Expression, regulation, and function of P2X4 purinergic receptor in human cervical epithelial cells

George I. Gorodeski

Department of Obstetrics and Gynecology, University MacDonald Women's Hospital, University Hospitals of Cleveland, and Departments of Reproductive Biology and Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Micromolar concentrations of ATP stimulate biphasic change in transepithelial conductance across CaSki cultures on filters, an acute transient increase (phase I response; triggered by P2Y2 receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability (phase II response). Phase II response is mediated by augmented calcium influx and protein kinase C-dependent increase in tight junctional resistance. The objective of the study was to determine the role of P2X4 receptor as a mediator of phase II response. Human cervical epithelial cells express P2X4 receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms by Northern blot analysis) and P2X4 protein. Depletion of vitamin A reversibly downregulated P2X4 receptor mRNA and protein and ATP-induced calcium influx. Depletion of vitamin A abrogated phase II response, and the effect could be partially reversed only with retinoic acid receptor (RAR)-selective retinoids but not retinoid X receptor (RXR) agonists. Depletion of vitamin A also abrogated protein kinase C increase in tight junctional resistance, and the effect could not be reversed with retinoids. Depletion of vitamin A also abrogated phase I increase in permeability and reversibly downregulated P2Y2 receptor mRNA and ATP-induced calcium mobilization. However, in contrast to phase II response, both RAR and RXR agonists could fully reverse those effects. These results suggest that phase II response is mediated by a P2X4 receptor mechanism.

P2 purinergic receptor; cervix; epithelium; paracellular permeability; transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EPITHELIAL CELLS of the uterine cervix regulate secretion of cervical plasma, which is important for human reproduction and women's health (15). Cervical epithelia, like other types of secretory epithelia, are organized as layers of confluent cells that restrict free movement of fluid and solutes from the blood into the lumen. Molecules can move across the epithelia either through the cells (transcellular route) or via the intercellular space (paracellular route). Human cervical cells form relatively leaky types of epithelia, and their overall permeability properties are determined by the paracellular route.

Movement of fluid and solutes in the paracellular route is determined by the resistance of the lateral intercellular space (RLIS) and by the resistance of the intercellular tight junctions (RTJ) in series (28, 45, 52). In cultured human cervical epithelia RLIS and RTJ can be independently regulated (24, 27, 28). Experimentally, micromolar concentrations of ATP stimulate acute biphasic change in paracellular permeability across cultured human cervical epithelia, an initial increase in permeability due to decreased RLIS followed by a slower, sustained decrease in permeability due to increased RTJ (22). Effects triggered by micromolar concentrations of ATP usually involve activation of P2Y and/or P2X purinergic receptors (24, 34, 44). In the female reproductive tract purinergic receptors regulate endometrial (2), myometrial (2, 4, 14, 41, 46, 48), and fallopian tube (9, 50) functions. The permeability effects of ATP in cervical epithelial cells suggest that extracellular ATP is a potent regulator of transepithelial transport of solutes and fluid across the human cervical epithelium (22, 24, 26, 27, 52).

Functional studies indicated that the effects of ATP in the cervix could be described in terms of activation of P2 receptor(s) located on the apical (luminal) cell surface (22, 24-26). More recent studies showed expression of P2Y2 (19) and P2X4 receptors (18) and suggested activation of RLIS and RTJ, respectively. The possibility that P2X4 receptor triggers an acute decrease in permeability was suggested on the basis of the finding that ATP-induced decrease in permeability is triggered by calcium influx (25) and the knowledge that P2X receptors are ligand-gated ionotropic channels that, on activation, trigger calcium influx (44).

P2X receptors are multimeric, fast-response, membrane-bound, ligand-operated K+-, Na+-, and Ca2+ (and perhaps other cations)-permeable channels. In vivo they can be activated by extracellular ATP both from nerve terminals and from local tissue sources (24, 34, 44). At present, all seven cloned P2X receptor isoforms share a common structural motif characterized by two transmembrane spanning domains connected by an extracellular loop (24, 34, 44). P2X receptors are expressed in excitable and nonexcitable cells and can mediate a variety of physiological actions in a cell-specific manner. These include smooth muscle contractility, neuroendocrine secretion, modulation of synaptic transmission, transmission of pain perception, and regulation of cell functions such as metabolism, DNA synthesis and proliferation, differentiation, and necrotic and apoptotic death (8, 11, 12, 24, 33-35, 40, 44). Stimulation of P2X receptors and augmented calcium influx can activate secondary signaling of phosphatidylinositides (36), ion transporters (47), protein kinases (37), and nonpurinergic calcium channels; cytoskeleton changes involving cytoskeletal proteins and microtubule transport of membrane-bound organelles (32, 36); myofilament contraction (3); exocytosis and synaptic vesicle release of neurotransmitters (48); release of nitric oxide (13); and modulation of growth factors (1).

Recent studies show expression of P2X receptors in rat uterine tissues (4, 48) and expression of P2X4 receptor in human cervical epithelial cells (18), but relatively little is known about function of these receptors in these tissues. The objective of the present study was to test the hypothesis that the ATP-induced decrease in permeability (increased RTJ) is mediated by a P2X4 receptor mechanism.


    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 a histologically normal cervix. After removal of the uterus, 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 (28), were grown and maintained in regular medium enriched with 8% fetal calf serum as described previously (28). In some experiments, cells were shifted to a medium enriched with fetal calf serum that was delipidized (retinoid-free medium) (20, 29). Human ectocervical epithelial cells (hECE) and murine 3T3 fibroblasts were cultured as described previously (21). For transport experiments, cells were plated and grown on filters (28) and all drugs were added to the luminal and subluminal solutions.

Determinations of changes in cytosolic calcium in dispersed cells. Changes in cytosolic calcium were determined in cells on filters as described previously (30). Briefly, cells were plated on Millicel-CM filters for 4-5 days, harvested, and loaded with 1 µM fura 2. Cuvettes containing cells (106 cells/1.5 ml medium) were used in a filter fluorimeter, and changes in cytosolic calcium were determined as described previously (30).

Measurements of transepithelial electrical conductance. Measurements of transepithelial electrical conductance (GTE), including calibrations, controls, and conditions for optimal determinations of GTE across low-resistance epithelia, were performed as described previously (27). Briefly, changes in GTE were determined in Ussing chambers from successive measurements of the transepithelial electrical current (Delta 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 transepithelial potential difference (Delta PD; lumen negative) as GTE = Delta I/Delta PD. In CaSki cells determinations of GTE correlate well with changes in fluxes across the paracellular pathway (28).

RNA isolation. RNA isolation and preparation of poly(A)+ RNA were described previously (19). For Northern blots we used 20 µg of total RNA or 10 µg of poly(A)+.

Reverse transcriptase-polymerase chain reaction. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as described previously (19). We used 1.5 µg of total RNA. The following oligonucleotide primers were used: human P2Y2 receptor (43): forward (sense) 5'-CTC TAC TTT GTC ACC ACC AGC GCG-3' (nucleotides 750-773), reverse (antisense) 5'-TTC TGC TCC TAC AGC CGA ATG TCC-3' (nucleotides 1364-1387); human P2X4 purinergic receptor (GenBank accession no. AF000234): forward (sense) 5'-CTC TGC TTG CCC AGG TAC TC-3' (nucleotides 705-725), reverse (antisense) 5'-CCA GCT CAC TAG CAA GAC CC-3' (nucleotides 1059-1039). The sequences used to amplify glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment were GAPDH forward (sense) 5'-TGA AGG TCG GAC TCA ACG GAT TTG GT and GAPDH reverse (antisense) 5'-GTG GTG GAC CTC ATG GCC CAC ATG-3'. The sequences used to amplify the primers were synthesized by the Molecular Biology Core Laboratory at Case Western Reserve School of Medicine and were prepared as 10 µM stocks. The following PCR conditions were applied: P2Y2 receptor: 94°C for 5 min, followed by 30 cycles of 1-min 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 (expected cDNA length 632 bp); P2X4 receptor: 94°C for 45 s, 35 cycles of 1 min at 94°C, 45 s at 60°C, and 1 min at 72°C, followed by 10 min at 72°C (expected cDNA length 355 bp); GAPDH: 94°C for 5 min, 30 cycles of 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C (expected cDNA length 932 bp). Samples were cooled at 4°C (soak file) and frozen at -80°C.

To validate that RT-PCR can yield semiquantitative estimates of changes in mRNA, the following steps were taken. 1) The cDNAs of the P2Y2 and P2X4 receptors and GAPDH were amplified in parallel tubes, and changes in P2Y2 and P2X4 receptors RNA were determined relative to the changes in GAPDH RNA. 2) Parallel experiments were routinely done using DNase I before RT, to exclude amplification of genomic cDNA contaminants. 3) In control experiments, GAPDH was amplified using different doses of GAPDH cDNA to determine the semiquantitative changes in the amplification rates (19). 4) For the P2Y2 and P2X4 receptors and GAPDH we used PCR cycles that resulted in synthesis reaction that did not reach plateau, indicating amplification conditions for log-phase synthesis (17).

Northern blot analysis. Northern blot analysis using total RNA or mRNA was performed as described previously (19). RNA was separated on a 1% denaturing agarose gel and transferred to a Hybond-N nylon membrane (Amersham, Cleveland, OH), crosslinked by brief exposure to ultraviolet light, or baked at 80°C. The cDNA probes of the P2X4 receptor (see below) and 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) sodium dodecyl sulfate (SDS), and 100 µg/ml denatured salmon sperm DNA at 65° 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.

Western blot analysis. Western blot analysis was performed as described previously (21). Briefly, aliquots of cell lysate (normalized to 10 µg protein) were loaded on 12% SDS-PAGE gels and then transferred to nitrocellulose. Blots were probed with 1.5 µg/ml of rabbit anti-P2X4 antibody. Preadsorption controls were performed by incubating the antibodies to 50 µg/ml of the P2X4 control antigen peptide for 1 h at room temperature. Anti-rabbit peroxidase-conjugated secondary antibody was used for visualization (ECL kit, Amersham). To determine expression of actin, membranes were deblotted by serial washings and blotted with 2 µg/ml of mouse anti-actin antibody.

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

Cloning and sequencing of partial P2Y2 and P2X4 receptor PCR fragments. RT-PCR of CaSki cells total RNA using oligonucleotide primers specific to the P2Y2 and P2X4 receptors yielded single bands of 632- and 355-bp fragments, respectively. The fragments were extracted from a 1% agarose gel and purified using a Qiagen Gel extraction kit (Qiagen, Chatsworth, CA) according to the manufacturer's protocol and cloned into the HincII site of pGEM3z vector (Promega, Madison, WI). After transformation of DH5alpha cells, plasmid DNA was prepared from single colonies using a miniprep kit (Promega) with the manufacturer's protocol. The subcloned partial P2Y2 and P2X4 receptor DNA sequences were then sequenced in both directions using the P2Y2 and P2X4 receptor forward and reverse primers described in Reverse transcriptase-polymerase chain reaction. Sequencing was performed by the core molecular biology facility at Case Western Reserve University with an ABI373A automated sequencer. The sequences were checked against the GenBank databases using the NCBI BLAST network services and were confirmed to be homologous to the P2Y2 purinergic receptor (accession no. U07225/HSU07225) and the P2X4 purinergic receptor (accession no. AF000234).

45Ca2+ influx. Cells on filters were shifted for 1 min at 37°C to modified Ringer buffer composed of (in mM) 120 NaCl, 5 KCl, 10 NaHCO3 (before saturating with 95% O2-5% CO2), 1.2 CaCl2, 1 MgSO4, 5 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid HEPES, pH 7.4, with 0.1% bovine serum albumin in volumes of 4.7-5.2 ml in the luminal and subluminal compartments. For experiments, fresh buffer supplemented with 45CaCl (2 µCi/ml) was added for 3 min of preincubation at 37°C. Agonists were added to the solution from concentrated (1,000×) stocks to the luminal and subluminal compartments. At the end of incubation, the medium was removed and the filter was dipped three times in 3 liters of ice-cold Ca2+ free buffer to remove extracellular isotope. Fresh buffer (0.5 ml) containing 0.3% (vol/vol) Triton X-100 was then added to the filter to release the isotopes into the medium. After 5 h, the detergent extracts were removed. One-milliliter aliquots of buffer were used to rinse the filter. Ten-microliter aliquots were separated for DNA measurements, and radioactivity was determined in the remaining solution and expressed per milligram of DNA.

Cellular levels of DNA. Cellular levels of DNA were measured as described previously (28).

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 (Dynamic Microsystems, Silver Spring, MD) and analyzed with ANOVA.

Chemicals and supplies. Anocell (Anocell-10) filters were obtained from Anotec. Fura 2-AM was obtained from Molecular Probes (Eugene, OR). TTNPB and SR11217, synthetic RAR-selective and RXR-selective synthetic retinoids, respectively (29), were synthesized in the Department of Chemistry, Allergen, Inc. (Irvine, CA) and were a gift of Dr. R. Eckert (Dept. of Physiology and Biophysics, Case Western Reserve Univ. School of Medicine, Cleveland, OH). All other chemicals, unless specified otherwise, were obtained from Sigma (St. Louis, MO). All retinoids were prepared as 1,000× stocks in dimethyl sulfoxide and stored in the dark at -20°C. Monoclonal IgM mouse anti-actin antibody that recognizes human beta -actin was obtained from Zymed Laboratories (San Francisco, CA). Polyclonal rabbit anti-P2X4 raised against the purified peptide (C)KKYKYVEDY EQGLSGEMNQ [P2X4370-388, corresponding to residues 370-388 of rat P2X4 (49) with additional NH2-terminal cysteine] as well as the P2X4 control antigen peptide were obtained from Alomone (Jerusalem, Israel).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ATP phase I and phase II responses. Micromolar concentrations of ATP stimulate biphasic change in GTE across CaSki cultures on filters, an acute transient increase (phase I response) followed by a slower decrease in permeability (phase II response) (Fig. 1; Table 1). The biphasic change in permeability is the result of activation of two distinct and independent mechanisms. Phase I response is the result of calcium mobilization-dependent cell volume decrease and decrease in RLIS; phase II response is mediated by augmented calcium influx via voltage-dependent, dihydropyridine-sensitive calcium channels followed by release of diacylglycerol and activation of protein kinase C-dependent increase in RTJ (Fig. 1; Refs. 18, 22, 24, 25). It was previously suggested that phase I response is triggered by activation of P2Y2 receptor (18, 19). The objective of the present study was to determine the role of P2X4 receptor as a mediator of phase II response.


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Fig. 1.   Effects of extracellular ATP (50 µM) on transepithelial electrical conductance (GTE) in cultures of human cervical epithelial CaSki cells on filters. Circles show an actual recording. Solid lines describe the ATP-induced changes in GTE in terms of 2 independent effects (phases I and II) that differ in time course, direction, and magnitude.


                              
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Table 1.   Retinoids modulate ATP effects on cytosolic calcium and GTE

Expression of P2Y2 and P2X4 receptor mRNA and protein. With oligonucleotide primers complementary to the human P2X4 receptor, a single cDNA fragment (355 bp) was amplified by RT-PCR from human CaSki cells (Fig. 2). Similar results were obtained in extracts of human endocervix and ectocervix as well as in lysates of cultured hECE (Ref. 21; Fig. 2) but not in extracts of murine 3T3 fibroblasts (not shown). Polyacrylamide gel analysis of this product revealed a single band of 355 bp (not shown), which corresponded to an expected length based on the predicted sequence of the partial length of the human P2X4 receptor. 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 99% (sense and antisense) with the human P2X4 (the differences were sequence errors; not shown). Figure 2 also shows, for comparison, the expression of a 632-bp cDNA fragment corresponding to the human P2Y2 receptor in extracts of human endocervix and ectocervix and in lysates of CaSki and hECE cells; the latter result confirms our previous study (19).


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Fig. 2.   Human cervical cells express mRNA of P2X4 receptor (A) and P2Y2 (B) receptors. RT-PCR results using total RNA extracted from tissues of normal human ectocervix (EctoCx) and endocervix (EndoCx) and from cultured normal human ectocervical (hECE) and CaSki cells. Oligonucleotide primers complementary to cloned human P2Y2 and P2X4 receptors were used to amplify single cDNA fragments of 355 and 632 bp, respectively. The experiments were repeated 5 times. M, markers.

Expression of the P2X4 mRNA receptor in human CaSki cells was also analyzed by hybridization of the cloned segment of the P2X4 receptor to total RNA and poly(A)+ RNA. As shown in Fig. 3, Northern blot analysis revealed that the radiolabeled cDNA of the partial-length P2X4 receptor hybridized to poly(A)+ RNA species of 1.4, 2.2, and 4.4 kb. The 1.4-kb band was most abundant, whereas the other two were present at lower levels. Similar results (not shown) were obtained using total RNA. These results indicate that human cervical epithelial cells express P2X4 receptor mRNA.


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Fig. 3.   Expression of P2X4 receptor RNA in CaSki cells and regulation by retinoids: Northern blot analyses using poly(A+). Cells were grown in filters in regular medium (RM) or shifted to retinoid-free medium (RFM) for 6 days and replated on filters in the same medium for 6 additional days. Two days before experiments cells were treated with 10 nM all-trans-retinoic acid (RM + RA, RFM + RA) or vehicle (RFM). The r-RNA is from the total RNA extract. The experiment was also repeated 3 times with total RNA, and the results are summarized in Table 3.

Expression of the P2X4 receptor protein in human CaSki cells was analyzed with the use of polyclonal rabbit anti-P2X4 raised against the purified peptide (C)KKYKYVEDY EQGLSGEMNQ (P2X4370-388, corresponding to residues 370-388 of rat P2X4 with additional NH2-terminal cysteine) (49). Western immunoblot analysis of cells lysates revealed a single band of 57 kDa (Fig. 4). No bands could be detected when the primary antibody used for Western blot was preincubated with the control antigen peptide (not shown). These results indicate that human cervical epithelial cells express the P2X4 receptor.


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Fig. 4.   Human cervical cells express P2X4 receptor protein: Western immunoblots with polyclonal rabbit anti-P2X4. Experiments were done as in Fig. 3 (see METHODS for details). The experiment was repeated 3 times, and the results are summarized in Table 5.

Effects of retinoids. In human cervical epithelial cells, retinoids modulate expression of P2Y2 receptor mRNA as well as phase I and phase II responses (19, 20, 29). To determine the effects of retinoids on expression and function of the P2X4 receptor, we incubated cells in retinoid-free medium to deplete vitamin A. Depletion of vitamin A resulted in a number of effects: calcium mobilization induced by ATP was abolished without significantly affecting baseline cytosolic calcium (Fig. 5A); baseline permeability decreased and phase I response was abolished (Fig. 5B); and expression of P2Y2 receptor mRNA relative to GAPDH mRNA decreased (Fig. 6; Table 2). Treatment with 10 nM all-trans-retinoic acid (RA) reversed the effects of vitamin A depletion: it restored ATP-induced calcium mobilization (Fig. 5A), baseline permeability, and the ATP-induced phase I response (Fig. 5B), and it upregulated P2Y2 receptor mRNA/GAPDH mRNA. The effects of retinoids on expression of P2Y2 receptor mRNA were observed in cells grown on filters for 2 or 6 days (Fig. 6; Table 2).


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Fig. 5.   Retinoid effects on ATP-induced changes in cytosolic calcium (Cai; A) and GTE (B). A: cells were grown on filters in RM (C, control) or shifted to RFM for 6 days and plated on filters in the same medium for 6 additional days. Two days before experiments cultures were treated with 10 nM all-trans-RA or vehicle (RFM). Dispersed fura 2-loaded CaSki cells were treated with ATP (50 µM) or histamine (25 µM) (arrows), and changes in Cai were determined as described in METHODS. B: experiments were done as in A without treatment with fura 2-AM. Changes in GTE were determined as described in METHODS. Experiments were repeated 3-5 times, and the results are summarized in Table 1.



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Fig. 6.   Expression of P2X4 and P2Y2 receptor RNA in CaSki cells and regulation by retinoids: RT-PCR experiments. Cells were grown either on regular culture dish (CD) or on filters (F). Cells were grown in RM or shifted to RFM for 6 days and replated on filters in the same medium for 2 (F-2) or 6 (F-6) additional days. Two days before experiments cultures were treated with 10 nM all-trans-RA or vehicle. The experiment was repeated 6 times, and the results are summarized in Table 2.


                              
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Table 2.   Retinoid effects on expression of P2X4 and P2Y2 receptor mRNA in CaSki cells: RT-PCR experiments

Incubation in retinoid-free medium abolished the calcium influx induced by ATP (Fig. 5A), and treatment with 10 nM RA restored the effect in full (Fig. 5A; Table 1). The changes in cytosolic calcium determined by fura 2 fluorescence measurements were confirmed with 45Ca2+ influx measurements: incubation in retinoid-free medium abolished the ATP-induced 45Ca2+ influx, whereas treatment with RA restored it (Table 1).

Incubation in retinoid-free medium abolished phase II response (Fig. 5B). Treatment with RA restored phase II response (Fig. 5B); however, in contrast to phase I response, the magnitude of the restored phase II response was only 50% compared with cells grown in regular medium (Table 1).

Incubation in retinoid-free medium decreased expression of mRNA and protein levels of the P2X4 receptor. RT-PCR experiments showed that the ratio expression of P2X4 receptor mRNA/GAPDH mRNA decreased in cells grown in retinoid-free medium compared with cells incubated in regular medium. (Fig. 6; Table 2). Northern blot analyses confirmed those results and showed that depletion of vitamin A decreased the density of the 1.4-, 2.2-, and 4.4-kb bands compared with control cells (Fig. 3; Tables 3 and 4). Incubation in retinoid-free medium also decreased the ratio expression of P2X4 receptor protein/beta -actin compared with cells incubated in regular medium (Fig. 4; Table 5).

                              
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Table 3.   Retinoid effects on expression of P2X4 receptor mRNA in CaSki cells: Northern blot experiments


                              
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Table 4.   Retinoid effects on expression of P2X4 receptor mRNA in CaSki cells: RT-PCR experiments


                              
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Table 5.   Retinoid effects on expression of P2X4 receptor protein in CaSki cells: Western immunoblot experiments

RT-PCR experiments of lysates from cells grown in retinoid-free medium showed that treatment with RA increased the ratio expression of P2X4 receptor mRNA/GAPDH mRNA (Fig. 6; Table 2). Northern experiments in cells grown in regular medium showed that treatment with 10 nM RA significantly increased the expression of the P2X4 receptor 1.4-, 2.2-, and 4.4-kb bands relative to GAPDH. Similar results were obtained in cells grown in retinoid-free medium (Fig. 3; Table 3). The effects of retinoids on expression of the P2X4 receptor mRNA were observed in cells grown on filters for 2 or 6 days (Fig. 6; Table 2).

Western immunoblots in cells grown in regular medium showed that treatment with 10 nM RA significantly increased the ratio expression of the 57-kDa band P2X4 receptor protein/beta -actin. Similar results were obtained in cells grown in retinoid-free medium (Fig. 4; Table 5).

Role of RAR and RXR. Previous studies showed that selective agonists of both the RA receptor (RAR) and the retinoid X receptor (RXR) could upregulate P2Y2 receptor mRNA (19). RAR and RXR agonists could also restore phase I response, but only RAR-selective agonists could restore (in part) phase II response (29). To study the specificity of retinoid effects on the expression of P2X4 receptor, we studied the effects of TTNPB (RAR-selective ligand) and SR11217 (RXR-selective ligand; Ref. 29) on expression of P2X4 receptor mRNA and compared the results with effects on the P2Y2 receptor mRNA.

Similar to effects shown in Fig. 6, the ratio expression of P2Y2 and P2X4 receptor mRNA/GAPDH mRNA in cells grown in retinoid-free medium decreased compared with cells incubated in regular medium (Fig. 7; Tables 3 and 4). Treatment of cells grown in retinoid-free medium with the RAR-selective drug TTNPB increased the expression of P2Y2 and P2X4 receptor mRNA/GAPDH mRNA (Fig. 7; Table 4). Treatment with the RXR-selective drug SR11217 increased the expression of P2Y2 receptor/GAPDH mRNA but had no effect on the P2X4 receptor mRNA (Fig. 7; Table 4). These results indicate that RAR and RXR agonists can upregulate P2Y2 receptor mRNA, but only RAR agonists can upregulate P2X4 receptor mRNA.


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Fig. 7.   Expression of P2X4 and P2Y2 receptors RNA in CaSki cells and regulation by RA receptor (RAR)- and retinoid X receptor (RXR)-selective retinoids: RT-PCR experiments. Cells were grown on filters in RM or shifted to RFM for 6 days and replated on filters in the same medium for 6 additional days. Two days before experiments cultures were treated with 10 nM SR11217 (SR1), 10 nM TTNPB, or vehicle (RFM). The experiment was repeated 3 times, and the results are summarized in Table 4.

Retinoid effects on signaling distal to purinergic receptors. In cells grown in retinoid-free medium, treatment with ATP did not elicit changes in cytosolic calcium (Fig. 5A) and failed to stimulate changes in GTE (Fig. 5B). Our main hypothesis was that vitamin A deprivation downregulates the P2Y2 and P2X4 receptors. Another possibility is that vitamin A deficiency abrogates signal mechanisms distal to the receptors. To test these hypotheses it was necessary to bypass the P2 receptors and to trigger directly phase I and phase II signaling distal to the P2 receptors.

In the first experiment cells grown in retinoid-free medium were treated with histamine. In human cervical epithelial cells histamine stimulates phase I-like transient increase in GTE that is mediated by calcium mobilization-induced volume decrease and decrease in RLIS (25). A similar response was observed in cells grown in retinoid-free medium (Fig. 5; Table 1), indicating that the machinery of the calcium mobilization-induced volume decrease is not significantly affected by vitamin A deficiency.

The second experiment tested whether depletion of vitamin A abrogates kinase C-dependent modulation of RTJ. The experiments utilized diC8, a cell-permeable analog of diacylglycerol 1,2-dioctanoyl-sn-diglycerol (diC8), which can mimic protein kinase C-induced increase in RTJ (24). Similar to effects in cells grown in regular culture medium, diC8 decreased GTE also in cells grown in retinoid-free medium, as well as in cells grown in retinoid-free medium that were treated with RA, TTNPB, or SR11217 (Fig. 8; Table 1). In cells grown in retinoid-free medium, the magnitude of decrease in GTE was significantly smaller (by about half) than in cells grown in regular culture medium, regardless of treatment with any of the three retinoids (Fig. 8; Table 1). These results indicate that deprivation of vitamin A attenuates the decreases in paracellular permeability induced by diC8.


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Fig. 8.   Modulation of diC8-induced changes in GTE by retinoids. Experiments were done as in Fig. 5. Two days before experiments cultures were treated with all-trans-RA, TTNPB, or SR11217 (all at 10 nM) or with vehicle. Arrows indicate addition of 10 µM diC8. The experiment was repeated 3 times, and the results are summarized in Table 1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In cultured human cervical epithelial cells ATP stimulates calcium influx, protein kinase C-dependent increase in RTJ, and decrease in permeability (phase II response; Ref. 25). This signaling pathway differs from the ATP-induced increase in permeability (phase I response), which is mediated by calcium mobilization-dependent cell volume decrease and decrease in RLIS (24, 25). A previous study suggested that phase I response is triggered by activation of P2Y2 receptor (19). The present results support the hypothesis that activation of P2X4 receptor triggers the phase II response.

Two groups of experiments tested this hypothesis. First, we showed that human cervical epithelial cells express P2X4 receptor mRNA and protein. Human cervical epithelial cells also express P2X7 receptor mRNA and protein, but the selective P2X7 receptor agonist 2',3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (Bz-ATP; Ref. 44) did not stimulate changes in cytosolic calcium or in GTE in the first 5-10 min after its addition (not shown), indicating that phase I and phase II responses are not mediated by P2X7 receptor. In addition, human cervical epithelial cells express only insignificant amounts of mRNA for P2X1, P2X2, P2X3, P2X5, and P2X6 receptors (unpublished data). This profile differs from that of rat uterine and cervix tissues, in which all seven P2X isoform proteins could be identified (4, 14). The present experiments showed expression of a 57-kDa P2X4 receptor, similar to previous studies in the rat (38). This molecular mass is higher than that predicted from the amino acid sequence (44 kDa), and it can be explained by N-glycosylation occurring at consensus sequences in the extracellular loop (6).

The present experiments show that human cervical epithelial cells express at least three distinct transcripts of 1.4 (most abundant)-, 2.2-, and 4.4-kb mRNA fragments that cross-hybridize with the partial-length P2X4 receptor cDNA. At present it is unknown 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 1.4-kB band and the parallel changes in all three bands in response to vitamin A deprivation and 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.

The second group of experiments utilized the retinoid paradigm to dissect the effects of retinoids on expression and function of P2X4 and P2Y2 receptors. Vitamin A and the retinoids modulate growth and differentiation of human cervical epithelial cells and regulate permeability properties (7, 10, 20, 29, 39). Depletion of vitamin A abolished phase I and phase II responses, abrogated calcium signaling, and downregulated densities of P2Y2 receptor mRNA, P2X4 receptor mRNA, and P2X4 receptor protein. These results indicate that permeability responses related to ATP depend on the continuous stimulation of expression of P2Y2 and P2X4 receptors by vitamin A and the retinoids. Treatment with retinoids restored effects of retinoid depletion on permeability and expression of P2 receptors, but the potency, efficacy, and specificity of retinoid effects differed for the responses. Treatment with RA restored phase I response in full, but the amplitude of phase II response could be restored only in part (present study); shorter incubations and lower concentrations of RA were required to restore phase I response than to reverse phase II response (29); and phase I response could be restored by ligands that bind to either RAR or RXR, but only RAR agonists had an effect on phase II response (present results and Ref. 29).

The distinct permeability responses to RAR- and RXR-selective agonists also correlated with expression of P2X4 and P2Y2 receptors. Both RAR- and RXR-selective agonists could upregulate cellular levels of P2Y2 receptor mRNA; in contrast, only RAR agonists could upregulate cellular levels of P2X4 receptor mRNA and protein. The similarity of the responses to RAR and RXR ligands suggests that retinoids regulate distinctive signaling pathways induced by P2X4 and P2Y2 receptors and supports the hypothesis that P2X4 triggers phase II response whereas P2Y2 receptor triggers phase I response.

In vitamin A-depleted cells treatment with RA could restore phase II response to only half its magnitude (present results and Ref. 29). A possible explanation is that retinoids modulate not only expression of the P2X4 receptor but also a signaling pathway downstream from the receptor. Two such key signaling steps that are necessary and sufficient to decrease permeability are augmented calcium influx and protein kinase C modulation of the tight junctions (25). Treatment with RA restored ATP-induced calcium influx in full. Because augmented calcium influx is a signaling step distal to the P2X4 receptor, it is likely that failure to restore phase II response is not the result of insufficient upregulation of the P2X4 receptor. Protein kinase C modulation of RTJ is a signaling step downstream from calcium influx (25). Experiments using diC8 to activate protein kinase C showed that diC8 produced a smaller decrease in permeability in vitamin A-deprived cells than in cells grown in regular medium, and treatment with retinoids could not restore the effect. These results suggest that depletion of vitamin A is more critical for protein kinase C than for expression of the P2X4 receptor. The mechanism by which depletion of vitamin A results in only partial restoration of protein kinase C activity is at present unknown.

The concept that intact phase II response depends on "proximal" and "distal" groups of signaling, P2X4 receptor and protein kinase C-dependent modulation of RTJ, respectively, is supported by comparing ATP effects on permeability in cells 2 and 6 days in culture. P2X4 receptor mRNA is equally expressed and retinoid regulated in both types of culture conditions (present results). In contrast, the magnitude of phase II response is greater in day 6 than in day 2 cells (24), suggesting that >2 days in culture are required for the cells to develop the tight junctional machinery necessary to respond to ATP.

The experiments with histamine confirmed that retinoids exert different effects on phase I and phase II signaling pathways. Depletion of vitamin A abrogated ATP-induced phase I response but not histamine-induced (phase I like) increase in GTE. Histamine and ATP phase I response share a common signaling pathway and paracellular effector mechanisms downstream from the histamine and P2Y2 receptors (25). Both induce calcium mobilization-dependent chloride secretion and osmotic water loss, resulting in cell volume decrease and decrease in RLIS (31). Lack of modulation of histamine increase in GTE therefore indicates that retinoids do not affect calcium mobilization-dependent decrease in RLIS. Collectively, the results show that retinoids modulate distinct checkpoints along the ATP signaling pathway: P2Y2 and P2X4 receptor mRNA and protein kinase C-dependent increase in RTJ.

P2X receptors are ligand (ATP)-gated calcium channels that on activation by ATP permit influx of calcium (44). Augmented calcium influx is both necessary and sufficient to induce phase II response, and the present results suggest that phase II response is triggered by activation of P2X4 receptor. Our hypothesis therefore stipulates that phase II response depends on ATP stimulation of calcium influx via P2X4 receptor-calcium channels. Although each individual channel may respond to ATP with a time constant in the range of milliseconds (42, 44), the measured influx of calcium across the cultured epithelium is the summation of calcium influx over the entire population of the cultured cells, resulting in a time constant in the range of 2 min (18). It is possible that ATP activation of the P2X4 receptor and the ensuing calcium influx stimulate secondary signaling of augmented calcium influx via different types of calcium channels. This speculation is supported by a previous report that phase II response-related calcium influx occurs via voltage-dependent, dihydropyridine-sensitive receptor-operated calcium channels (25). However, the pharmacological profile of ATP action for activation of both phase II changes in GTE and augmented calcium influx suggests interaction with only one class of effectors (18, 24).

Phase II response could be attenuated by pretreatment with pertussis toxin (24), suggesting that the P2 receptor that mediates phase II response is coupled to a pertussis toxin-sensitive G protein. Present knowledge excludes P2X receptors from interaction with G proteins (44). In contrast, the current thinking is that P2X receptors, like other ion channels including those for acetylcholine, glutamate, GABA, glycine, and the epithelial sodium channel, assemble as homo- or heterooligomeric complexes in which different subunits associate to generate channels with unique functional properties (51). Pharmacological studies of heterologous expression of P2X receptor subunits in systems devoid of endogenous P2 receptor activity such as HEK 293 cells or Xenopus oocytes (5, 51) showed that the P2X4 isoform coassembles in a restricted fashion and only participates in forming stable assemblies with itself or the P2X5 or P2X6 isoforms but not with the P2X1, P2X2, or P2X3 isoforms (5). However, relatively little is known about P2X receptor structure and function in intact human cells, and conclusions should be drawn carefully from biochemical experiments to the physiological milieu, because native receptors do not necessarily match up well with individual subunits expressed recombinantly (51). The finding in cervical cells that phase II response is sensitive to pertussis toxin raises the possibility of chimeric complex formation between a P2X4 isoform and a pertussis toxin-sensitive P2 receptor (53), but more studies are required to test this possibility.

The present data may have clinical significance, given the potential role of ATP regulation of cervical paracellular permeability in human fertility, contraception, and health (16, 23, 25). Specifically, increases in RTJ may be important for understanding paracrine regulation of cervical plasma secretion by semen and, subsequently, sperm cell capacitation (23). Improving our understanding of purinergic regulation of permeability may provide clinicians with drugs that can target specific signaling pathways and effectors of paracellular resistance in the uterine cervix.


    ACKNOWLEDGEMENTS

The technical support of Kim Frieden, Brian De Santis, and Dipika Pal is acknowledged.


    FOOTNOTES

The study was supported by National Institutes of Health Grants HD-00977, HD-29924, and AG-15955.

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).

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. Section 1734 solely to indicate this fact.

Received 2 April 2001; accepted in final form 5 September 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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