distal to the ER, within the RLIS-associated signaling cascade because it attenuated estrogen-abrogation of the RLIS (1) but not of the RTJ (present results).
Estrogen modulated posttranscriptional expression of occludin: at low concentrations estrogen appeared to enhance synthesis of the predominant occludin 65-kDa isoform, whereas at higher concentrations, still within the physiological range for the woman, estrogen augmented turnover of the 65-kDa isoform and up-regulation of the 50-kDa isoform. A biphasic effect of estrogen on transendothelial paracellular permeability was previously described by us (48) and others (49). Ye et al. (49) also described biphasic regulation by estrogen of occludin mRNA in human endothelial cells. The physiological significance of the estrogen biphasic, posttranscriptional regulation of the occludin 65-kDa isoform in the epithelial cervical cells is at present unclear, and this subject is being further studied.
The present RT-PCR data rule out the possibility that the occludin 50-kDa form is genomically regulated or that it is a splice variant isoform. Instead, the Western data suggest that it is a product of occludin modulation because the increase in the 50-kDa form correlated with the estrogen modulation of the RTJ, suggesting that occludin modulation into the 50-Da isoform is the molecular mechanism of estrogen abrogation of the RTJ.
The molecular effect of estrogen action on the occludin structure was evaluated by comparing the effects of estrogen on occludin with those of three proteases, proteinase K, plasmin, and MMP-2. When added to the bathing solution to degrade membrane proteins at extracellular domains, all three proteases abrogated acutely the RTJ and induced, within minutes, degradation of occludin. The results with proteinase K confirm previous reports in Madin-Darby canine kidney cells (50) and can be best explained based on the predicated structure of occludin and the immunoreactivity of the antioccludin antibody. The cytoplasmic N and C terminus segments of occludin connect with two extracellular loops that interconnect by a short cytoplasmic segment. A broad-spectrum protease, e.g. proteinase K, could proteolyze occludin extracellularly by cleaving occludin extracellular loops. Because the antioccludin antibody Zymed 33–1500 recognizes antigenic domains at the C terminus, the size of immunoreactive bands will depend on the cleavage sites. In control cells not exposed to proteinase K, we found immunoreactivity to the full-length occludin protein (65 kDa) (Fig. 11). Digestion of loop 1 (i.e. close to the N terminus) would yield a segment containing the C terminal; the fourth transmembrane segment, loop 2; the third transmembrane segment; the intracellular interconnecting segment; and the second transmembrane segment. This segment would be about 55 kDa, as was originally reported by Medina et al. (50) and confirmed in Fig. 11. The occludin immunoblot would therefore identify lesser expression of the 65-kDa band and de novo expression of a 55-kDa isoform (Fig. 11). Digestion of loop 2 (close to the C terminus) would yield a segment of about 45 kDa containing the C terminus and fourth transmembrane segment, and the immunoblot would reveal expression of the 65- and 45-kDa isoforms. If both loops were digested, the immunoblots would detect the 65-, 55-, and 45-kDa isoforms. The result in Fig. 11 suggests that in CaSki cells proteinase K digested occludin loops 1 and 2 in their entirety.
Treatment with plasmin revealed the expression of 65-, 55-, and 48-kDa forms, suggesting plasmin cleaved occludin loop 1 entirely and loop 2 only partially, leaving a 3-kDa protein stretch attached to the fourth transmembrane segment and the C terminus. The pattern obtained after treatment with MMP-2 was more complex, suggesting MMP-2 digested occludin loop 1 at two sites, leaving 5- and 3-kDa protein stretches attached to the second transmembrane segment; loop 2 in its entirety (i.e. the 45-kDa segment); and in some cells digesting loop 2 also in a proximal site of the loop, leaving an 8-kDa protein stretch attached to the fourth transmembrane segment.
In contrast to the effects of the three proteases, treatment with 10–100 nM 17ß-estradiol for 48 h augmented expression of only one low-molecular-weight form of occludin of about 50 kDa. Based on the results of the effects of the three proteases, we propose that estrogen induces modulation of occludin at the middle of extracellular loop 2, therefore resulting in the expression of a 50-kDa occludin isoform. Compared with the effect of the proteases, treatment with estradiol would have lesser effect on RTJ because loop 1 was presumably unaffected. This speculation is supported by our findings that treatment with estrogen decreased the RTE and abrogated the RTJ to a lesser degree than proteinase K, plasmin, or MMP-2 (compare Figs. 1 and 10).
The prevailing theory of occludin mechanism of action is that of homotypic contact across the intercellular spaces between extracellular loops of occludin of adjacent cells or heterotypic contact between occludin-claudin extracellular loops (16). It was suggested that hydrophobic segments of the extracellular loops form intercellular bridges that effectively block the free movement of water through the paracellular space (16). Furthermore, treatment of cells with peptides to occludin extracellular loops decreased paracellular resistance (51, 52, 53). The present results therefore suggest that disruption of the integrity of occludin extracellular loop 2 is detrimental to the occlusion of the intercellular space. Therefore, estrogen degradation of occludin extracellular loop 2 could be the mechanism leading to abrogation of intercellular occludin-occludin or occludin-claudin connections and decreased tight-junctional resistance.
The present experimental data at the cellular-molecular level correlate with clinical observations in women. In vivo, estrogen increases lubrication of the lower genital canal. This phenomenon can be explained based on the effects of estrogen on the RLIS (1, 2, 3, 4, 6) and RTJ (present study). For both mechanisms, estrogen’s net effect would be to decrease the transepithelial resistance and increase the permeability and promote the movement of water from the blood through the intercellular space into the lumen. In women tamoxifen blocks estrogen effect, and it causes dryness of the lower genital tract. The present data provide a novel mechanism for tamoxifen’s effect by blocking estrogen-dependent modulation of occludin extracellular loop 2. These data could be used in the future to design drugs that affect lubrication of the lower genital canal in women by targeting postestrogenic mechanism that regulate occludin and the RTJ.
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Acknowledgments |
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Footnotes |
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Abbreviations: ER, Estrogen receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hECE, human ectocervical epithelial cell; I, transepithelial electrical current; MMP, matrix-metalloproteinase; PD, transepithelial potential difference; RLIS, resistance of the lateral intercellular space; RTE, transepithelial electrical resistance; RTJ, resistance of the intercellular tight junction; uCl, relative mobility of Cl–; uNa, relative mobility of Na+; Vdil, dilution potential; ZO, zonula-occludens protein.
Received May 4, 2004.
Accepted July 21, 2004.
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and ß in the regulation of cervical permeability. Am J Physiol 278:C689–C696
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