Regulation by retinoids of P2Y2 nucleotide receptor mRNA in human uterine cervical cells

George I. Gorodeski1,2, Peter Burfeind1, Shu Uin Gan1, Dipika Pal1, and Fadi W. Abdul-Karim3

Departments of 1 Reproductive Biology, 2 Physiology and Biophysics, and 3 Pathology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Extracellular ATP stimulates acute changes in paracellular permeability across cultures of human uterine cervical epithelial cells [G. I. Gorodeski, D. E. Peterson, B. J. De Santis, and U. Hopfer. Am. J. Physiol. 270 (Cell Physiol. 39): C1715-C1725, 1996]. In this paper, we characterize mRNA for a P2Y2 nucleotide receptor in human cervical cells. Using oligonucleotide primers based on the sequence of human airway epithelium P2Y2 receptor, a single 632-bp cDNA band was identified in RT-PCR experiments in extracts of human endocervical and ectocervical tissues and in lysates of human cervical CaSki cells, but not in 3T3 fibroblasts. The nucleotide sequence was homologous to the corresponding human airway epithelium P2Y2 receptor. Northern blot analyses revealed hybridization of the P2Y2 receptor probe to a 2.0-kb mRNA fragment, as well as to 2.2-, 3.0-, and 4.6-kb species, indicating that human cervical cells express P2Y2 receptor mRNA. Incubation of CaSki cells in retinoid-free medium abolished the ATP-induced changes in permeability and decreased the expression of the P2Y2 receptor mRNA; treatment with retinoids restored the responses to ATP and upregulated the P2Y2 receptor mRNA, suggesting that the receptor mediates ATP-related changes in permeability. Treatment with actinomycin D decreased the expression of the P2Y2 receptor RNA, but the ratio density of the receptor RNA relative to glyceraldehyde-3-phosphate dehydrogenase RNA remained unchanged, suggesting that retinoids upregulate transcription of the receptor mRNA. We conclude that retinoid-dependent modulation of the P2Y2 receptor expression, and hence of the responses to ATP, may be an important mechanism for the regulation of secretion of cervical mucus in vivo.

extracellular adenosinetriphosphatase; P2U and P2N nucleotide receptors; vitamin A; paracellular permeability; transepithelial transport; cervical mucus

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE MAIN FUNCTION OF THE epithelium that covers the woman's uterine cervix is to regulate secretion of fluid and solutes, the cervical mucus, into the cervical canal. Recently, we described an in vitro model of human cervical epithelial cells that retains phenotypic characteristics of the endocervical epithelium (24). The cultured cervical epithelium is characterized by a relatively high degree of permeability (14), suggesting that transcervical transport occurs mainly via the paracellular pathway.

These studies also showed that micromolar concentrations of extracellular purines (ATP) or pyrimidines (UTP) modulate the paracellular permeability across cervical cultures acutely and reversibly (19, 23) and that the effects could be described in terms of activation of nucleotide receptors (20). In keeping with the latest nomenclature proposal, the results suggested activation of P2Y2 nucleotide receptors (1, 6, 7). These novel data may be relevant to the regulation of cervical mucus in vivo because epithelial cells express P2Y2-nucleotide receptors (7, 8, 27, 30, 31) and ATP can accumulate in the extracellular space at micromolar concentrations (5, 11) that are sufficient to activate the P2Y2 receptor mechanism. Our first objective in the present study was to determine whether human cervical cells express nucleotide receptor mRNA.

Vitamin A and related compounds, the retinoids, regulate the reproductive tract epithelia in females (9, 12, 15, 17, 18). Vitamin A deficiency results in dryness of mucous membranes (9) and may lead to squamous metaplasia and keratinization of the simple columnar epithelium of the endocervix (12). In addition, retinoids regulate the paracellular permeability across the cultured human cervical epithelium (15) and modulate the responses to extracellular ATP (22). Our second objective was to test the hypothesis that in human cervical cells the effects of extracellular ATP are mediated by nucleotide receptor and that retinoids modulate the responses to extracellular ATP by regulating the expression of the P2Y2 receptor. We characterized mRNA for a P2Y2 receptor in human endocervical and ectocervical tissues and in cultured human cervical epithelial cells. Our results also show that the expression of the receptor mRNA is regulated by retinoids, both retinoic acid receptor (RAR)-selective and retinoid X receptor (RXR)-selective agonists, and that changes in the cellular density of the P2Y2 mRNA receptor correlate with the responses to ATP.

    METHODS
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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 had histologically normal cervices. After removal of the uterus, the cervical tissues were washed, minced, and transferred to the lab in ice-cold saline.

Cell culture techniques. CaSki cells, which retain phenotypic characteristics of human endocervical cells (1), were grown and maintained on culture dishes in regular medium enriched with 8% FCS as was described (14, 19, 20, 23, 24). In some experiments, cells were shifted to a medium enriched with FCS that was delipidized (retinoid-free medium) (22). 3T3 fibroblasts were cultured as described (17, 18).

Electrophysiological experiments. Changes in paracellular resistance were determined in terms of changes in the transepithelial electrical conductance (GTE) as described (23). Changes in GTE were calculated continuously across filters mounted in a modified Ussing chamber from successive measurements of the short-circuit current (Isc) and the transepithelial potential difference (PD), switching between Isc and PD at a rate of 20 Hz: GTE = Delta Isc /Delta PD. The experimental design of the electrophysiological measurements, including calibrations and controls, the significance of the voltage and Isc, and the conditions for optimal determinations of GTE across the low-resistance CaSki epithelium were described and discussed (15, 23).

Isolation of RNA. We used well-established methods (4) with some modifications (16, 17). Tissues (endocervix and ectocervix) were pulverized and then homogenized by tissue tearer using the Qiagen kit (Qiagen, Chatsworth, CA) with lysis buffer plus 2-mercaptoethanol at 350 µl/30 mg. Total RNA from CaSki cells or from 3T3 fibroblasts was isolated with the Qiagen kit, using lysis buffer plus 2-mercaptoethanol at 350 µl/107 cells. The final total RNA pellets were resuspended in 50 µl of diethyl pyrocarbonate water and quantitated by measuring the optical density at 260 nm. Total RNA of human placenta was a generous gift from Dr. J. Ilan (Department of Reproductive Biology, Case Western Reserve University School of Medicine, Cleveland, OH). Poly(A)+ RNA was isolated from total RNA using an oligo(dT) cellulose affinity column (Poly[A] Quik column, Stratagene, La Jolla, CA), according to the manufacturer's protocol. For Northern blots, we used 20 µg total RNA and 7-10 µg poly(A)+.

RT-PCR was carried out in a Perkin-Elmer DNA thermal cycler (Cetus, Norwalk, CT) using an RT-PCR kit (Boehringer Mannheim, 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, 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), each primer at 1 µM, 0.01 mM dNTPs, 5 units Taq polymerase (Amersham), and 1.4 µM Taq antibody (Clonetech) in a Perkin-Elmer 460 DNA thermal cycler (Cetus). The following conditions were applied: 94°C for 5 min, followed by 30 cycles of 1 min of denaturation step at 94°C, 1 min of annealing step at 60°C, and 1 min of extension step at 72°C, followed by 7 min at 72°C. Samples were cooled at 4°C (soak file) and frozen at -80°C to facilitate removal of the mineral oil. The primers used to amplify the P2Y2 receptor were synthesized by the Molecular Biology Core Laboratory of the Case Western Reserve University School of Medicine and were prepared as 10 µM stocks. The following oligonucleotide primers were used, based on the reported corrected sequence of a P2U nucleotide receptor cloned from human airway epithelial cells (CF/T43) (32): forward (sense), 5'-CTC TAC TTT GTC ACC ACC AGC GCG-3' (nucleotides 750-773); and reverse (antisense), 5'-TTC TGC TCC TAC AGC CGA ATG TCC-3' (nucleotides 1364-1387). The sequences used to amplify the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment were GAPDH forward (sense), 5'-TGA AGG TCG GAC TCA ACG GAT TTG GT-3'; and GAPDH reverse (antisense), 5'-GTG GTG GAC CTC ATG GCC CAC ATG-3'. Amplified samples (20 µl) were analyzed on 1.5% agarose gel, stained with ethidium bromide, and photographed. Densitometry of bands was taken from the Polaroid negatives using an Ultrascan XL laser densitometer (Pharmacia). 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 digest of OX174 DNA (United States Biochemical, Cleveland, OH).

Northern blot analysis. Total RNA or mRNA was separated on a 1% denaturing agarose gel, transferred to a Hybond N nylon membrane (Amersham, Cleveland, OH), and cross-linked by brief exposure to ultraviolet light or baked at 80°C. The cDNA probes of the P2Y2 receptor (see below) or of GAPDH (Clontech Laboratories, Palo Alto, CA) were labeled with [32P]dCTP (NEN, Boston, MA) using the random hexanucleotide primer method and hybridized to Northern blots in 5× standard saline citrate (SSC), 5× Denhardt's solution, 10% (wt/vol) dextran sulfate, 0.1% (wt /vol) SDS, and 100 µg/ml denatured salmon sperm DNA at 65°C for 18 h. The filter was washed at room temperature for 15 min in 2× SSC, followed by 5-15 min in 0.5× SSC and 0.5% (wt /vol) SDS at 65°C. The filter was exposed to X-ray film for 6-24 h.

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

Cloning and sequencing of the partial P2Y2 nucleotide receptor PCR fragment. RT-PCR of CaSki cell total RNA using oligonucleotide primers specific to the P2U nucleotide receptor of human airway epithelial cells (32) yielded a single 632-bp fragment. The fragment was extracted from a 1% agarose gel, purified using the Qiagen gel extraction kit (Qiagen) according to the manufacturer's protocol, and cloned into the Hinc II site of the pGEM3z vector (Promega). Before transformation of DH5alpha cells, plasmid DNA was prepared from single colonies using a miniprep kit (Promega) with the manufacturer's procedure. The subcloned partial P2Y2 nucleotide receptor DNA sequence was then sequenced in both directions using the P2U forward and reverse primers described in the previous section. Sequencing was performed by the Molecular Biology Core Laboratory at Case Western Reserve University, using an ABI373A automated sequencer. The sequences were checked against the GenBank databases using the National Center for Biotechnology Information BLAST network services and was confirmed to be homologous to P2Y2 nucleotide receptor (accession no. U07225/HSU07225).

Statistical analysis of 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 V5.3 (1995; Dynamic Microsystems, Silver Spring, MD) and analyzed with ANOVA.

Chemicals and supplies. Anocell (Anocell-10) filters were obtained from Anotec (Oxon, UK). 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl]benzoic acid (TTNPB), SRI-11217, and SRI-11237 were synthesized in the Department of Chemistry, Allergen (Irvine, CA), and were a gift of Dr. R. Eckert (Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH). TTNPB is a potent synthetic RAR-selective retinoid, whereas SRI-11217 and SRI-11237 are RXR-selective synthetic retinoids (3). All other chemicals, unless otherwise specified, were obtained from Sigma Chemical (St. Louis, MO). All retinoids were prepared as 1,000× stocks in DMSO and stored in the dark at -20°C.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of retinoids on phase I and phase II responses to extracellular ATP and to other nucleotides. Extracellular ATP (50 µM) stimulates a biphasic change in GTE across CaSki cells, which is the composite of two effects (19, 23): an acute increase in the permeability of the lateral intercellular space (phase I response) and a subsequent, slower decrease of the tight junctional permeability (phase II response) (Fig. 1A). In cells incubated in retinoid-free medium, baseline GTE decreased, and the responses to ATP were abrogated (Fig. 1B). Treatment of cells with 10 nM all-trans-retinoic acid (tRA) restored baseline GTE and the phase I response to ATP (Fig. 1C); the amplitude of phase II response was restored only in part compared with cells grown in regular medium (Fig. 1, A and C). These results indicate that retinoids modulate the ATP-induced changes in permeability.


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Fig. 1.   Effects of retinoids on changes in transepithelial electrical conductance (GTE) across cultures of CaSki cells on filters in response to extracellular ATP. When indicated (arrows), ATP was added at a concentration of 50 µM to luminal and subluminal solutions from 1,000× concentrated stock (pH 7.2). Shown are tracings of changes in GTE. A: CaSki cells grown in regular medium. Early increase in GTE is phase I response, and late decrease in GTE is phase II response. B: cells were grown for 5 days in retinoid-free medium. C: cells were grown for 3 days in retinoid-free medium and then treated with 10 nM all-trans-retinoic acid (tRA) for 2 additional days. Experiments were repeated 4 times.

The effects of extracellular ATP on GTE across CaSki cells could be described in terms of activating P2Y2 nucleotide receptors (20). To determine whether retinoids also modulated the responses to other nucleotides, we repeated the experiment also with UTP, ATPgamma S, ADP, and AMP. In control cells grown in regular medium, some of these agents produced acute changes in GTE, but the effects differed in magnitude compared with ATP: in general, the order of efficacy was ATP approx  UTP > ATPgamma S > ADP and AMP, similar to our previous report (20). Incubation of cells in retinoid-free medium abolished the responses to all agonists, whereas treatment with 10 nM tRA restored the phase I responses and, in part, the amplitude of phase II responses (Table 1). These results indicate that retinoids also modulate the changes in permeability in response to other ligands of the P2Y2 receptor.

                              
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Table 1.   Retinoid effects on agonist-induced changes in GTE across CaSki cultures

Characterization of P2Y2 nucleotide receptor mRNA in human cervical cells. With the use of oligonucleotide primers complementary to a P2U nucleotide receptor cloned from human airway epithelial cells (CF/T43) (32), a single cDNA fragment (632 bp) was amplified by RT-PCR from human CaSki cells (Fig. 2A). Similar results were obtained in tissues of human endocervix and ectocervix but not in extracts of 3T3 fibroblasts (Fig. 2A). Polyacrylamide gel analysis of this product revealed a single band of 632 bp (Fig. 2B), which corresponded to the expected length based on the predicted sequence of the partial length of the P2U nucleotide receptor of human airway epithelial cells (32). This cDNA fragment was isolated, amplified, and purified, and the product was sequenced by the dideoxy chain termination method. Sequence analysis of the cloned segment revealed homologies of 98% (sense) and 96% (antisense) with the human airway epithelium P2U nucleotide receptor (32) (the differences were sequence errors; not shown).


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Fig. 2.   Characterization of P2Y2 nucleotide receptor mRNA in human cervical cells. A and B: RT-PCR results using total RNA extracted from CaSki cells, from tissues of human endocervix (Endo) and ectocervix (Ecto), and from 3T3 fibroblasts. Oligonucleotide primers complementary to a P2U nucleotide receptor cloned from human airway epithelial cells were used to amplify a single cDNA fragment of 632 bp. Samples were analyzed on either agarose gels (A) or polyacrylamide gels (B). S, sample; M, mock reaction [excluding oligo(dT) and avian myeloblastosis virus RT; see METHODS]. C: total RNA (tR) or poly(A)+ RNA (mR) extracts of CaSki cells or of human term placenta were separated on Northern gels and hybridized with radiolabeled cDNA of partial-length P2Y2 receptor. Probe hybridized to total RNA and mRNA species of 4.6, 3.0, 2.2, and 2.0 kb (CaSki) and of 4.6 and 2.0 kb (placenta). Experiments were repeated 3-6 times. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Expression of the P2Y2 mRNA receptor in human CaSki cells was also analyzed by hybridization of the cloned segment of the P2Y2 receptor to total RNA and mRNA. Northern analyses revealed that the radiolabeled cDNA of the partial-length P2Y2 receptor hybridized to total RNA and mRNA species of 2.0, 2.2, 3.0, and 4.6 kb (Fig. 2C; see also Figs. 5 and 6). The 2.0-kb band was most abundant, whereas the other three were present at lower levels, and their relative expression differed among the experiments. The 2.0- and 4.6-kb species, and perhaps the 2.2-kb species, were also present in total RNA and mRNA of human placenta (Fig. 2C). Collectively, these results indicate that human cervical cells express a P2Y2 receptor mRNA.

Regulation by retinoids of P2Y2 nucleotide receptor mRNA in human cervical cells. Treatment of CaSki cells with 10 nM tRA for 2 days had no effect on the expression of GAPDH mRNA, but it increased the expression of the P2Y2 receptor mRNA (the 632-bp band in RT-PCR assays; Fig. 3). Results of four densitometry experiments revealed that expression of the P2Y2 receptor mRNA relative to that of GAPDH mRNA increased 2.5 ± 0.4-fold with the addition of tRA, compared with control cells.


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Fig. 3.   Effects of retinoids on expression of P2Y2 receptor mRNA in CaSki cells: RT-PCR results. Two days after plating, cells were grown for 3 days in regular medium or in retinoid-free medium and then treated either with 10 nM tRA (+RA) or with vehicle (C; control) for 2 additional days. Experiments were repeated 4 times.

To better understand the effect of retinoids on P2Y2 mRNA receptor expression, cells were cultured for 3 days in retinoid-free medium and then treated with tRA for 2 additional days. Neither incubation in retinoid-free conditions nor treatment with tRA had any significant effect on the GAPDH mRNA (Fig. 3). In contrast, retinoids modulated the expression of the P2Y2 receptor mRNA. In cells grown in retinoid-free medium, the ratio expression of P2Y2 receptor mRNA to GAPDH mRNA decreased, significantly, 3.3 ± 0.4-fold compared with that in cells incubated in regular medium (Fig. 3). Treatment of cells cultured in retinoid-free medium with tRA increased the density of the P2Y2 receptor mRNA significantly: the ratio of P2Y2 receptor mRNA to GAPDH mRNA increased 10.2 ± 0.4-fold compared with cells grown in retinoid-free medium and by 3.5 ± 0.4-fold compared with cells grown in regular medium and not treated with tRA (Fig. 3).

To ascertain that the RT-PCR technique is sensitive in measuring changes in the expression of the P2Y2 receptor, the experiment was repeated using different amounts of cDNA for the PCR amplification. As is shown in Fig. 4 and summarized in Table 2, the quantity of the amplified products of both the GAPDH and the P2Y2 receptor cDNA was dependent on the amount of cDNA used for the amplification. Treatment with tRA had no effect on GAPDH RNA, but it increased P2Y2 receptor RNA (Fig. 4). Subsequently, and regardless of the amount of initial cDNA used for the PCR, the ratio of P2Y2 receptor RNA to GAPDH RNA increased following treatment with tRA by ~4.5-fold compared with retinoid-free conditions (Table 2).


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Fig. 4.   Effects of retinoids on expression of P2Y2 receptor mRNA in CaSki cells: RT-PCR results. Two days after plating, cells were shifted to retinoid-free medium and then treated either with 10 nM tRA (+RA) or with vehicle (C; control) for 2 additional days. For PCR, 375 ng (1), 75 ng (1:5), and 15 ng (1:25) of cDNA were used in each group, both for P2Y2 receptor cDNA and for GAPDH. Experiment was done twice, and results are summarized in Table 2.

                              
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Table 2.   Retinoid effects on expression of P2Y2 receptor mRNA in CaSki cells: RT-PCR experiments

To confirm these findings, we used Northern analyses to study the effects of retinoids on the expression of the P2Y2 receptor total RNA. Incubation in retinoid-free medium decreased the density of the 2.0-, 2.2-, 3.0-, and 4.6-kb bands but did not have a significant effect on the levels of GAPDH total RNA (Figs. 5 and 6). Densitometry of the bands revealed that incubation in retinoid-free medium decreased the 2.0-, 2.2-, and 4.6-kb products relative to GAPDH by 80-90% (Table 3). The 3.0-kb product was minimally expressed and therefore it was difficult to quantify the changes (Fig. 5). Treatment with tRA upregulated the 2.0-, 2.2-, and 4.6-kb bands to levels observed in cells incubated in regular medium (Figs. 5 and 6, Tables 3 and 4).


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Fig. 5.   Effects of retinoids on expression of P2Y2 receptor RNA in CaSki cells: Northern analysis. Two days after plating, cells were grown for 3 days in regular medium (C/RM) or in retinoid-free medium and then treated with vehicle (C; control) or with 10 nM of 1 of following retinoids for 2 additional days: tRA (RA), TTNPB, SRI-11217, or SRI-11237. Experiment was done twice, and results are summarized in Table 3.


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Fig. 6.   Effects of retinoids and of actinomycin D on expression of P2Y2 receptor RNA in CaSki cells: Northern analysis. Two days after plating, cells were grown for 5 days in regular medium (C/RM) or shifted to retinoid-free medium for 3 days. Latter were then treated with 10 nM of tRA (RA) or with vehicle (C; control) for 2 additional days. Actinomycin D (10 µM) was added for 1-4 h before extraction of RNA. Experiment was done twice.

                              
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Table 3.   Retinoid effects on expression of P2Y2 receptor mRNA in CaSki cells: Northern blot experiments

                              
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Table 4.   Effects of retinoic acid and of actinomycin-D on expression of 2.0-kb band of P2Y2 receptor mRNA in CaSki cells: Northern blot experiments

Role of RAR and of RXR. To study the specificity of retinoid effects on the P2Y2 receptor RNA, we compared the effects of tRA to those of other retinoids, all added at a concentration of 10 nM. TTNPB, an RAR-selective ligand, and SRI-11217 and SRI-11237, RXR-selective ligands, upregulated the 2.2- and 4.6-kb bands significantly and upregulated the 2.0-kb bands nearly to the control levels observed in cells grown in regular medium (Fig. 5, Table 3). Collectively, the results shown in Figs. 3-6 indicate that incubation of CaSki cells in retinoid-free medium decreases the density of the P2Y2 receptor mRNA and that agonists of both the RAR and RXR receptors can upregulate the P2Y2 receptor mRNA.

Mechanisms of the effects of retinoids. Retinoids modulate cell functions via several known mechanisms, including transcription regulation (13). The objective of the next experiment was to study the effects of the transcription inhibitor actinomycin D on the tRA-induced expression of the P2Y2 receptor RNA. Cells were shifted to retinoid-free medium for 3 days and then treated with 10 nM tRA or the vehicle (control) for 2 additional days. Actinomycin D (10 µM) was added for 1-4 h before extraction of total RNA. Treatment with actinomycin D decreased the expression of the 2.0-, 2.2-, 3.0-, and 4.6-kb bands of the P2Y2 receptor RNA, and of the GAPDH RNA, in a time-related manner, both in control cells and in cells treated with tRA (Fig. 6). Because the expression of the 2.2-, 3.0-, and 4.6-kb species was weak, we considered changes only in the 2.0-kb band for densitometric analysis. As is shown in Fig. 6 and summarized in Table 4, treatment with actinomycin D decreased the expression of the 2.0-kb transcript of the P2Y2 receptor RNA, and of the GAPDH RNA, with time. However, the density of the 2.0-kb band relative to GAPDH did not change over the 4-h period of incubation with actinomycin D either in control cells or in cells that were treated with tRA (Fig. 6, Table 4).

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Extracellular ATP and UTP stimulate acute changes in paracellular permeability across cultured human cervical epithelia (Ref. 19 and present study), and the responses can be modulated by retinoids (22). Previous studies (20, 21), using pharmacological criteria, described the effects in terms of activating P2Y2 receptor(s) [formerly designated P2U or P2N, (1, 6, 7)]. The present results describe, for the first time, the expression and regulation by retinoids of a nucleotide receptor mRNA in human cervical epithelial cells. On the basis of the experimental findings, we suggest that the newly described receptor mediates the ATP- and UTP-induced changes in transcervical paracellular permeability.

Cervical cells expressed at least four distinct transcripts that cross-hybridized with the partial-length P2Y2 receptor cDNA, but the most abundant was the 2.0-kb transcript. Other human tissues also express a number of different mRNA for the P2Y2 receptor (32), but no definitive conclusion was reached as to whether these are unprocessed or alternatively spliced forms of the receptor mRNA or whether they are products of distinct genes. The relative abundance of the 2.0-kb band, the variability in the expression of the 2.2-, 3.0-, and 4.6-kb transcripts among different experiments, and the parallel changes in all four bands in response to vitamin A deprivation and to retinoid treatment suggest that in CaSki cells the different bands represent intermediary steps in receptor mRNA processing. However, the possibility that these bands are alternatively processed forms of the same gene, or products of different genes, has not been ruled out and is currently being studied.

Despite the important role that nucleotide receptors may have for cell function in vivo, only a few studies have focused on regulation of the responses to extracellular nucleotides and of the cellular content of the nucleotide receptors. Hung et al. (26) reported that treatment of chondrocytes isolated from chick embryo sternum with nanomolar concentrations of retinoic acid stimulated responsiveness to ATP in the form of increases in cytosolic calcium. The authors hypothesized that retinoic acid upregulated a nucleotide receptor and that the effect plays a role in chondrocyte differentiation. Martin et al. (29) reported that phorbol esters or inflammatory activators downregulate P2U nucleotide receptor mRNA expression during in vitro differentiation of human myeloid leukocytes.

Retinoids modulate changes in paracellular permeability across human cervical cells induced by extracellular ATP. It was suggested that retinoids modulate a proximal step (or steps) in the ATP-dependent stimulus (22), possibly the ATP receptor. The present results support this hypothesis and indicate that retinoids control the expression of P2Y2 receptor mRNA in CaSki cells. Incubation of cells in retinoid-free medium decreased the expression of the P2Y2 receptor mRNA, whereas treatment with physiological levels of retinoic acid increased it. These results correlated with the effects of retinoids on paracellular permeability, namely that incubation in retinoid-free medium abrogated the phase I and phase II responses to ATP and UTP, whereas treatment with retinoic acid restored the effects (present results and Ref. 22). These findings suggest that the retinoid-induced changes in permeability are the result of modulation of the P2Y2 receptor expression.

The effects of retinoids on gene expression are mediated via interaction with the nuclear retinoid receptors. The nuclear retinoid receptor family consists of six members, RAR alpha , beta , and gamma  and RXR alpha , beta , and gamma  (13), that function as ligand-activated trans-acting factors. RAR and RXR receptors are expressed in human cervical cells (2, 3, 25), and their cellular levels can be modulated by retinoids (3). The effects of retinoids on the transcervical permeability responses to ATP are also mediated via the retinoid receptor mechanism (15, 22). The present results extend these findings and indicate that both RAR-selective ligands (e.g., TTNPB) and RXR-selective ligands (e.g., SRI-11217 and SRI-11237) can upregulate the P2Y2 receptor. Collectively, these findings suggest that the retinoid regulation of the P2Y2 receptor and the responses to ATP are downstream from the retinoid receptor(s). RAR-selective ligands are important regulators of gene expression and differentiation in cervical cells (2, 25). In contrast, RXR ligands have been shown to be relatively inactive (3, 25). This does not appear to be the case for the effects on paracellular resistance (15), on the ATP-induced increase in permeability (22), and on the expression of the P2Y2 receptor (present study), as SRI-11217 and SRI-11237 (selective ligands of the RXR) were effective regulators in CaSki cells. Our results therefore suggest a role for RXR-specific receptors in regulation of cervical cell nucleotide receptors and paracellular permeability.

One of our objectives was to study the mechanisms by which retinoids modulate expression of the P2Y2 receptor. In cells incubated in retinoid-free medium and treated with tRA, actinomycin D decreased significantly the expression of both the P2Y2 receptor RNA and the GAPDH RNA, but the density of the P2Y2 receptor relative to GAPDH did not change significantly. These results support the hypothesis that retinoids upregulate P2Y2 receptor mRNA by stimulating transcription rather than by increasing the stability of the message.

Nucleotide receptor regulation of paracellular permeability and the effects of retinoids may be important for understanding regulation of transcervical transport in vivo, because even small changes in transepithelial permeability may have profound effects on secretion of fluid and solutes and subsequently on the production of cervical mucus (14). Under baseline conditions, the extracellular levels of ATP are too low to effectively modulate transcervical paracellular resistance. However, under certain conditions, ATP can reach micromolar concentrations that are sufficient to activate the cervical cell P2Y2 receptor and to modulate permeability (5, 11). These include sudden breakage of intact cells, exocytotic secretion of ATP from nerve endings or from nonneuronal cells (such as platelets, mast cells, and basophilic leukocytes), release of ATP from T lymphocytes following stimulation of cytolysis-dependent pore-forming proteins, release of ATP from the putative ATP-binding cassette ("ABC") transporters (possibly the multidrug resistance gene; Ref. 11), and release of ATP in seminal fluid after intercourse (28). Cervical epithelial cells therefore can be exposed to episodic release of ATP into the extracellular space, which can activate the P2Y2 receptor, modulate paracellular permeability, and affect transcervical transport.

Our findings that retinoids modulate transcervical permeability and the expression of P2Y2 receptor in cervical cells may be important for the overall understanding of cervical mucus production in vivo. Vitamin A deficiency leads to dryness of mucous membranes, including the endocervix in women (9, 10, 12). According to our current understanding of transepithelial transport, these phenomena are the result of diminished transudation of fluids from the blood into the lumen across the cervical epithelium. In contrast to vitamin A deficiency, treatment with vitamin A or with retinoids reverses the condition (9, 10, 12). Retinoids increase baseline transcervical permeability (15) and upregulate the ATP-induced increase in permeability (22), possibly as a result of increasing the P2Y2 receptor (present results). This would tend to increase the permeability and augment secretion of cervical fluid. Retinoids also upregulate the ATP-induced late decrease in permeability (22), a response that may have an opposite effect on cervical mucus. However, the latter effect is less sensitive to retinoids than the ATP-related increase in permeability (22), and the overall effect of retinoids would therefore tend to increase transport across the cervical epithelium.

Plasma levels of vitamin A in the human are tightly regulated (9, 12), and changes in cervical permeability in vivo are unlikely to be determined by changes in plasma levels of vitamin A per se. Possible explanations are changes in the rate of vitamin A conversion to more active metabolites (e.g., tRA) or the status of tissue retinoid receptors. The present results suggest an additional mechanism, notably regulation of transcription of P2Y2 receptor mRNA and modulation of the expression of the P2Y2 receptor in cervical cells.

    ACKNOWLEDGEMENTS

This study was supported by National Institute of Child Health and Human Development Grants HD-00977 and HD-29924 to G. I. Gorodeski.

    FOOTNOTES

Present address of P. Burfeind: Institute of Human Genetics, Georg-August-University, Gosslerstr. 12D, D-37073 Göttingen, Germany.

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: G. I. Gorodeski, University MacDonald Women's Hospital, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106.

Received 8 April 1998; accepted in final form 3 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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