Departments of 1Physiology and Biophysics and 2Cell Biology and 3Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 8 October 2004 ; accepted in final form 22 March 2005
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ABSTRACT |
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purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy
P2X receptor channels function as extracellular zinc- and ATP-gated, Ca2+-permeable, nonselective cation channels (23). We (13, 14, 16) and others (3, 9) have shown that human and rodent airway, gastrointestinal, and renal epithelia, epithelial cells from glands, specialized hair cell neuroepithelia from the inner ear, retinal pigment epithelia, and vascular endothelia express multiple subtypes of the P2XR gene family. In particular, epithelia and endothelia share abundant expression of P2X4 and P2X5 at the mRNA level, to the notable exclusion of other subtypes (3, 9, 13, 14, 16). P2X6 expression was not assessed by this degenerate RT-PCR approach used in previous studies by our laboratory (13, 14, 16).
Thus we hypothesized that multiple P2X receptor Ca2+ entry channel subtypes may contribute to the extracellular zinc- and ATP-induced Ca2+ signal in airway epithelia. We show here molecular, biochemical, and functional evidence, strengthened by novel small interference RNA (siRNA) approaches, for the cooperative involvement of P2X4 and P2X6 receptor channels in sustained Ca2+ signaling in human airway epithelia.
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MATERIALS AND METHODS |
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Identification of mRNA expression of P2X4 and P2X5 in IB3-1 cells and P2X6 expression in IB3-1 and 16HBE14o cells. Total RNA was extracted from epithelial cells by TRIzol reagent and reverse transcribed to cDNA as described previously (16). Standard RT-PCR of P2XR cDNA was performed (16). The specific primers for amplification of P2X4, P2X5, P2X6, and the splice variant form of P2X6 are as follows: P2X4 primers: forward primer 5'-AAC AGG CAG GTG CGT AGC TT-3', reverse primer 5'-TGA TGT CAC ACA GCA CGG TC-3' (PCR product length is 631 bp); P2X5 primers: forward primer 5'-GTG GTC ACC AAC CTG ATT GTG-3', reverse primer 5'-GGC TGC GTC TCG GTA ATA TCT-3' (PCR product length is 608 bp); P2X6 primers: forward primer 5'-GGG ATC GTG GTC TAT GTG GTA-3', reverse primer 5'-GGT TCA TAG CGG CAG TGC TTA-3' (PCR product length is 533 bp); splice variant of P2X6 specific primers: forward primer 5'-ATG CCC AGA GGT GTA AAA ACA-3', reverse primer same as above (PCR product length is 243 bp).
Gel-excised PCR products were purified with a QIAquick gel extraction kit (Qiagen, CA). The PCR products were ligated into the pGEM-T vector system (Promega, WI). Transformation was performed with JM109 competent cells (Promega), and the transformants were plated on LB-agar plates containing ampicillin (100 µg/ml), 5-bromo-4-chloro-3-indolyl -D-galactopyranoside (40 µg/ml), and isopropylthiogalactoside (100 µg/ml) for ampicillin selection of successful transformants and for blue/white selection of successful PCR product insertion into the pGEM-T vector. White colonies were picked and inoculated in LB medium containing ampicillin (100 µg/ml) and were grown up on a shaker in a 37°C warm room overnight. The replicated plasmid was isolated with a Qiagen plasmid purification mini kit. The purified PCR product insert was sequenced by a DNA sequencing core at the University of Alabama at Birmingham.
Western blot analysis. Human airway epithelial cells were lysed in a RIPA buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM pH 8.0 Tris-Cl, and a Complete mini protease inhibitor cocktail tablet. Twenty micrograms of protein were loaded and run on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. P2X4 rabbit polyclonal antibody (Alomone Laboratories) was diluted at 1:500. P2X5 and P2X6 rabbit polyclonal antibodies (Santa Cruz Biotechnology) were diluted at 1:200. The secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG diluted at 1:3,000. Enhanced chemiluminescence was used to visualize the secondary antibody.
Peptide-N-glycosidase F treatment.
Protein lysates were mixed with the denaturation solution (0.1% SDS and 0.05 M -mercaptoethanol) and heated at 100°C for 5 min to denature the protein. The deglycosylating enzyme, peptide-N-glycosidase F (PNGase F; Prozyme), was then added to the protein lysate. The final concentration was 200 IUB mU/ml. The lysates were incubated on a rocker overnight at 37°C. Treated and untreated protein was then loaded and run on a 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Subsequent methods were identical to the Western blot methods described above.
siRNA fragment construction and transient transfection. siRNA fragments targeted to the mRNAs for P2X4, P2X5, and P2X6 were designed with the help of the Ambion website. Three separate fragments, identified as the best sequences from the Ambion website software, were selected, synthesized, and purchased from Ambion. The three best fragments were each studied in parallel by Western blot and by fura-2 analysis to select the most efficacious fragment of the three. The sequences of the most effective fragments for the human mRNA of each subtype are as follows.
Sense and antisense P2X4 siRNA fragments were 5'-GGAAAACUCCCUCUUCGUCtt-3' and 5'-GACGAAGAGGGAGUUUUCCtc-3'. They recognize the mRNA sequence corresponding to the beginning of exon 3 derived from the transcription of the genomic P2X4 sequence.
Sense and antisense P2X5 siRNA fragments were 5'-GAGUGCUGUCAUCACCAAAtt-3' and 5'-UUUGGUGAUGACAGCACUCtg-3'. They recognize the mRNA sequence corresponding to the beginning of exon 5 derived from the transcription of the genomic P2X5 sequence.
Sense and antisense P2X6 siRNA fragments were 5'-GGAGAAGAGCUACAACUUCtt-3' and 5'-GAAGUUGUAGCUCUUCUCCtg-3'. They recognize the mRNA sequence corresponding to the middle of exon 9 derived from the transcription of the genomic P2X6 sequence.
Scrambled siRNA fragments were also made in parallel to these P2XR fragments and were used as a negative control.
RNAi fragments were transiently transfected into IB3-1 cells with LipofectAMINE and PLUS reagents in OptiMEM-1 medium (Invitrogen/Life Technologies) in a manner similar to that described previously for transfection of cDNA-bearing plasmids (18). Initially, Western blot analysis and fura-2 AM measurement of intracellular Ca2+ concentration ([Ca2+]i) were performed 2 days after a single 6-h transfection of siRNA on day 0. This produced "knockdown" of both protein and Ca2+ entry that averaged 3350%; however, there was some variability that hinged on transient transfection efficiency (e.g., the percentage of cells positively transfected). As published previously (17), transfection efficiency averaged 75% in IB3-1 cell cultures. This efficiency was assessed in parallel with green fluorescent protein (GFP) reporter gene transfection. We also attempted to mix siRNA fragments (two different fragments targeted to one P2XR subtype or two different fragments targeted to two different P2XR subtypes). However, this led to less knockdown rather than additive reduction. We believe that mixing multiple siRNA fragments may lead to duplex formation between fragments designed for multiple mRNA targets because we are mixing them with cationic lipids to deliver them to the cells. Therefore, we transfected the same siRNA fragment on consecutive days (days 0 and 1 before analysis on day 2). Transfection efficiency averaged 85% in IB3-1 cell cultures when monitored with GFP. Data shown in RESULTS derive from this method. We performed GFP transfection to gauge transfection efficiency, Western blot analysis for P2XR protein, and fura-2 analysis of extracellular zinc-induced Ca2+ entry in each experimental data set. We are developing methods for knocking down two P2XR subtypes within the same cell based on our results below; however, these developing methods are beyond the scope of the current manuscript.
Fura-2 fluorescence imaging of cytosolic free Ca2+. [Ca2+]i was measured with dual excitation wavelength fluorescence microscopy after cells were loaded with fura-2 AM (TefLabs) as described previously (23). A standard protocol was used for all siRNA fragment-transfected cultures. Cells were first perfused with standard Ringer solution containing (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1.5 CaCl2, and 10 HEPES at pH 7.3. After 100 s to allow a steady-state baseline, the solution was changed to an optimized saline solution for Ca2+ entry described previously (23). In this Ca2+ entry solution, N-methyl-D-glucamine (NMDG) was used as a substitute cation for Na+. This Na+-free solution was also modified to contain 3 mM CaCl2 and 0 mM MgCl2, and the pH was adjusted to 7.9 with 10 mM HEPES included. These conditions remove Na+ and Mg2+ that may compete with Ca2+ to enter the pore or block Ca2+ entry into the pore, respectively (23, 24). Alkalinization of the pH to 7.9 (and possibly the removal of Na+) allows zinc to bind with higher affinity to airway epithelial P2XRs (11, 23, 24). After 200 s in this solution, 20 µM ZnCl2 was added to the same Ca2+ entry solution to stimulate P2XRs. ATP was omitted as a coagonist in these studies so as not to contaminate Ca2+ signals with P2Y receptor-mediated Ca2+ mobilization from intracellular stores. Solutions without zinc were reperfused to fully reverse the responses.
Data analysis. Data are expressed as means ± SD and were tested for significance with unpaired Student's t-test (P < 0.05 was considered significant). We also did ANOVA analysis with a Tukey's test. We calculated the area under the curve at 500-s duration after it reached the peak. The scrambled siRNA-treated group performed on the same day is treated as 100%, and the specific P2XR siRNA-treated group is calculated as a percentage of the scrambled siRNA group. Bio-Rad Quantity One software was used to quantify protein expression of scrambled siRNA fragment and P2XR-specific siRNA-treated cell cultures via densitometric methodology.
Materials. All chemicals not described in detail above were obtained from Sigma-Aldrich.
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RESULTS |
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Because of the lack of specific P2X4 receptor agonists and antagonists, we used ivermectin to initially link activation of P2X4 and Ca2+ entry. Ivermectin is a recently described specific allosteric modulator of P2X4 that potentiates ATP stimulation, allowing discrimination from other P2X receptor channels (5). Ivermectin augmented the sustained Ca2+ entry plateau induced by 100 µM ATP in direct fura-2 measurements of [Ca2+]i (Fig. 1A). Summary data of ivermectin augmentation of the ATP-induced Ca2+ entry plateau above basal or baseline [Ca2+]i in IB3-1 CF human airway epithelial cells are shown in Fig. 1B. These initial data suggested that P2X4 might be involved in extracellular ATP-induced Ca2+ entry. However, because the potentiation effects of ivermectin were only modest, we speculated that additional P2X receptor subtypes like P2X5 and P2X6, which are less well-described P2XR channels with regard to pH and zinc sensitivity (10), may also be expressed in airway epithelia and play a role in Ca2+ entry.
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P2X5 and P2X6 protein expression in airway epithelial cells. Our laboratory showed previously (23) that non-CF and CF airway epithelial cells express P2X4 protein. To provide evidence that P2X5 and P2X6 receptor channel proteins are also expressed in human airway epithelial cells, we performed Western blot analysis. Protein lysates from IB3-1 and 16HBE14o cells were probed with an anti-P2X5 polyclonal antibody and an anti-P2X6 polyclonal antibody. Figure 3, A and B, show the positive results for P2X5 and P2X6 receptor channel protein in both cell lines. Bands of the expected size were blotted. A rabbit IgG negative control is shown in Fig. 3C and indicates that the signal is due solely to primary antibody binding. These data show that human airway epithelial cells also express P2X5 and P2X6 protein. We also used P2X4 and P2X6 blocking peptides to block the protein signal and illustrate the specificity of these subtype-specific antibodies (Fig. 3, G and H).
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Molecular definition of epithelial P2XR subtypes mediating Ca2+ entry triggered by extracellular zinc. To examine further the relative roles of P2X4, P2X5, and P2X6 in the zinc-induced Ca2+ entry mechanism, IB3-1 cells were transiently transfected on consecutive days with the most effective P2X4 siRNA fragment. Compared with scrambled siRNA fragment-transfected controls, transient transfection of P2X4 siRNA constructs inhibited the sustained Ca2+ entry plateau induced by zinc (Fig. 4A). Summary data for the zinc-induced cell Ca2+ increase for both scrambled siRNA fragment- and P2X4 siRNA fragment-transfected cells are shown in Fig. 4B. The area under the curve is calculated as the cytosolic Ca2+ increase measured in scrambled siRNA-treated cultures and is assigned a 100% value. The area under the Ca2+ entry curve of the P2X4 siRNA-transfected cells is reduced by 55% compared with the scrambled siRNA-transfected controls. RNA interference also caused significant knockdown of P2X4 protein in parallel immunoblotting for P2X4 in IB3-1 CF cells (Fig. 4C). An average of 67% protein knockdown of the glycosylated form of P2X4 protein and 85% protein reduction of the nonglycosylated forms was observed. Together, these data suggest that P2X4 plays a significant role in extracellular zinc-induced sustained Ca2+ entry. However, despite almost complete reduction of both forms of the P2X4 protein, zinc-induced Ca2+ entry was reduced only partially. This result suggested that an additional P2XR subtype or subtypes were involved.
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DISCUSSION |
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Unfortunately, we were unable to transiently transfect multiple siRNA fragments directed against both P2X4 and P2X6. We actually observed less inhibition of Ca2+ entry and protein when siRNA fragments against both subtypes were mixed together. We believe that the fragments likely form duplexes with each other within the cationic lipid-based micelles, diminishing their effectiveness. New strategies are being developed to account for this problem and knock down multiple mRNA targets within the same cell.
P2X4 is a widely expressed zinc- and ATP-gated receptor channel (16, 23). It is well known to be a slowly desensitizing and/or inactivating subtype. Its activation by agonists is inhibited at acidic pH and potentiated modestly at alkaline pH (11, 20). P2X6 is more of a mystery. Heterologous expression of this subtype has often led to the conclusion that it is not a functional receptor channel itself (6, 17). In this light, P2X6 may be a modulatory subunit that potentiates the function of P2X4. This is analogous to the roles of - and
-epithelial Na+ channels (ENaCs) that modulate the activity of the
-ENaC subunit when coexpressed together (8, 19). P2XRs and ENaCs share very similar topologies and could share similar epithelial cell biology.
However, a recent study showed that the glycosylated form of P2X6 is indeed a functional channel (4), whereas the nonglycosylated form is not. Our biochemistry in epithelia suggests that only the nonglycosylated form is expressed. However, this does not mean that P2X6 fails to reach the cell surface. RNAi targeted to P2X6 attenuated zinc-induced Ca2+ entry most markedly. We hypothesize currently that P2X4 could complex with P2X6 early in the biosynthetic pathway and facilitate the trafficking of P2X6-P2X4 complexes to the cell surface. Because P2X6 has been difficult to express as a functional receptor channel, its biophysical properties are understood poorly. Moreover, the effects of zinc and extracellular pH on its activity are unknown. In the study of the functional and glycosylated P2X6, its properties of desensitization and inactivation appear to be quite similar to those of P2X4. It also has a much lower EC50 of activation by the physiological agonist ATP (4) vs. other poorly inactivating subtypes like P2X2 and P2X4. ATP analogs also appear to have different effects on P2X6 vs. P2X4. Further examination of the chemistry and potency of different nucleotide analogs on zinc-induced Ca2+ entry is in progress, based on this very recent paper. However, many of these analogs and antagonists, when dissolved in vehicle, turn the vehicle color yellow. This interferes with our fura-2 measurements. Nevertheless, we believe that P2X6, like P2X4, is also potentiated by extracellular zinc and by external alkaline pH, thereby explaining why epithelial zinc- and ATP-induced Ca2+ entry is potentiated markedly by both variables.
For this and for other reasons mentioned above, it was siRNA that defined dual roles of P2X4 and P2X6 in zinc-induced Ca2+ entry in airway epithelial cells. This is the first study in which we have performed two consecutive transient transfections in as many days before analysis. This approach enhanced transient transfection efficiency from previous studies (18). More importantly, however, it allowed each cell in the culture two chances to be exposed to and absorb siRNA fragment. This was an important factor in the consistency and degree of knockdown in these studies. Interestingly, siRNA to P2X4 and siRNA to P2X6 each reduced zinc-induced Ca2+ entry by more than half. This result suggests key roles for both subtypes and, possibly, collaboration between them in mediating zinc-induced Ca2+ entry. Expression studies with tagged P2X4 and P2X6 constructs have shown that they coassemble to form heteromultimers (6, 17). In fact, P2X4 prefers to coassemble with P2X5 and P2X6, to the exclusion of other subtypes (18). P2X4 and P2X6 coassembly has also been suggested in native neurons (12). We hypothesize that P2X4 and P2X6 may coassemble in airway epithelia and collaborate to confer the unique properties observed with zinc- and ATP-induced Ca2+ entry in airway epithelia. This latter hypothesis is under investigation and is a difficult task with regard to the study of two native P2XR subtype proteins endogenous to airway epithelia or any cell or tissue. These studies, along with immunocytochemistry and immunohistochemistry designed to demonstrate colocalization, are in progress.
Novel facets of epithelial P2X receptor Ca2+ entry channel function have emerged from this and previous work (23, 24). First, zinc alone is sufficient as an agonist for epithelial P2X receptor channels. Despite exhaustive research of the previous literature, to our knowledge, the effect of biometals alone on P2X receptors independent of nucleotide has not been investigated. Zinc-induced, P2X receptor-mediated Ca2+ entry is potentiated markedly by alkaline pH. This is a novel result. Only P2X4 is potentiated by alkaline pH, and the effect is only modest. Zinc-induced, P2X receptor-mediated Ca2+ entry failed to show any desensitization of P2X receptor properties or inactivation of P2X receptor channel function. The latter property is ideal for a possible therapeutic target. In a previous study, we showed (23, 24) that we could reacquire sustained Ca2+ entry after only 2 min of washout of zinc or zinc and ATP. We also showed (23) that the sustained Ca2+ signal was maintained for at least 1 h in the presence of the coagonists. Moreover, we showed (24) that we could reverse and reacquire at least three times the rescue of Cl secretion mediated by P2X receptor channel-mediated Ca2+ entry in in vivo electrical assays from multiple CF mouse models.
If coassembled or, at least, coexpressed on the airway surface, these P2XRs provide at least two membrane surface targets for putative CF therapy with a zinc- and nucleotide-based formulation. If one or more P2X receptor subtypes are also expressed in the basolateral membrane oral therapy with zinc salts is possible, and it is already approved by the Food and Drug Administration. It is important to note that the polarity of P2XR homo- or heteromultimers may be different in the luminal vs. the serosal membrane. Finally, Silberberg and colleagues (7) postulated that a "P2X cilium" is critical for extracellular ATP, external calcium, and extracellular sodium-dependent regulation of ciliary beat in freshly isolated rabbit respiratory epithelia. We agree, and we hypothesize that the P2X cilium may be a heteromultimer of P2X4 and P2X6.
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DISCLOSURES |
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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