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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Protease-activated receptor regulation of Cl^– secretion in Calu-3 cells requires prostaglandin release and CFTR activation

Departments of ¹Physiology and ²Animal Science, University of Minnesota, St. Paul, Minnesota; and ³Department of Veterinary Pharmacology, Seoul National University, Seoul, Republic of Korea

Submitted 16 September 2005 ; accepted in final form 20 November 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl^– secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (I_sc: thrombin, 21 ± 2 µA; SLIGRL, 83 ± 22 µA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca²⁺-chelating agent BAPTA-AM (50 µM) abolished the increase in I_sc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 µM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I_sc was observed. In addition, basolateral treatment with the PGE₂ receptor antagonist AH-6809 (25 µM) significantly inhibited the effects of SLIGRL on I_sc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca²⁺-activated KCNN4 K⁺ channel, and the KCNQ1 K⁺ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl^– conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl^– secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl^– secretion requires activation of CFTR and at least two distinct K⁺ channels located in the basolateral membrane.

cystic fibrosis transmembrane conductance regulator; KCNQ1; calcium-activated potassium channels; KCNN4; cAMP

Each PAR possesses a unique proteolytic cleavage site within the extracellular NH₂-terminal domain. Activation of PARs involves enzymatic cleavage of the NH₂ terminus, exposing a tethered ligand that is then available to bind to the second extracellular loop of the cleaved receptor to induce self-activation (22). Once activated, this process is irreversible. At present, the resulting upstream NH₂-terminal fragment has no known function. After PAR stimulation, G proteins belonging to the G_q/11 subfamily are activated. The G_q subunit stimulates PLC- to produce inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG), which in turn increases intracellular Ca²⁺ concentration ([Ca²⁺]) and PKC activity (14, 17).

PAR receptor activation by trypsin or thrombin or with PAR-2-selective activating peptides has been shown to regulate ion transport function in epithelial cells from airways, colon, small intestine, pancreatic duct, and renal collecting duct (1–3, 5, 9, 11, 13, 20, 21, 23, 26, 31). In most cases stimulation of PARs localized to the basolateral membrane produced a rise in intracellular Ca²⁺ and an increase in anion secretion (2, 3, 11, 20, 21, 23, 26). In porcine jejunum however, PAR-2 receptors were also identified on submucosal neurons, so that basolateral stimulation with trypsin or with a PAR-2-activating peptide produced a neurogenic anion secretory response that was dependent on release of endogenous opiate peptides and prostaglandins (16). In native bovine pancreatic duct epithelial cells, however, basolateral stimulation with trypsin or with PAR-2 agonists did not affect transport function, and apical stimulation produced inhibition of bicarbonate secretion (1). Moreover, the predominant effect of basolateral PAR-2 stimulation of human bronchial epithelial cells was inhibition of epithelial Na⁺ channel (ENaC)-dependent Na⁺ absorption, caused by a decrease in basolateral membrane K⁺ permeability (13).

Human Calu-3 cells, originally derived from adenocarcinoma cells, possess properties similar to those of airway gland serous cells (30). Stimulation with agonists that increase cAMP results in large increases in anion secretion that depend on the activity of CFTR Cl^– channels located in the apical membrane and basolateral K⁺ channels including KCNQ1 and the Na-K-2Cl cotransporter (NKCC)1 (7, 8, 10, 12, 15, 30, 33, 36). In addition, previous studies showed that Ca²⁺-mobilizing agonists such as carbachol produce transient increases in anion secretion (25). The proposed mechanism involves stimulation of basolateral Ca²⁺-activated K⁺ channels resulting in an increase in driving force for Cl^– exit across the apical membrane through basally active CFTR Cl^– channels (10, 12, 36). In the present study we investigated the effects of PAR agonists on Cl^– transport across monolayer cultures of Calu-3 cells. Our objectives were to identify the PAR subtypes expressed in these cells and to determine the mechanism by which PAR activation increases Cl^– secretion.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials BAPTA-AM, insulin, indomethacin, nonessential amino acids, and high-purity-grade salts were purchased from Sigma (St. Louis, MO). DMEM, Dulbecco's PBS, FBS, normal goat serum, collagenase type 1, kanamycin, penicillin-streptomycin, puromycin, and fungizone were purchased from GIBCO-BRL (Grand Island, NY). 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP), thrombin, and clotrimazole were purchased from Sigma. SLIGRL was purchased from Tocris (Ellisville, MO). AH-6809 and prostaglandins were obtained from Cayman (Ann Arbor, MI) and Biomol (Plymouth Meeting, PA), respectively.

Methods Quantitative RT-PCR. Calu-3 monolayers used to generate the functional data shown in Figs. 3 and 6 were placed in TRIzol (GIBCO-BRL) after the transport experiments. Total RNA was isolated with the protocol provided by the manufacturer. RT reactions were diluted 1:600 for each quantitative RT-PCR (QRT-PCR) reaction. SYBR Green master mix (1:2) and passive reference dye (1:200) were purchased from Stratagene. GAPDH was used as a control, and each transfected cell line was within 1 threshold cycle (C_T) value of GAPDH results obtained from nontransfected control monolayers. Percent reduction in mRNA was calculated with the following equation:

View larger version (13K): [in this window] [in a new window]   Fig. 3. Effects of the PAR-2-activating peptide SLIGRL on Isc and apical membrane Cl– current. A: representative traces showing the effects of basolateral addition of 1 µM SLIGRL on Isc in the presence and absence of 10 µM indomethacin (n = 6). B: bar graph comparing effects on peak Isc responses of basolateral SLIGRL (1 µM) before and after pretreatment with 10 µM indomethacin, 25 µM AH-6809, and 50 µM BAPTA-AM (n = 6 for each condition). The graph also shows the effect of 1 µM SLIGRL on the peak apical membrane Cl– current before and after treatment with 10 µM indomethacin (n = 6 for both conditions). C: representative traces showing the effect of basolateral SLIGRL (1 µM) on apical membrane Cl– currents in the presence and absence of 10 µM indomethacin (n = 6).

View larger version (15K): [in this window] [in a new window]   Fig. 6. QRT-PCR data comparing effects of short hairpin RNA (shRNA) expression on CFTR mRNA levels in Calu-3 cells. A: no difference was observed for CFTR and GAPDH mRNA between nontransfected control cells (CT for CFTR = 23.1; CT for GAPDH = 18.4) and cells transfected with the shCFTRALTER construct (CT for CFTR = 23.0; CT for GAPDH = 18.8, n = 3). Single peak PCR product melting temperatures were obtained for each condition (GAPDH nontransfected controls = 86.9°C, GAPDH shCFTRALTER cells = 86.9°C; CFTR nontransfected controls = 83.6°C; CFTR shCFTRALTER cells = 83.6°C). B: analysis of CFTR knockdown between shRNA-expressing cells and shCFTRALTER-expressing cells. Two separate PCR primer sets targeting the NH2-terminal and COOH-terminal regions of CFTR were compared (n = 4). Note that nearly identical shifts in CT values were obtained (CT for CFTR shRNA: primer 1 = 32.5 cycles, primer 2 = 32.4 cycles; CFTRALTER shRNA primer 1 = 23.4 cycles, primer 2 = 23.7 cycles). Single peak PCR product melting temperatures were obtained under each condition (CFTR shRNA: primer 1 = 80.8°C, primer 2 = 77.3°C; CFTRALTER shRNA: primer 1 = 81.2°C, primer 2 = 77.7°C).

shCFTR (21-mers; GC CONTENT: 42.86%). The sequence for shCFTR is 5'-GAAGAAGAGGTGCAAGATACACTTCCTGTCATGTATCTTGCACCTCTTCTTC-3'. The 21-mer complementary sequences are specific for human CFTR, corresponding to base pair positions 4420–4440 of the complete CFTR nucleotide sequence (GenBank accession no. AY585334).

shCFTR_ALTER (21-mers; GC CONTENT: 42.86%). The sequence for shCFTR_ALTER is 5'-GCATGCAGAAGTGTAAAGCTACTTCCTGTCATAGCTTTACACTTCTGCATGC-3'. The 21-mer complementary sequences possess 4 base pair mutations of shCFTR (underlined in bold along with the hairpin loop; complements in regular underline) and distantly correspond to base pair positions 446–466 of the complete CFTR nucleotide sequence. A BLAST search confirmed that this short hairpin CFTR_ALTER sequence does not match nucleotide sequences from any cloned cellular proteins known to date.

Standard RT-PCR and gel electrophoresis. Standard RT-PCR results were obtained with primers based on the human gene products and conditions described in Table 1. Primers were developed with Primer-3 software, and BLAST searches were performed to ensure that the primers developed were specific for their respective targets. GAPDH was used as a control. PCR products were confirmed by sequence analysis.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Figure 1A shows the RT-PCR products obtained at 30 cycles with primers designed against the four known PAR subtypes. Only PAR-1 and PAR-2 subtypes could be detected. Subsequent analysis by QRT-PCR consistently identified PAR-1 and PAR-2 mRNA below 30 cycles as well as PAR-3 at cycle numbers above 30. Each of these PCR reactions possessed a single peak melting temperature (see Fig. 1 for C_T values and PCR product melting temperatures). Identification of the PAR-4 subtype by QRT-PCR was inconclusive given the presence of multiple melting temperature peaks and the relatively high cycle numbers necessary to detect a signal. The results of these experiments indicated that Calu-3 cells express mRNA for at least three of the cloned PAR subtypes, with PAR-2 mRNA in greatest abundance.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Expression of protease-activated receptor (PAR) mRNA in Calu-3 cells. A: PAR-1 and PAR-2 receptors were detected by RT-PCR at 30 cycles (see Table 1 for primer sequences). Identification was confirmed by sequencing the PCR products. B: quantitative RT-PCR (QRT-PCR) also detected PAR-1 and PAR-2 while revealing low levels of expression for PAR-3 (n = 4, 3, 3 for PAR-1, -2, and -3, respectively). The threshold cycle (CT) values were 25.6, 20.1, and 29.7, and the single product melting temperatures were 82.5, 82.3, and 84.4 for PAR-1, -2, and -3, respectively.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Effects of the PAR-1 agonist thrombin on short-circuit current (Isc). A: representative trace showing that basolateral addition of 10 µM thrombin to the basolateral solution produced a rapid increase in Isc that returned to baseline within 3–4 min (n = 6). B: apical addition of thrombin and the PAR-2-activating peptide SLIGRL do not produce significant effects on Isc. An increase in Isc was observed after basolateral (blm) thrombin (10 µM) that was significantly diminished when monolayers were pretreated with 10 µM indomethacin (Indo) (n = 6 for control and indomethacin-treated conditions).

2

2

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effects of exogenous prostaglandins on Isc. A: basolateral addition of 1 nM PGE2 produced a rapid increase in Isc followed by a sustained oscillation lasting >10 min. B: basolateral concentration-response effects of PGE2, PGD2, PGI2, and PGF2 on Isc. EC50 values were PGE2, 20 ± 5 nM (R2 = 0.90); PGD2, 1.4 ± 3 µM (R2 = 0.95); PGI2, 1.0 ± 0.2 µM (R2 = 0.97); and PGF2, 0.15 ± 0.03 µM (R2 = 0.95); n = 6 for each prostaglandin. Isc, change in Isc; [PGX], prostaglandin concentration.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Expression of PGE2 receptors in Calu-3 cells. QRT-PCR data showing expression of EP2 and EP4 receptor mRNA in Calu-3 cells. CT values were 30.0 and 26.1, respectively, and single peak PCR product melting temperatures (EP2 = 77.8°C and EP4 = 82.8°C) were obtained for each receptor (n = 3). EP1 and EP3 receptor mRNAs were not detected.

The effects of CFTR silencing on I_sc and apical membrane Cl^– current after stimulation with either 8-CPT-cAMP or SLIGRL are shown in Fig. 7. The effect of SLIGRL on the peak I_sc response was reduced by 95% (Fig. 7, A and C). Moreover, analysis of apical membrane Cl^– conductance shown in Fig. 7B revealed that CFTR silencing reduced the SLIGRL- and 8-CPT-cAMP-stimulated conductances by 93% and 94%, respectively. These findings strongly support the assertion that PAR-2 activation produces Cl^– secretion that is dependent on activation of CFTR.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Effect of CFTR silencing on 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP)- and SLIGRL-stimulated Cl– transport. A: representative tracings comparing the Isc responses elicited by basolateral addition of 1 µM SLIGRL from control monolayers and monolayers expressing shCFTR (n = 6). B: current-voltage relationships comparing the effects of 1 µM SLIGRL and 2.5 µM 8-CPT-cAMP on apical membrane Cl– conductance in control and shCFTR-expressing monolayers (n = 6 for each condition). C: bar graph showing peak apical membrane Cl– current responses to 10 µM 8-CPT-cAMP and 1 µM SLIGRL from control and shCFTR-expressing monolayers (n = 6 for each condition).

View larger version (14K): [in this window] [in a new window]   Fig. 8. Effects of SLIGRL and 8-CPT-cAMP on basolateral membrane K channel activity. A: basolateral addition of 1 µM SLIGRL produced a biphasic effect on basolateral membrane K+ current. Pretreatment with 10 µM indomethacin abolished the second peak of the current response but did not significantly reduce the first peak (n = 6 for control and indomethacin-treated conditions). B: representative tracing showing the effect of 10 µM 8-CPT-cAMP on K+ efflux across the basolateral membrane (n = 6). The peak current was of similar magnitude to the second peak elicited by SLIGRL. C: bar graph showing the effects of 10 µM indomethacin on the first and second K+ current peaks observed after stimulation with 1 µM SLIGRL (n = 6). Pretreatment with the KCNN4 K+ channel blocker clotrimazole (10 µM) significantly inhibited the first peak observed after 1 µM SLIGRL stimulation (n = 6). *P < 0.05, significantly different from control peak.

View larger version (10K): [in this window] [in a new window]   Fig. 9. QRT-PCR results comparing KCNN4 and KCNQ1 mRNA expression in Calu-3 cells. A: the CT value for KCNQ1 was 27.4 cycles (n = 3), and a single peak melting temperature was obtained (84.0°C). B: the CT value for KCNN4 was 24.0 cycles (n = 3), and a single peak melting temperature was obtained (86.5°C).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

An important observation of the present study was that PAR-dependent activation of Cl^– secretion was abolished when cyclooxygenase activity was blocked with indomethacin. This result strongly supports the conclusion that PAR regulation of Cl^– secretion occurs indirectly through stimulation of arachidonic acid synthesis and prostaglandin release from the epithelium. A similar inhibitory response of the PGE₂ receptor antagonist AH-6809 on the SLIGRL-stimulated I_sc was also consistent with this conclusion and suggested that PGE₂ was the major prostaglandin involved. QRT-PCR analysis provided evidence showing that Calu-3 cells express mRNA for the EP₂ and EP₄, but not EP₁ or EP₃, receptor subtypes, and analysis of concentration-response relationships for several prostaglandins indicated that Calu-3 cells were most sensitive to PGE₂. These findings were consistent with the results of a recent study that identified EP₄ receptor mRNA in Calu-3 cells and demonstrated that activation of the receptor produced both an increase in intracellular cAMP concentration and Cl^– efflux (6). Moreover, a role for prostaglandins in PAR-dependent stimulation of Cl^– secretion was previously identified in human bronchial epithelial cells, human colonic epithelia, rat jejunum, and porcine ileum (16, 21, 23, 34). Studies with human bronchial epithelial cells showed that 50% of the increase in Cl^– secretion observed after PAR stimulation was inhibited by indomethacin (21). Thus prostaglandins appear to play a critical role in PAR-mediated regulation of transport function in several epithelial cell types and, in the case of Calu-3 cells, allow for activation of essential signal transduction pathways necessary for eliciting Cl^– secretion.

PAR-2 activation with SLIGRL produced an increase in apical membrane Cl^– conductance that was associated with an elevation in I_sc. The current-voltage relationship was nearly linear and identical to that obtained from monolayers stimulated with 2.5 µM 8-CPT-cAMP. The increase in apical Cl^– permeability was abolished after pretreatment with indomethacin, again demonstrating a dependence on cyclooxygenase activity and release of prostaglandins from the epithelium. To test whether the Cl^– conductance activated by PAR-2 stimulation was CFTR, an RNA silencing experiment was performed in which shRNAs targeting CFTR were constitutively expressed in Calu-3 cells. QRT-PCR revealed that CFTR mRNA expression was dramatically inhibited in cells expressing the shRNA construct compared with nontransfected control cells or cells transfected with an altered shRNA sequence that did not recognize CFTR. In addition, functional studies showed that apical Cl^– currents elicited by either 8-CPT-cAMP or SLIGRL were also markedly reduced in cells expressing the shRNA sequence. These findings provide strong support for the conclusion that PAR-2 activation increases the activity of CFTR. In a previous study investigating muscarinic receptor regulation of Cl^– secretion in Calu-3 cells, it was concluded that the apical conductance responsible for Cl^– efflux was CFTR (25). The proposed mechanism involved mobilization of intracellular Ca²⁺, which stimulated Ca²⁺-activated K⁺ channels located in the basolateral membrane, resulting in an increase in driving force for Cl^– efflux through basally active CFTR channels in the apical membrane. We propose that the results of the present study extend these earlier findings by suggesting that CFTR activation above basal activity is necessary for Ca²⁺-mobilizing agonists to elicit Cl^– secretion and that secondary effects on prostaglandin synthesis and release are responsible for the increase in CFTR activity.

PAR stimulation of Cl^– secretion also involved an increase in basolateral K⁺ conductance, and the results suggest activation of at least two separate K⁺ conducting pathways. This result may provide some explanation for the apparent phases of the I_sc response, where the initial I_sc increase correlates with the rise of the K⁺ current associated with the first peak and the delay in I_sc recovery corresponds to the more sustained K⁺ current associated with the second peak. Addition of 8-CPT-cAMP also produced an increase in basolateral K⁺ current with a longer duration than observed after SLIGRL stimulation. Pretreatment with indomethacin abolished the second current peak elicited by SLIGRL but did not significantly affect the first peak, which in a subsequent experiment was shown to be significantly inhibited after pretreatment with clotrimazole, an inhibitor of KCNN4 K⁺ channels. No significant effect of clotrimazole was observed on the second peak or on the 8-CPT-cAMP response. QRT-PCR analysis indicated that Calu-3 cells express mRNA for a Ca²⁺-activated K⁺ channel (KCNN4) and KCNQ1, which is known to be regulated by cAMP in epithelial cells (4, 12, 18, 19, 29, 32, 35). This result was consistent with RT-PCR data from an earlier study that reported the presence the same K⁺ channel subtypes as well as the KCNE3 -subunit in Calu-3 cells (10). Although the results of the present study do not definitively identify the K⁺ channel subtypes involved in Cl^– secretion elicited by PAR activation, we speculate that both of these channels play a critical role in providing the necessary driving force for Cl^– efflux across the apical membrane. The PAR-2 activation of K⁺ channels reported in this study contrasts with previous results in human bronchial epithelial cells that indicated inhibition of basolateral membrane K⁺ current after basolateral stimulation with SLIGRL. This disparity is likely due to differences in the K⁺ channel subtypes that are expressed in primary bronchial epithelial cells compared with Calu-3 cells (13).

Figure 10 presents a model to explain PAR regulation of Cl^– secretion in Calu-3 cells. We propose that activation of basolateral PARs results in an increase in PLC activity leading to release of IP₃ and stimulation of PKC isoforms within the cell. IP₃, in turn, stimulates the release of Ca²⁺ from intracellular stores, which then activates PLA₂ and increases production of arachidonic acid. Arachidonic acid serves as a rate-limiting substrate for cyclooxygenase, so that an increase in its production leads to increased synthesis and release of prostaglandins. PGE₂, presumably acting at EP₂ and EP₄ receptors, stimulates adenylyl cyclase and increases intracellular cAMP, which then leads to activation of CFTR in the apical membrane as well as K⁺ channels (perhaps KCNQ1) located in the basolateral membrane. The transient nature of Cl^– secretion elicited by PAR-2 stimulation is presumably the result of a decrease in prostaglandin release that occurs in parallel with the fall in intracellular [Ca²⁺]. An additional effect of Ca²⁺ is stimulation of Ca²⁺-activated K⁺ channels also located in the basolateral membrane, which contributes to the electrical driving force needed for Cl^– secretion. Thus the results of this study offer additional insight into the mechanism by which a Ca²⁺-dependent agonist stimulates Cl^– secretion in Calu-3 cells and provide an explanation of how CFTR activity is indirectly stimulated through the effects of Ca²⁺ mobilization on prostaglandin synthesis and release.

View larger version (30K): [in this window] [in a new window]   Fig. 10. Model showing the proposed mechanism for PAR regulation of Cl– secretion in Calu-3 cells. See DISCUSSION for description. IP3, inositol triphosphate; AC, adenylyl cyclase; COX, cyclooxygenase; AA, arachidonic acid, V, voltage.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. M. O'Grady, Depts. of Physiology and Animal Science, 495 Animal Science/Veterinary Medicine Bldg., 1988 Fitch Ave., Univ. of Minnesota, St. Paul, MN 55110 (e-mail: ograd001{at}umn.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.

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