Cystic fibrosis transmembrane conductance regulator activation by cAMP-independent mechanisms

Zhaoping He1, Sasikala Raman1, Yi Guo1, and William W. Reenstra1,2

Departments of 1 Clinical Science and 2 Pediatrics, Alfred I. duPont Hospital for Children, Thomas Jefferson University, Wilmington, Delaware 19803

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Recent studies have demonstrated that several compounds with diverse structures can activate wild-type cystic fibrosis transmembrane conductance regulator (CFTR) by non-receptor-mediated mechanisms. Some of these compounds have been shown to enhance cAMP-dependent activation of Delta F508-CFTR. This study was undertaken to compare the mechanisms by which genistein, IBMX, milrinone, 8-cyclopentyl-1,3-dipropylxanthine (CPX), the benzimidazolone NS004, and calyculin A increase CFTR activity. Our studies demonstrate that, in transfected NIH-3T3 cells, maximal enhancements of forskolin-dependent Delta F508-CFTR activity are greatest with genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, and calyculin A do not increase cellular cAMP. Because forskolin and calyculin A increase in vivo phosphorylation of cAMP binding response element (CREB), the inability of milrinone, genistein, CPX, and NS004 to increase CREB phosphorylation suggests that they do not stimulate protein kinase A or inhibit phosphatase activity. Our data suggest that the mechanisms by which genistein and NS004 activate CFTR differ. We also demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement of cAMP-dependent CFTR activity is not due to an increase in cellular cAMP and may involve a mechanism like that of genistein.

genistein; 3-isobutyl-1-methylxanthine; in vivo phosphorylation; adenosine 3',5'-cyclic monophosphate

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

CYSTIC FIBROSIS (CF) is the most common genetic disease in the Caucasian population, afflicting ~1 in 2,500 live births in the United States (32). The disease is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cAMP-dependent chloride channel that is expressed in the apical membrane of many epithelial cells (7, 24). The loss of channel activity in CF is due to the absence of CFTR in the apical membrane, or the presence of inactive forms (31). The most common mutation, Delta F508-CFTR, accounts for ~65% of all CF mutations, and at least one copy is present in 90% of all CF patients (20). It is well established that posttranslational processing of Delta F508-CFTR is defective and that little protein reaches the apical membrane (8). We have recently established that cAMP-dependent activation of Delta F508-CFTR is also defective (16).

CFTR can be activated by a mechanism that involves protein kinase A (PKA)-dependent phosphorylation on multiple sites and the subsequent hydrolysis of ATP at two nucleotide binding domains, NBD1 and NBD2 (1, 4). Recently, a number of compounds have been shown to activate the CFTR by mechanisms that do not involve the activation of adenylate cyclase. The first compound described was IBMX, an inhibitor of phosphodiesterase (9). At millimolar concentrations, IBMX potentiates cAMP-dependent activation of wild-type CFTR and Delta F508-CFTR. A more specific phosphodiesterase inhibitor, milrinone, has also been shown to activate both forms of the CFTR (18, 19). It has been proposed that these agents activate CFTR by increasing cAMP levels (18). This seems unlikely for three reasons: 1) IBMX inhibits phosphodiesterase at lower concentrations than those required for CFTR activation (33); 2) when several phosphodiesterase inhibitors were compared, CFTR activation was poorly correlated with cAMP levels (18, 19); and 3) the ~50-fold increase in the activity of cAMP-activated Delta F508-CFTR observed by Drumm et al. (9) would require an equally large increase in cellular cAMP. Another xanthine, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been shown to activate both wild-type CFTR and Delta F508-CFTR at nanomolar concentrations (2, 5, 10). Because CPX has been shown to bind to NBD1, it has been proposed that CPX activates CFTR by a mechanism that involves a direct interaction (5). CPX has also been shown to potentiate cAMP-dependent activation of Delta F508-CFTR in a manner that is very similar to that of IBMX (14). The benzimidazolone NS004 has also been shown to activate both wild-type CFTR and Delta F508-CFTR and to potentiate cAMP-dependent activation of Delta F508-CFTR (13). However, there is little known about the mechanism of action. Inhibition of phosphodiesterase activity with calyculin A and okadaic acid has also been shown to activate wild-type CFTR (15, 23, 34). It has been proposed that, in the absence of phosphatase activity, a basal level of PKA activity can phosphorylate and activate CFTR (23).

Genistein has been shown to activate wild-type CFTR (11, 16, 21, 23, 26, 30) and to potentiate cAMP-dependent activation of Delta F508-CFTR (16). Studies in several systems have shown that genistein acts by a mechanism that involves increases in cellular cAMP that are far smaller than those that would be required for activation with a cAMP-dependent agonist (17, 21). However, CFTR activation by genistein requires PKA-dependent phosphorylation of CFTR and is thought to be due to an inhibition of CFTR dephosphorylation (23). It has been suggested that genistein activates CFTR by a mechanism that involves binding to CFTR (16, 29). This study was undertaken to compare the mechanism of genistein-dependent CFTR activation with that of other compounds that have been reported to activate CFTR or potentiate cAMP-dependent channel activation. We used transfected NIH-3T3 cell lines stably expressing wild-type CFTR or Delta F508-CFTR. This has allowed us to study both forms of the CFTR in cells with similar background properties, and the high level of CFTR expression in NIH-CFTR cells was essential for phosphopeptide mapping studies. We have examined channel activation, in vivo phosphorylation, cellular cAMP, and in vivo phosphorylation of CREB.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell culture. NIH-3T3 cells that stably express wild-type CFTR (NIH-CFTR) and Delta F508-CFTR (NIH-508) were obtained from Dr. Richard Mulligan and grown as described previously (3, 16). Both cell lines were cultured at 37°C with 5% CO2 in DMEM supplemented with 100 µg/ml penicillin, 50 µg/ml streptomycin, 50 µg/ml gentamicin, and 10% newborn calf serum. Three days before all experiments, NIH-508 cells were shifted to 27°C, and 1 day before all experiments, media were supplemented with 5 mM butyrate. For I-efflux studies, cAMP measurements, and cAMP binding response element (CREB) phosphorylation, cells were grown on 35-mm plates. For in vivo phosphorylation studies, cells were grown on 60-mm plates.

I efflux. CFTR activity was assayed by measuring the rate of I efflux. Measurements were performed as previously described (16, 28). Cells, at 37°C, were incubated for 30 min with efflux buffer (141 mM NaCl, 3 mM KCl, 2 mM KH2PO4, 0.9 mM MgCl2, 1.7 mM CaCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4) containing 5 µCi/ml carrier-free 125I (sodium salt). Cells were washed five times with efflux buffer to remove extracellular 125I. The loss of intracellular 125I was determined by replacing the bathing solution with efflux buffer every 60 s for 10 min. Agonist or vehicle was present at all times after 4 min. Intracellular 125I was calculated at each time point, and rates of I efflux (r) were determined as follows: r = ln (It1/It2)/(t1 - t2), where It1 and It2 are intracellular 125I at successive time points t1 and t2, respectively (28). Rates of I efflux were time dependent, and all comparisons were based on maximal values for the time-dependent rates (peak rates).

In vivo labeling of CFTR with 32Pi. In vivo labeling of the CFTR was performed as previously described (35). Cells were incubated for 150 min in phosphate-free efflux buffer containing 2.0 mCi/ml [32P]orthophosphate (3,000 Ci/mmol). Cells were washed with phosphate-free efflux buffer and stimulated with agonist for 2 min. After stimulation, cells were lysed with 4°C RIPA buffer (100 mM NaCl, 50 mM NaF, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml aprotinin, 1 mM orthovanadate, and 50 mM Tris · HCl, pH 7.5). Lysates were cleared by centrifugation (100,000 g for 20 min), and CFTR was immunoprecipitated from a supernatant containing 3.8 mg/ml protein with 7.5 µg/ml anti-CFTR monoclonal antibody (Genzyme, anti-COOH-terminal). The CFTR was resolved by SDS-PAGE and visualized by autoradiography. Protein was determined by bicinchoninic acid (BCA) assay (27).

Tryptic digestion and two-dimensional peptide mapping. Two-dimensional phosphopeptide mapping of in vivo-labeled CFTR was performed as described previously (35). After separation by SDS-PAGE, gel slices containing 32P-CFTR were rehydrated and suspended in 50 mM NH4HCO3 (pH 7.4) containing 0.1% SDS and 5% beta -mercaptoethanol. BSA (20 µg) was added to the clarified supernatant and protein precipitated with 17% TCA. Pellets were washed with 100% ethanol, suspended in 50 mM NH4HCO3, and digested at 37°C with 25 units N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-trypsin (Worthington). Samples were diluted with water and lyophilized to dryness four times. Peptides were then spotted onto 20 × 20-cm thin-layer chromatography plates (EM Science) and separated by electrophoresis for 30 min at 1,000 V in 100 mM (NH4)2CO3, pH 8.9, and then by ascending chromatography in n-butanol-pyridine-water-acetic acid, 15:10:12:3 (vol/vol/vol/vol). Phosphopeptides were detected with the Storm 860 Phosphorimager (Molecular Dynamics) and identified by comparison with previously published studies (4) and maps provided by Dr. Jonathan Cohn.

Measurement of cellular cAMP. As previously described, cellular cAMP was determined by radioimmunoassay with an Amersham kit (35). Briefly, confluent NIH-CFTR cells were preincubated for 10 min at 37°C with efflux buffer. Cells were stimulated with agonist for 2 min, and the reaction was terminated with 500 µl of 70% ethanol at 4°C. Cellular cAMP was extracted into 70% ethanol, and extracts were evaporated to dryness at 70°C. The residue was dissolved in 600 µl of 50 mM sodium acetate, pH 5.8, and total cAMP was determined by radioimmunoassay. Values were normalized to total cell protein as determined by BCA assay (27).

In vivo phosphorylation of CREB. Confluent NIH-CFTR cells were washed with PBS and incubated at 37°C with DMEM plus agonist for 20 min. Cells were washed with cold PBS and lysed with 0.35 ml of CFTR-RIPA buffer. Lysates were cleared by centrifugation (100,000 g for 20 min). Protein, 40 µg in SDS sample buffer, was electrophoresed and transblotted onto nitrocellulose membranes. CREB and phospho-CREB were detected with anti-CREB antibodies and anti-phospho-CREB antibodies (Upstate Biochemicals) and visualized using an enhanced chemiluminescence kit (Amersham). For quantification, blots were developed using a Vistra enhanced chemifluorescence kit (Amersham) and scanned with a Storm 860 (Molecular Dynamics).

Materials. Forskolin, genistein, and calyculin A were obtained from Alexis. IBMX and milrinone were obtained from Sigma. CPX was obtained from RBI. NS004 was a gift from Dr. Valentin Gribkoff of Bristol-Meyers Squibb. Stock solutions were dissolved in DMSO so that the final concentration of DMSO in all buffers was <= 0.2%. Radiochemicals were obtained from New England Nuclear-DuPont.

Statistics. Calculated values are presented as means ± SE. Comparisons were made among paired data by Student's t-test with P < 0.05 taken as significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Recent reports have suggested that genistein (16), IBMX (9), CPX (10), milrinone (18), and NS004 (13), compounds with rather diverse structures, can enhance cAMP-dependent activation of Delta F508-CFTR. This study compared the effects of these compounds on wild-type CFTR and Delta F508-CFTR to determine if they act by a common mechanism. We have used the I-efflux assay (17, 28), which measures the rate of 125I loss from agonist-stimulated cells to assay CFTR activity. As shown in Fig. 1A, genistein, IBMX, CPX, milrinone, and NS004 stimulated I efflux in NIH-CFTR cells expressing wild-type CFTR. All agonists caused statistically significant increases in I efflux, but the rates were less than those obtained with forskolin (Table 1). Forskolin, genistein, milrinone, CPX, NS004, and calyculin A were used at concentrations that gave maximal changes in the rate of I efflux, but the response to IBMX was not saturated at 2 mM. In all cases, the agonist-dependent effects appeared to be due to stimulation of the CFTR because none of the agonists in Table 1 stimulated I efflux in mock transfected cells (data not shown). However, it is possible that CFTR activation could stimulate I efflux via another chloride channel (25). The data in Fig. 1B and Table 1 also demonstrate that genistein, IBMX, milrinone, CPX, and NS004 did not increase wild-type CFTR activity in the presence of 10 µM forskolin. When Delta F508-CFTR activity was stimulated with forskolin, genistein, IBMX, milrinone, CPX, and NS004, there were small increases in channel activity with all compounds tested (Fig. 1C and Table 1). The increases were statistically significant for all agonists but NS004 and CPX. Genistein, IBMX, milrinone, CPX, and NS004 enhanced forskolin-dependent activation of Delta F508-CFTR (Fig. 1D and Table 1). The largest effects were obtained with genistein, IBMX, and NS004.


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Fig. 1.   Time courses for agonist-dependent I efflux from NIH-CFTR and NIH-508 cells. I efflux from NIH-CFTR cells (A and B) and from NIH-508 cells (C and D) was stimulated with vehicle (open circle ), 10 µM forskolin (bullet ), 50 µM genistein (), 2 mM IBMX (), 100 µM milrinone (triangle ), 100 µM 8-cyclopentyl-1,3-dipropylxanthine (CPX; black-triangle), and 10 µM NS004 (black-down-triangle ). Agonists were added separately in A and C, and with exception of vehicle and 10 µM forskolin, with 10 µM forskolin in B and D. I efflux was assayed as described in METHODS with agonist present when indicated by bar. Traces are representative of data in Table 1.

                              
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Table 1.   Agonist-dependent stimulation of wild-type and Delta F508-CFTR activity

The effects of calyculin A on wild-type CFTR and Delta F508-CFTR are also shown in Table 1. Calyculin A, an inhibitor of protein phosphatases 1 and 2A, activates wild-type CFTR by a mechanism that is distinct from that of genistein (23). With wild-type CFTR, calyculin A increased channel activity but did not cause a significant enhancement in forskolin-dependent activity. With Delta F508-CFTR, calyculin A increased the rate of I efflux. When Delta F508-CFTR was stimulated with forskolin plus calyculin A, there was no significant increase in channel activity beyond that seen with calyculin A alone.

Differences in the mechanisms by which genistein, NS004, and calyculin A activate wild-type CFTR were examined by assaying the additive effects of these agonists. As shown in Fig. 2, the rate of I efflux with saturating concentrations of calyculin A, genistein, and NS004 were two- to fourfold greater than the control rate. The addition of calyculin A with either genistein or NS004 caused a significant increase in the rate of I efflux when compared with the rates with either agonist alone. These results suggest that neither genistein nor NS004 activate wild-type CFTR by inhibiting protein phosphatases 1 or 2A. The rate of I efflux when genistein and NS004 were added together is also shown in Fig. 2. The significant increase in rate when both agonists are present suggests that genistein and NS004 activate the CFTR by different mechanisms. An additional test was made to determine if an agonist could increase the activity of Delta F508-CFTR in the presence of forskolin plus genistein. As shown in Table 2, CPX, IBMX, milrinone, and calyculin A did not produce significant increases in Delta F508-CFTR activity beyond that seen with forskolin plus genistein. As with wild-type CFTR, the addition of NS004 caused a significant increase in Delta F508-CFTR activity.


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Fig. 2.   Additive effects of genistein, calyculin A, and NS004 on I efflux from NIH-Delta F508 cells. Relative rates of I efflux were obtained from a comparison of rate in presence and absence of agonist. Data are presented as means ± SE (n >=  5). * Rates in presence of 2 agonists were significantly greater than those for either agonist alone.

                              
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Table 2.   Agonist-dependent stimulation of Delta F508-CFTR

To examine the role of kinase-dependent phosphorylation in CFTR activation, we have examined the in vivo phosphorylation of wild-type CFTR. After agonist-dependent activation, immunoprecipitation, and tryptic digestion, two-dimensional phosphopeptide maps of wild-type CFTR were obtained. As shown in Fig. 3A, stimulation with forskolin leads to phosphorylation of several peptides, with the most prominent being peptides that contain serine-795 and -737. These and the other indicated peptides have been assigned by comparison with previously published peptides maps (4). Previous studies have reported that serine-660, -700, -737, -795, and -813 are the most highly phosphorylated serines (4, 12, 22). In our studies, we observed PKA-dependent phosphorylation of serine-660, -700, and -813, but at levels that were less intense than that of serine-737 and -795. There appears to be less phosphorylation of these serines than serine-768, a serine that has been reported to be phosphorylated by PKA under in vitro, but not in vivo, conditions (12). We have no explanation for the differences between these studies and previously published studies, other than that the experimental conditions were different. We have assayed in vivo phosphorylation under the same experimental conditions that gave maximal stimulation of CFTR activity, a protocol that has not been universally followed in other laboratories.


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Fig. 3.   Phosphopeptide maps for in vivo phosphorylated wild-type cystic fibrosis transmembrane conductance regulator (CFTR). In vivo phosphorylated CFTR was immunoprecipitated and digested with trypsin, and phosphopeptides were separated by 2-dimensional peptide mapping. NIH-CFTR cells were stimulated with 10 µM forskolin (A), 2 mM IBMX (B), 50 µM genistein (C), 100 nM calyculin A (D), or 10 µM NS004 (E). Peptides were identified by comparison with previously published maps, and phosphorylated serine is indicated. Origin is in bottom right. Electrophoresis was performed in horizontal axis and ascending chromatography in vertical axis. Maps are representative of 3 or more experiments for each condition.

Stimulation with IBMX (Fig. 3B) produced peptide maps that were identical to those obtained with forskolin. The same peptides were phosphorylated when cells were stimulated with genistein (Fig. 3C), although the relative levels of phosphorylation appeared to be different, with phosphorylation of serine-768 being approximately equal to that of serine-795 and serine-737. Stimulation with the phosphatase inhibitor calyculin A (Fig. 3D) caused phosphorylation of the same peptides that were phosphorylated with forskolin, but serine-768 and -795 were more highly phosphorylated than serine-737, and there was extensive phosphorylation of serine-660 and -712. Stimulation with NS004 (Fig. 3E) led to CFTR phosphorylation on peptides containing serine-737 and -795, but the greatest amount of phosphorylation occurred on a nonidentified peptide, peptide A, that was not phosphorylated with any of the other treatments. These results suggest that genistein, IBMX, and calyculin A all activate the CFTR by a mechanism that involves PKA-dependent phosphorylation. Although the peptide map for NS004 clearly shows phosphorylation on sites for PKA-dependent phosphorylation, the appearance of an additional phosphopeptide suggests that phosphorylation by another kinase may be involved in NS004-dependent activation.

Several studies from this laboratory have suggested that genistein activates both wild-type CFTR and Delta F508-CFTR by inhibiting channel dephosphorylation (16, 23). Two mechanisms are possible: 1) genistein inhibits a phosphatase that dephosphorylates the CFTR; or 2) genistein binds to phosphorylated CFTR and thereby inhibits channel dephosphorylation. Recent studies, demonstrating a direct interaction between genistein and CFTR, are most consistent with the second mechanism but do not exclude inhibition of phosphatase activity. To help distinguish between these possibilities, we have examined in vivo phosphorylation of CREB. As shown in Fig. 4A, where in vivo phosphorylated CREB was probed with a polyclonal antibody for CREB in the top panel and phospho-CREB in the bottom panel, stimulation of NIH-CFTR cells with forskolin (lane 2) or calyculin A (lane 8) increased CREB phosphorylation. Thus we predicted that agents that increase PKA activity, or agents that inhibit phosphatase activity, would cause an increase in CREB phosphorylation. As shown in Fig. 4A, lane 4, IBMX increased CREB phosphorylation. This is consistent with IBMX-dependent inhibition of phosphodiesterase activity. In contrast, genistein, milrinone, CPX, and NS004 did not cause an increase in CREB phosphorylation (Fig. 4, A and B). Because milrinone is a specific inhibitor of type III phosphodiesterase, it is most likely that there is little type III phosphodiesterase activity in NIH-3T3 cells or that other phosphodiesterases are able to compensate for the loss of this activity. With regard to genistein, CPX, and NS004, the data suggest that the effects of these agents on CFTR activity were not due to increases in PKA activity or inhibition of phosphodiesterase activity.


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Fig. 4.   In vivo phosphorylation of cAMP binding response element (CREB). A: NIH-CFTR cells were activated with vehicle (lane 1), 10 µM forskolin (lane 2), 50 µM genistein (lane 3), 2 mM IBMX (lane 4), 100 µM milrinone (lane 5), 100 µM CPX (lane 6), 10 µM NS004 (lane 7), or 100 nM calyculin A (lane 8). Western blots of cell lysates were probed with anti-CREB (top panel) and anti-phospho-CREB (bottom panel). B: CREB phosphorylation as a percentage of control is plotted for indicated agonists. Data are presented as means ± SE (n >=  4).

To confirm that genistein, CPX, and NS004 do not activate CFTR by increasing cellular cAMP, agonist-dependent levels of cellular cAMP were determined in NIH-CFTR cells. In agreement with previous studies (16), both forskolin and IBMX increased cellular cAMP (Table 3). However, the increase in cAMP with IBMX, 3.4-fold, was far less than the 20-fold increase with forskolin. Milrinone, CPX, NS004, and calyculin A had no effect on cellular cAMP levels, whereas there was a small, 30%, increase with genistein. Because it has been suggested that IBMX enhances maximal forskolin-dependent CFTR activity by increasing cellular cAMP (9), we assayed cAMP levels in the presence of 10 µM forskolin plus 2 mM IBMX or 50 µM genistein. As shown in Fig. 5, neither IBMX nor genistein increased cAMP levels beyond those observed with 10 µM forskolin. In addition, 100 µM forskolin, shown previously not to increase wild-type CFTR, or Delta F508-CFTR, activity beyond that seen with 10 µM forskolin (16), failed to produce a greater increase in cAMP than 10 µM forskolin.

                              
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Table 3.   Agonist-dependent changes in cAMP


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Fig. 5.   Agonist-dependent stimulation of cAMP in NIH-CFTR cells. Cells were exposed to indicated agonists for 2 min. cAMP levels were determined by radioimmunoassay and are presented as means ± SE (n >=  5).

In a second test of the hypothesis that IBMX increases cAMP-dependent CFTR activity by increasing cellular cAMP, we used the membrane-permeant form of cAMP 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) (6) to stimulate I efflux in NIH-508 cells. As shown in Fig. 6, CPT-cAMP, like forskolin and IBMX, produced a small increase in Delta F508-CFTR activity. The addition of IBMX had synergistic effects on both CPT-cAMP and forskolin-dependent Delta F508-CFTR activity. However, the rate of I efflux in the presence of CPT-cAMP and forskolin was no greater than that with forskolin alone. These studies suggest that although IBMX can activate CFTR activity by increasing cellular cAMP, its synergistic effects on Delta F508-CFTR activity in the presence of agents that maximally elevate cellular cAMP must involve another mechanism.


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Fig. 6.   Synergistic effect of IBMX on cAMP-dependent activation of Delta F508-CFTR. NIH-508 cells were exposed to indicated agonists, and relative rates of I efflux were obtained by comparison with rate in absence of agonist. Data are presented as means ± SE (n >=  6). Where indicated, rates were significantly greater than those for forskolin (FSK) and IBMX (*) or 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) and IBMX (**).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Recent work has demonstrated that IBMX, genistein, CPX, milrinone, NS004, and calyculin A can activate the wild-type CFTR or Delta F508-CFTR by mechanisms that do not involve the activation of adenylate cyclase (9, 10, 13, 16, 17, 20, 21, 23, 30). Because these compounds also enhance cAMP-dependent activation of Delta F508-CFTR, it has been suggested that they might be of therapeutic value in the treatment of CF (16). These studies were undertaken to examine the mechanisms by which these compounds activate wild-type CFTR and Delta F508-CFTR and to determine if they act by common mechanisms.

Agonist-dependent effects on CFTR activity. When assayed by I efflux, agonist-dependent activation of wild-type CFTR was observed with all agonists. However, there was considerable variation in the maximal levels of CFTR activity. When added with forskolin, no agonist was able to increase wild-type CFTR activity beyond that obtained with forskolin alone. Our results are qualitatively similar to previous findings with the exception that we did not observe the potentiation of forskolin-dependent activity by IBMX that others have seen in the oocyte system (9). This may be because of a limitation of the I-efflux assay system, because in NIH-CFTR cells we have not seen potentiation of forskolin-dependent CFTR activity by genistein when activity is assayed by I efflux but have observed potentiation when activity is assayed in cell-attached patches (16).

For Delta F508-CFTR, increases in the rates of I efflux were seen with forskolin, genistein, IBMX, calyculin A, and milrinone. These increases in Delta F508-CFTR activity were similar to previous results from several laboratories (10, 16, 18). However, we have no explanation for the absence of activation with CPX and NS004. Although it has been suggested that micromolar concentrations of CPX are inhibitory, we have never observed activation of Delta F508-CFTR, or wild-type CFTR, at the nanomolar concentrations described by others (10). When Delta F508-CFTR was activated with forskolin, four agonists (genistein, IBMX, CPX, and NS004) caused synergistic increases in channel activity. These effects were similar to those seen previously (9, 13, 14, 16). If the mechanism of Kelley and co-workers (18, 19) is correct and milrinone stimulates channel activity by enhancing the forskolin-dependent increase in cAMP, the absence of a significant effect with milrinone may reflect a low level of phosphodiesterase III in NIH-3T3 cells. This is consistent with the lack of milrinone-dependent increase in cellular cAMP. Because calyculin A increased the rate of I efflux when added with saturating genistein or NS004, neither genistein nor NS004 is likely to inhibit a calyculin A-sensitive phosphatase. Because NS004 increased Delta F508-CFTR activity in the presence of saturating concentrations of genistein, both genistein and NS004 are also likely to activate CFTR by different mechanisms.

Agonist-dependent effects on in vivo phosphorylation. Studies of in vivo CFTR phosphorylation were undertaken to determine if the sites of in vivo phosphorylation were the same for each agonist. Phosphorylation during stimulation with forskolin, a PKA-dependent agonist, provided an assay for PKA-dependent phosphorylation. Calyculin A caused CFTR to be phosphorylated on the same sites that were phosphorylated during stimulation with forskolin. Although the relative magnitude of phosphorylation at some sites differed with calyculin A and forskolin, the similarity in the overall patterns suggests that under basal conditions, PKA is the only kinase that phosphorylates CFTR, or that calyculin A-inhibitable phosphatases are selective for PKA-specific phosphorylation sites. Both IBMX and genistein caused phosphorylation of the same sites as forskolin, suggesting that, in the presence of these agonists, CFTR is phosphorylated by PKA. This is consistent with IBMX activating CFTR by an inhibition of phosphodiesterase activity and the resulting increase in cAMP causing an activation of PKA (9). However, alternative mechanisms are consistent with the phosphopeptide maps because genistein and calyculin A produce very similar phosphopeptide maps without activating PKA (23). Because genistein does not activate PKA (23) or induce the phosphorylation of CREB, PKA-dependent CFTR phosphorylation in the presence of genistein must be due to an inhibition of CFTR dephosphorylation. However, because CREB is not phosphorylated and the effects of genistein and calyculin A on both wild-type CFTR (23) and Delta F508-CFTR activity are additive, genistein cannot inhibit a calyculin A-sensitive phosphatase. The most likely explanation for genistein-dependent in vivo phosphorylation of CFTR is that genistein binds to CFTR and that this inhibits dephosphorylation (16); inhibition of a calyculin A-insensitive phosphatase that dephosphorylates CFTR, but not CREB, is also a possible mechanism.

In contrast to forskolin, genistein, IBMX, and calyculin A, CFTR activation with NS004 led to phosphorylation on PKA-dependent sites and on at least one site that is not phosphorylated by PKA. This suggests that NS004 stimulates CFTR phosphorylation by a kinase that is distinct from PKA. In previous studies, we have shown that the unidentified phosphopeptide in Fig. 3E is also phosphorylated during stimulation with protein kinase C (PKC)-dependent agonists (35). It is therefore possible that NS004 activates CFTR through a PKC-dependent mechanism. However, the data do not distinguish between activation of PKC and inhibition of a phosphatase that reverses PKC-dependent phosphorylation.

Agonist-dependent effects on cAMP. Forskolin-dependent levels of intracellular cAMP were similar to those obtained previously (17). Milrinone, CPX, and NS004 did not increase cellular cAMP or stimulate CREB phosphorylation. Genistein produced a small increase in cellular cAMP that was similar to results from previous studies (17). Although there was a small increase in cAMP with genistein, there was no increase in CREB phosphorylation. This result is most consistent with genistein-dependent stimulation of CFTR by a cAMP-independent mechanism; however, it is possible that genistein increases cAMP levels in a cellular compartment that is associated with CFTR. Forskolin and IBMX, presumably via an increase in intracellular cAMP, and calyculin A, by inhibition of phosphatase activity, caused in vivo phosphorylation of CREB. The absence of CREB phosphorylation with genistein and NS004 demonstrates that neither agonist inhibits calyculin A-sensitive phosphatases.

Mechanism of IBMX-dependent CFTR stimulation. Dumm et al. (9) reported that millimolar concentrations of IBMX increased forskolin-dependent activation of Delta F508-CFTR by as much as 50-fold. Similar, albeit smaller, effects were seen with wild-type CFTR. These observations led to the widely accepted hypothesis that the increase in CFTR activity with IBMX is due to inhibition of phosphodiesterase activity and a consequent increase in cAMP. However, if the relationship between cAMP concentration and CFTR activity were linear (recent studies suggest that it shows saturation; T.-C. Hwang, personal communication), this mechanism requires that IBMX increases cellular cAMP by 50-fold beyond that achieved with 10 µM forskolin. Although, in the absence of agonist-dependent stimulation of adenylate cyclase, IBMX-dependent inhibition of phosphodiesterase activity increases cellular cAMP, our data demonstrate that IBMX does not increase cAMP when adenylate cyclase was stimulated with forskolin. Because in NIH-3T3 cells IBMX can increase the maximal forskolin-dependent Delta F508-CFTR activity without increasing cellular cAMP, our data demonstrate that IBMX increases Delta F508-CFTR activity by a cAMP-independent mechanism. It should also be noted that previous studies of the dose-response relationship for forskolin-dependent cAMP formation and wild-type CFTR activity (17) suggest that the increase in cAMP produced by 2 mM IBMX would not cause a detectable increase in CFTR activity were it produced with forskolin. Thus, even in the absence of high levels of cAMP, much of the stimulatory effect of IBMX is likely to be because of a cAMP-independent mechanism. The conclusion that IBMX does not increase forskolin-dependent Delta F508-CFTR via a cAMP-dependent mechanism was confirmed by the observation that CPT-cAMP does not increase forskolin-dependent activation of Delta F508-CFTR. The fact that in the presence of cAMP-dependent agonists IBMX activates CFTR by a cAMP-independent mechanism may necessitate the reevaluation of several studies that have used IBMX-dependent stimulation to make mechanistic interpretations about PKA-dependent channel activity. Our data also suggest that genistein and IBMX may use a common mechanism to activate CFTR. Both genistein and IBMX increase forskolin-dependent CFTR activity by a cAMP-independent mechanism. They also induce CFTR phosphorylation at the same sites. Moreover, at millimolar concentrations, IBMX increases the open time of wild-type CFTR in a fashion that is very similar to that observed with micromolar concentrations of genistein (Hwang, personal communication).

In summary, our data demonstrate that maximal cAMP-dependent activation of CFTR can be increased by a cAMP-independent mechanism. Our work provides additional evidence for genistein-dependent CFTR activation by a mechanism that does not involve the inhibition of phosphatase activity. Our data suggest that IBMX and genistein may activate CFTR by the same mechanism and that this may involve binding to CFTR. Even though we were unable to observe activation of Delta F508-CFTR by CPX, recent studies suggesting that CPX binds to CFTR at NBD1 are consistent with CPX acting by a similar mechanism (5). Our data also suggest that NS004 activates CFTR by a mechanism that is different from that of genistein. The ability of these compounds to increase cAMP-dependent activation of Delta F508-CFTR suggests that it may be possible to design therapeutic drugs that would increase cAMP-dependent activation. This approach, perhaps in concert with mechanisms for circumventing the processing defect in Delta F508-CFTR, may provide therapeutic benefit to the 90% of all CF patients that have at least one copy of Delta F508-CFTR.

    ACKNOWLEDGEMENTS

We thank Dr. Suzanne Fish for critical reading of the manuscript and Drs. Tzyh-Chang Hwang and Jonathan Cohn for helpful comments and sharing data before publication.

    FOOTNOTES

This work was supported by grants from the Cystic Fibrosis Foundation and the Nemours Foundation.

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: W. W. Reenstra, Dept. of Clinical Science, Alfred I. duPont Hospital for Children, PO Box 269, Wilmington, DE 19899-0269.

Received 4 May 1998; accepted in final form 6 July 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Anderson, M. P., H. A. Berger, D. R. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 67: 775-784, 1991[Medline].

2.   Arispe, N., J. Ma, K. A. Jacobson, and H. B. Pollard. Direct activation of cystic fibrosis transmembrane conductance regulator channels by 8-cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX). J. Biol. Chem. 273: 5727-5734, 1998[Abstract/Free Full Text].

3.   Berger, H. A., M. P. Anderson, R. J. Gregory, S. Thompson, P. W. Howard, R. A. Mauer, R. Mulligan, A. E. Smith, and M. J. Welsh. Identification and regulation of the cystic fibrosis transmembrane conductance regulator-generated chloride channel. J. Clin. Invest. 88: 1422-1431, 1991[Medline].

4.   Cheng, S. H., D. P. Rich, J. Marshall, R. J. Gregory, M. J. Welsh, and A. E. Smith. Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66: 1027-1036, 1991[Medline].

5.   Cohen, B. E., G. Lee, K. A. Jacobson, Y.-C. Kim, Z. Huang, E. J. Sorscher, and H. B. Pollard. 8-Cyclopentyl-1,3-diproplyxanthine and other xanthines differentially bind to the first nucleotide binding fold (NBF-1) domains of the cystic fibrosis transmembrane conductance regulator. Biochemistry 36: 6455-6461, 1997[Medline].

6.   Connolly, B. J., P. B. Willits, B. H. Warrington, and K. J. Murray. 8-(4-Chlorophenyl)thio-cyclic AMP is a potent inhibitor of the cyclic GMP-specific phosphodiesterase (PDE VA). Biochem. Pharmacol. 44: 2303-2306, 1992[Medline].

7.   Crawford, I., P. C. Maloney, P. L. Zeitlin, W. B. Guggino, S. C. Hyde, H. Turley, K. C. Gatter, A. Harris, and C. F. Higgins. Immunocytochemical localization of the cystic fibrosis gene product CFTR. Proc. Natl. Acad. Sci. USA 88: 9262-9366, 1991[Abstract].

8.   Denning, G. M., M. P. Anderson, J. F. Amara, J. Marshall, A. E. Smith, and M. J. Welsh. Processing of mutant cystic fibrosis transmembrane conductance regulator is temperature-sensitive. Nature 358: 761-764, 1992[Medline].

9.   Drumm, M. L., D. J. Wilkinson, L. S. Smit, R. T. Worrell, T. V. Strong, R. A. Frizzell, D. C. Dawson, and F. S. Collins. Chloride conductance expressed by Delta F508 and other mutant CFTRs in Xenopus oocytes. Science 254: 1797-1799, 1991[Medline].

10.   Eidelman, O., C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. A1-adenosine-receptor antagonists activate chloride efflux from cystic fibrosis cells. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992[Abstract].

11.   French, P. J., J. Bijman, A. G. Bot, W. E. M. Boomars, B. J. Scholte, and H. R. De Jonge. Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism. Am. J. Physiol. 273 (Cell Physiol. 42): C747-C753, 1997[Abstract/Free Full Text].

12.   Gadsby, D. C., and A. C. Nairn. Regulation of CFTR channel gating. Trends Biochem. Sci. 19: 513-518, 1994[Medline].

13.   Gribkoff, V., G. Champigny, P. Barbry, S. Dworetzky, N. Meanwell, and M. Lazdunski. The substituted benzimidazolone NS004 is an opener of the cystic fibrosis chloride channel. J. Biol. Chem. 269: 10983-10986, 1994[Abstract/Free Full Text].

14.   Haws, C. M., I. B. Nepomuceno, M. E. Krouse, H. Wakelee, T. Law, Y. Xia, H. Nguyen, and J. J. Wine. Delta F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines. Am. J. Physiol. 270 (Cell Physiol. 39): C1544-C1555, 1996[Abstract/Free Full Text].

15.   Hwang, T.-C., M. Horie, and D. C. Gadsby. Functionally distinct phospho-forms underlie incremental activation of PKA-regulated Cl- conductance in mammalian heart. J. Gen. Physiol. 101: 629-650, 1993[Abstract].

16.   Hwang, T.-C., F. Wang, S. Zeltwanger, I. C.-H. Yang, and W. W. Reenstra. Potentiation of Delta F508 channel function by genistein binding to CFTR. Am. J. Physiol. 273 (Cell Physiol. 42): C988-C998, 1997[Abstract/Free Full Text].

17.   Illek, B., H. Fischer, G. F. Santos, J. H. Widdicombe, T. E. Machen, and W. W. Reenstra. cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein. Am. J. Physiol. 268 (Cell Physiol. 37): C886-C893, 1995[Abstract/Free Full Text].

18.   Kelley, T. J., L. Al-Nakkash, C. U. Cotton, and M. L. Drumm. Activation of endogenous Delta F508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition. J. Clin. Invest. 98: 513-520, 1996[Abstract/Free Full Text].

19.   Kelley, T. J., L. Al-Nakkash, and M. L. Drumm. CFTR-mediated chloride permeability is regulated by type III phosphodiesterases in airway epithelial cells. Am. J. Respir. Cell. Mol. Biol. 13: 657-664, 1995[Abstract].

20.   Kerem, B.-S., J. M. Rommens, J. A. Buchanan, D. Markiewicz, T. K. Cox, A. Chakravarti, M. Buchwald, and L.-C. Tsui. Identification of the cystic fibrosis gene: genetic analysis. Science 245: 1073-1085, 1989[Medline].

21.   Lehrich, R. W., and J. N. Forrest, Jr. Tyrosine phosphorylation is a novel pathway for regulation of chloride secretion in shark rectal gland. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F594-F600, 1995[Abstract/Free Full Text].

22.   Picciotto, M. R., J. A. Cohn, G. Bertuzzi, P. Greengard, and A. C. Nairn. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 267: 12742-12752, 1992[Abstract/Free Full Text].

23.   Reenstra, W. W., K. Yurko-Mauro, A. Dam, S. Raman, and S. Shorten. CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases. Am. J. Physiol. 271 (Cell Physiol. 40): C650-C657, 1996[Abstract/Free Full Text].

24.   Riordan, J. R., J. M. Rommens, B.-S. Kerem, N. Alon, R. Rozmahel, Z. Grzelczak, J. Zielenski, S. Lok, N. Plavsic, J.-L. Chou, M. L. Drumm, M. C. Iannuzzi, F. S. Collins, and L.-C. Tsui. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245: 1066-1073, 1989[Medline].

25.   Schweibert, E. M., M. E. Eagan, T. H. Hwang, S. B. Fulmer, S. S. Allen, G. R. Cutting, and W. B. Guggino. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 81: 1063-1073, 1995[Medline].

26.   Sears, C. L., F. Firoozmand, A. Mellander, F. G. Chambers, I. G. Eromar, A. G. M. Bot, B. Scholte, H. R. De Jonge, and M. Donowitz. Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 monolayers. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G874-G882, 1995[Abstract/Free Full Text].

27.   Smith, P. K., R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150: 76-85, 1985[Medline].

28.   Venglarik, C. J., R. J. Bridges, and R. A. Frizzell. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells. Am. J. Physiol. 259 (Cell Physiol. 28): C358-C364, 1990[Abstract/Free Full Text].

29.   Wang, F., S. Zeltwanger, I. C.-H. Yang, A. C. Nairn, and T.-C. Hwang. Actions of genistein on cystic fibrosis transmembrane conductance regulator channel gating: evidence for two binding sites with opposite effects. J. Gen. Physiol. 111: 477-490, 1998[Abstract/Free Full Text].

30.   Weinreich, F., P. G. Wood, J. R. Riordan, and G. Nagel. Direct action of genistein on CFTR. Pflügers Arch. 434: 484-491, 1997[Medline].

31.   Welsh, M. J., and A. E. Smith. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73: 1251-1254, 1993[Medline].

32.   Welsh, M. J., L.-C. Tsui, T. F. Boat, and A. L. Beaudet. The Metabolic and Molecular Basis of Inherited Disease (7th ed.). New York: McGraw-Hill, 1995, p. 3799-3863.

33.   Yamamoto, T., S. Yamamoto, J. C. Osborne, V. C. Manganiello, M. Vaughan, and H. Hikada. Complex effects of inhibitors on cyclic GMP-stimulated cyclic nucleotide phosphodiesterase. J. Biol. Chem. 258: 14173-14177, 1983[Abstract/Free Full Text].

34.   Yang, I. C.-H., T.-H. Cheng, F. Wang, E. M. Price, and T.-C. Hwang. Modulation of CFTR chloride channels by calyculin A and genistein. Am. J. Physiol. 272 (Cell Physiol. 41): C142-C155, 1997[Abstract/Free Full Text].

35.   Yurko-Mauro, K. A., and W. W. Reenstra. Prostaglandin F2alpha stimulates CFTR activity by PKA- and PKC-dependent phosphorylation. Am. J. Physiol 275 (Cell Physiol. 44): C653-C660, 1998[Abstract].


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