Departments of 1 Clinical Science and 2 Pediatrics, Alfred I. duPont Hospital for Children, Thomas Jefferson University, Wilmington, Delaware 19803
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 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
F508-CFTR is defective and that little protein reaches the apical
membrane (8). We have recently established that cAMP-dependent
activation of
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 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
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
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
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
F508-CFTR and to
potentiate cAMP-dependent activation of
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 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
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture.
NIH-3T3 cells that stably express wild-type CFTR (NIH-CFTR) and
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% -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 F508-CFTR. This
study compared the effects of these compounds on wild-type CFTR and
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
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
F508-CFTR (Fig.
1D and Table 1). The largest effects
were obtained with genistein, IBMX, and NS004.
|
|
The effects of calyculin A on wild-type CFTR and 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
F508-CFTR, calyculin A increased
the rate of I efflux. When
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 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
F508-CFTR activity beyond that seen with forskolin plus genistein. As with wild-type CFTR, the
addition of NS004 caused a significant increase in
F508-CFTR activity.
|
|
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.
|
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 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.
|
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 F508-CFTR, activity
beyond that seen with 10 µM forskolin (16), failed to produce a
greater increase in cAMP than 10 µM forskolin.
|
|
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 F508-CFTR activity. The addition of
IBMX had synergistic effects on both CPT-cAMP and forskolin-dependent
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
F508-CFTR activity in the presence of agents that maximally elevate cellular cAMP
must involve another mechanism.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent work has demonstrated that IBMX, genistein, CPX, milrinone,
NS004, and calyculin A can activate the wild-type CFTR or 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
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
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).
ForAgonist-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
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.
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 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
F508-CFTR activity without increasing cellular
cAMP, our data demonstrate that IBMX increases
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
F508-CFTR via a
cAMP-dependent mechanism was confirmed by the observation that CPT-cAMP
does not increase forskolin-dependent activation of
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).
![]() |
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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 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
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
14.
Haws, C. M.,
I. B. Nepomuceno,
M. E. Krouse,
H. Wakelee,
T. Law,
Y. Xia,
H. Nguyen,
and
J. J. Wine.
F508-CFTR channels: kinetics, activation by forskolin, and potentiation by xanthines.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1544-C1555,
1996
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 F508 channel function by genistein binding to CFTR.
Am. J. Physiol.
273 (Cell Physiol. 42):
C988-C998,
1997
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
18.
Kelley, T. J.,
L. Al-Nakkash,
C. U. Cotton,
and
M. L. Drumm.
Activation of endogenous F508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.
J. Clin. Invest.
98:
513-520,
1996
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
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
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
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
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
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
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
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
35.
Yurko-Mauro, K. A.,
and
W. W. Reenstra.
Prostaglandin F2 stimulates CFTR activity by PKA- and PKC-dependent phosphorylation.
Am. J. Physiol
275 (Cell Physiol. 44):
C653-C660,
1998[Abstract].