Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
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Previous studies in rat bile
canalicular membrane vesicles and WIF-B9 cells revealed that
cAMP-induced trafficking of ATP-binding cassette (ABC) transporters to
the canalicular membrane and their activation require phosphoinositide
3-kinase (PI3-K) products. In the present studies, canalicular
secretion of fluorescein isothiocyanate-glycocholate in WIF-B9 cells
was increased by cAMP and a decapeptide that enhances PI3-K activity;
these effects were inhibited by wortmannin. To determine the
mechanism(s) whereby cAMP activates PI3-K, we examined signal
transduction pathways in WIF-B9 and COS-7 cells. cAMP activated PI3-K
in both cell lines in a phosphotyrosine-independent manner. PI3-K
activity increased in association with p110 in both cell lines. The
effect of cAMP was KT-5720 sensitive, suggesting involvement of protein
kinase A. Expression of a dominant-negative
-adrenergic receptor
kinase COOH terminus (
-ARKct), which blocks G
signaling, decreased PI3-K activation in both cell lines. cAMP increased GTP-bound
Ras in COS-7 but not WIF-B9 cells. Expression of dominant-negative Ras
abolished cAMP-mediated PI3-K, which suggests that the effect is
downstream of Ras and G
. These data indicate that cAMP activates PI3-K in a cell type-specific manner and provide insight regarding mechanisms of PI3-K activation required for bile acid secretion.
bile secretion; heterotrimeric G protein; G; protein kinase
A; Ras
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INTRODUCTION |
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CAMP REGULATES critical biological functions through activation of several signaling pathways (44). In addition to activation of adenylyl cyclase isoforms and protein kinase A (PKA), cAMP-activated signaling pathways include A-kinase anchor proteins, phosphodiesterases, cAMP regulatory element-binding protein (CREB), CREB-binding proteins (CBP/p300) (39), and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs), which activate small GTPases such as Rap1 and Rap2 (5, 22). Our previous studies (11, 31, 32, 42) suggest cross talk between cAMP and phosphoinositide 3-kinase (PI3-K) in regulation of bile acid secretion across the canalicular membrane. The mechanism is unknown and is the long-range goal of our studies.
Administration of dibutyryl cAMP (DBcAMP) to rats increases biliary secretion of taurocholate, which results from increased trafficking of bsep, the canalicular ATP-dependent bile acid transporter, from intracellular sites to the canalicular membrane (11, 24). The concentration of bsep in the canalicular membrane increased approximately threefold, concomitant with a similar increase in PI3-K activity and ATP-dependent transport of taurocholate (21). Administration of wortmannin in vivo or after addition to canalicular membrane vesicles inhibited each response to DBcAMP; however, addition of phosphoinositide products of PI3-K but not of phosphatidylinositol (PI)4-P, the enzyme's substrate, to canalicular membrane vesicles restored bsep activity in the presence of wortmannin (21). In studies using a polarized rat hepatoma-human fibroblast hybrid cell line (WIF-B9), administration of a decapeptide (PI3-K peptide; Refs. 15, 18), which enters cells and activates PI3-K, increased canalicular bile acid secretion in a wortmannin-inhibitable manner (32, 42, 53). These previously published studies in rat liver, canalicular membrane vesicles, and WIF-B9 cells suggest interactions between cAMP and PI3-K signal transduction pathways as related to bsep trafficking and activity in the canalicular membrane. Studies in other systems suggest similar cross talk. For example, wortmannin inhibits cAMP activation of Akt (2, 3, 13, 28, 54, 56) in a cell type-specific manner, as well as serum- and glycocorticoid-induced protein kinase (Sgk) (35).
The mechanism(s) responsible for cAMP and PI3-K cross talk have not been identified in any system and were the objective of the present studies. WIF-B9 and COS-7 cells were chosen for study on the basis of the following rationale. Preliminary studies in freshly isolated rat hepatocytes confirmed that cAMP increased PI3-K activity (54); however, primary cultures lack cellular polarity, which prevents correlation with bsep function or trafficking. Hepatocytes that retain tight junctions during preparation (so-called hepatocyte doublets) were not studied because they retain a hemi-canaliculus from adjacent cells that complicates investigation of canalicular membrane trafficking (24). Furthermore, transient transfection in primary hepatocytes is unpredictable for biochemical studies. Because transport studies in WIF-B9 cells yielded results similar to those observed in rat liver (42, 53), we chose to study the relation between cAMP administration and PI3-K activation in polarized WIF-B9 cells and in nonpolarized COS-7 cells as a control.
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MATERIALS AND METHODS |
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Materials. DBcAMP, wortmannin, pertussis toxin (PTX), phosphatidylserine (PS), PI, PI 3,4-bisphosphate (PI 3,4-P2), and glycocholate were purchased from Sigma (St. Louis, MO). KT-5720, PP2, and forskolin were obtained from Calbiochem (San Diego, CA).
Cell culture. COS-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (Life Technologies, Rockville, MD). WIF-B9 cells, an immortalized cell line obtained from a rat hepatoma cell line-human fibroblast fusion that forms functional bile canaliculus (20, 45), were grown in F-12 medium (Life Technologies) supplemented with 5% FBS. Jurkat cells were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) with 10% FBS. All cells were cultured at 37°C in a humidified atmosphere with 5% (COS-7 and Jurkat) or 7% (WIF-B9) CO2. Cells were starved in 1% bovine serum albumin (Sigma) for 40 h for COS-7 and 16 h for WIF-B9 cells and treated with DBcAMP or forskolin for the indicated time period. When indicated, cells were incubated with inhibitors KT-5720 for 30 min, PTX overnight, or PP2 for 1 h before treatment with DBcAMP.
Expression of dominant-negative constructs.
COS-7 cells were transfected with RasN17 (gift of Dr. L. A. Feig,
Tufts University, Boston, MA) by using Lipofectamine Plus (Life
Technologies) according to the manufacturer's instructions and
cultured for 24 h. Cells were induced to quiescence and subjected to treatment with DBcAMP. For expression of dominant-negative G,
cells were infected with adenovirus vector alone or adenovirus expressing the
-adrenergic receptor kinase COOH terminus (
-ARKct) (gift of Dr. R. J. Lefkowitz, Duke University Medical Center, Durham, NC) at 100 multiplicities of infection (MOI) (7,
26). Medium was replaced with complete medium 2 h after
infection. Cells were starved after 24 h and subjected to the
experiments as indicated. The expression of
-ARKct was tested by
immunoblotting with a
-ARK antiserum that recognizes the COOH
terminus of
-ARK2 (gift of Dr. R. J. Lefkowitz).
PI3-K activity assay.
Cells were washed with ice-cold phosphate-buffered saline (PBS) three
times and lysed in lysis buffer (50 mM HEPES, pH 7.5, 0.5 mM EGTA, 5 mM
sodium orthovanadate, 10% glycerol, and 1% NP-40) with protease
inhibitors (0.5 mM PMSF, 5 µg/ml pepstatin A, 10 µg/ml aprotinin,
and 10 µg/ml leupeptin). Cell lysates were incubated on ice for 5 min
and spun at 4°C for 10 min at 12,000 g to remove insoluble
fractions. The supernatant was frozen immediately in aliquots by
immersion in liquid nitrogen and stored at 80°C until being used.
PI3-K activity assay was performed in immunoprecipitates by using 200 µg of protein from cell lysates after incubation with anti-p85
antibody (no. 06-497; Upstate Biotechnology, Lake Placid, NY) or
350 µg of protein incubated with anti-phosphotyrosine (anti-P-tyr)
antibody (4G10; Upstate Biotechnology) or anti-p110
, -
, or -
antibodies (H-201, H-198, and H-199, respectively; Santa Cruz
Biotechnology, Santa Cruz, CA) at 4°C for 2 h followed by incubation with 30 µl of 50% suspension of protein A beads (Sigma) for 1 h. After the beads were washed once with lysis buffer and three times with PBS, they were sedimented by brief centrifugation and
PI3-K activity was quantified as previously described (32, 50). Briefly, the reaction was performed in a total volume of 50 µl containing 150 µM ATP, 25 mM MOPS (pH 7.0), 5 mM
MgCl2, 1 mM EGTA, and 0.2 mg/ml sonicated lipids: PS-PI-PI
4,5-bisphosphate (PI 4,5-P2) (1:1:1 vol/vol/vol) in
sonication buffer (25 mM MOPS, pH 7.0, 1 mM EGTA) with 25 µCi of
[
-32P]ATP per assay (NEN, Boston, MA). After
incubation at 37°C for 20 min, the reaction was stopped with 90 µl
of methanol-1 N HCl (1:1 vol/vol) and lipids were extracted twice in
100 µl of chloroform. The organic layer was combined, dried under
nitrogen gas flow, and analyzed by TLC (Whatman, Clifton, NJ).
Polyphosphoinositides were resolved in water-acetic
acid-n-propanol (34:1:65 vol/vol/vol), and 32P
label was detected by autoradiography. TLC spots corresponding to PI
3,4,5-trisphosphate (PIP3) products were scraped and eluted in scintillation liquid, and 32P incorporation into
PIP3 was quantified by liquid scintillation counting. To
compare the results obtained from different experiments, whole cell
lysates from HEK-293 cells were diluted 1,000-fold (50)
and used as control (designated as 1 unit of PI3-K activity).
Immunoblotting. Twenty micrograms of cell lysates obtained as described in PI3-K activity assay were separated by SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA), and probed with antibodies to Akt and phospho-Akt (pAkt) (Cell Signaling Technology, Beverly, MA), total ERK1/2 (Upstate Biotechnology), and phospho-ERK1/2 (pERK1/2, Santa Cruz Biotechnology). The membranes were probed with appropriate horseradish peroxidase-coupled secondary antibodies (Cell Signaling) followed by enhanced chemiluminescence (ECL) reaction (NEN, Boston, MA). The densitometric analysis was performed with the NIH Image program, and the relative intensity was calculated relative to total protein content. Normal rabbit IgG (Santa Cruz Biotechnology) was used as a negative control.
Ras-GTP assay. GTP-bound Ras was quantified as described previously (12). Briefly, the Ras-binding domain of Raf was expressed as a glutathione S-transferase (GST)-fusion protein in Escherichia coli and affinity purified on glutathione Sepharose 4B beads (Amersham Pharmacia Biotech, Piscataway, NJ). Ten micrograms of immobilized fusion protein were incubated with four hundred micrograms of whole cell lysates for 2 h at 4°C. The beads were washed once with lysis buffer and three times with PBS. After addition of 30 µl of sample buffer, the beads were heated to 95°C for 5 min and centrifuged for 2 min at 12,000 g in a microcentrifuge column (Bio-Rad Laboratories, Hercules, CA) to remove the beads. The flow-through was separated by SDS-PAGE followed by immunoblotting with anti-Ras antibodies (Upstate Biotechnology). To confirm that an equal amount of total Ras protein was present in each sample, cell lysates were also immunoblotted with anti-Ras antibodies.
Bile acid transport in WIF-B9 cells. With a previously described technique (42), monolayers of WIF-B9 cells were grown on glass coverslips, mounted in a Dvorak-Stotler culture chamber that was thermostabilized at 37°C, and promptly viewed by confocal laser scanning microscopy. Cells were perfused with modified F-12 supplemented with 5% FBS and 12 mM HEPES (pH 7.0) with a perfusion pump (model 351; Sege Instruments, Cambridge, MA) at 0.3 ml/min. Images were obtained with an Odyssey XL confocal system (Noran Instruments, Middletown, WI) and an inverted Nikon Diaphot microscope equipped with a Nikon ×60 oil-immersion Planchromat lens (NA 1.4). After a baseline fluorescent image was acquired, the chamber was perfused for 20 min with a second solution containing 0.5 µM glycocholic acid conjugated with fluorescein isothiocyanate (FITC-GC), which exhibits the requisite structural features of conventional bile acids and is a substrate for the bile acid transporter (16, 25). Excitation was at 488 nm with a krypton-argon laser. The microscope was calibrated daily by adjusting the gain and offset to obtain constant values for an intensity calibration curve. Linearity of the signal as a function of the concentration of the fluorescent probe was verified. Quantification was performed with Inter Vision computer software by measuring fluorescence intensity in the perinuclear cytoplasmic and canalicular regions, which were delimited by boundaries drawn on the first image of the series. Similar studies were performed in WIF-B9 cells that were preincubated for 10 min at 37°C with DBcAMP (300 µM) or rhodamine-linked PI3-K peptide (5, 20 µM) (15, 18, 42) and washed twice with PBS before perfusion with FITC-GC. Cells were also incubated in wortmannin (100 nM) for 10 min at 37°C before addition of DBcAMP, PI3-K peptide, or FITC-GC. Control cells for experiments using wortmannin were exposed to DMSO (0.01%).
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RESULTS |
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cAMP and PI3-K peptide increased FITC-GC secretion in a
wortmannin-inhibitable manner in WIF-B9 cells.
As shown in Table 1, cAMP and PI3-K
peptide significantly increased FITC-GC secretion into bile canaliculi
of WIF-B9 cells 2.0- and 2.1-fold, respectively; both responses were
inhibited by prior incubation with wortmannin (100 nM). In a previous
study (42), canalicular secretion of FITC-GC by WIF-B9
cells was time dependent, saturable, stimulated by preincubation of
cells with taurocholate (100 µM) or PI3-K peptide (5 µM), and
decreased by preincubation with wortmannin (100 nM). The present study
demonstrated that, like taurocholate, cAMP and PI3-K peptide
significantly increased FITC-GC secretion in WIF-B9 cells. The
stimulatory effects of cAMP and PI3-K peptide were both inhibited by
wortmannin (100 nM), which suggests that cAMP may enhance bile acid
secretion through activation of PI3-K. These studies provided the
impetus to identify the biochemical mechanism(s) whereby cAMP and PI3-K interact with one another as regards bile acid secretion.
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cAMP activated PI3-K in a time- and dose-dependent manner in COS-7
and WIF-B9 cell lines.
PI3-K activity was quantified before and after stimulation with the
cell-permeant cAMP analog DBcAMP. Within 15 min, cAMP activated
p85-associated PI3-K in both cell lines (Fig.
1, A and C for
COS-7 and B and D for WIF-B9 cells). Growth
factors such as PDGF produced maximal activation within 10 min
(50); in contrast, the maximal response (2.0- to 2.5-fold)
to cAMP was observed at 60 min; p85-associated PI3-K activation
returned to basal levels after 4 h in both cell lines. Stimulation
with forskolin (50 µM, 30 min) also activated PI3-K by 2.8 ± 0.5- and 2.3 ± 0.2 (mean ± SE)-fold in COS-7 and WIF-B9
cells, respectively (Fig. 1E for COS-7, Fig. 1F
for WIF-B9 cells). The increase in association with p85 was not smaller
than that observed by growth factors; EGF (10 ng/ml, 15 min) in COS-7
and insulin (10 ng/ml, 15 min) in WIF-B9 induced 2.4 ± 0.5- and
2.6 ± 0.4 (mean ± SE)-fold increase, respectively. The cAMP
effect on PI3-K was dose dependent (Fig. 1,G and
H). The calculated ED50 was 67.0 and 56.2 µM
in COS-7 and WIF-B9 cells, respectively. The increase in PI3-K activity resulted from modification in intrinsic catalytic activity, because the
amount of immunoreactive p110 subunits associated with p85 did not vary
after cAMP stimulation (data not shown). In whole lysates, PI3-K
activity increased 1.5-fold in both cell lines within 1 h (data
not shown).
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cAMP-mediated PI3-K activation was independent of phosphotyrosine.
cAMP stimulation did not generate tyrosine-phosphorylated proteins, in
contrast to the effect of EGF and insulin in COS-7 and WIF-B9 cells,
respectively (Fig. 2A).
Phosphotyrosine-associated PI3-K activity did not change after cAMP
stimulation at any time period, in contrast to the effect of EGF and
insulin, which induced robust activation in COS-7 and WIF-B9 cells
(Fig. 2B). These observations indicate that cAMP-mediated
PI3-K activation is phosphotyrosine independent. The rapidity of PI3-K
activation by cAMP suggests that transcription is unaffected.
Therefore, on the basis of evidence from other pathways that involve
cAMP, we investigated the role of PKA, G, and Ras in
cAMP-mediated activation of PI3-K in COS-7 and WIF-B9 cells.
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cAMP activated Akt and ERK1/2 in COS-7 cells.
To determine the effect of cAMP on downstream targets of PI3-K,
we quantified activation of Akt by determining phosphorylation of Akt
on Ser473 (Fig. 3, A and
C). In COS-7 cells, Akt
phosphorylation was detected 15-30 min after addition of cAMP. The
effect of cAMP on Akt phosphorylation was significant [relative
intensity at 15 min (mean ± SE): 2.3 ± 0.3;
P < 0.05 vs. basal level], although substantially
smaller than the response to EGF (9.0-fold). This effect was wortmannin inhibitable (Fig. 3A), suggesting that cAMP-mediated Akt
activation, although modest and transient, is PI3-K dependent. In
contrast, Akt activation was not detectable in WIF-B9 cells at any time (Fig. 3C). EGF also failed to activate Akt, possibly because
WIF-B9 cells lack the EGF receptor. WIF-B9 cells possess a functional insulin receptor, and Akt was activated by insulin (13.7-fold). Similar
events were observed in ERK1/2 activation by cAMP, namely, cAMP
transiently activated ERK1/2 by 2.5-fold in COS-7 cells (Fig. 3B), consistent with a previous report (9).
There was no activation of ERK1/2 in WIF-B9 cells (Fig. 3D).
Insulin also activated ERK1/2 in WIF-B9 cells. Higher concentrations of
cAMP (up to 1 mM) did not activate Akt or ERK1/2 in WIF-B9 cells (data
not shown).
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p110 Catalytic subunit-specific PI3-K activity and expression of
each subunit.
To determine why cAMP did not activate Akt in WIF-B9 cells despite cAMP
activation of p85-associated PI3-K, we characterized expression of
PI3-K catalytic subunits by measuring catalytic subunit-associated
PI3-K activity and protein expression by Western blotting. cAMP
increased p110-associated PI3-K activity in COS-7 cells (3-fold) and
in WIF-B9 cells (2.5-fold) (Fig.
4A). p110
-associated PI3-K
activity increased 1.7-fold in COS-7 cells, whereas basal and
cAMP-stimulated activities in immunoprecipitates were substantially lower in WIF-B9 cells. PI3-K activity associated with p110
was undetectable in both cell lines even in the presence of cAMP. These
results are not due to an inhibitory effect of anti-p110
antibodies
on PI3-K because p110
-associated PI3-K activity was detected in
Jurkat cells. Immunoblotting revealed p110
expression in cell
lysates and p85 immunoprecipitates in COS-7 and WIF-B9 cells (Fig.
4B). In contrast, p110
was expressed only in COS-7 cells
in concordance with the activity assay data described above. No protein
was detected in immunoprecipitates with normal rabbit IgG, which
confirmed specificity of the results. Expression of p110
was
detected only in Jurkat cells, an acute T cell leukemia cell line,
confirming that expression of p110
, originally cloned from a bone
marrow cDNA library (49), is relatively restricted to
hematopoietic cells. The class 1A PI3-K catalytic subunit, p110
, was
not tested because its expression is restricted to leukocytes
(52). These results reveal that COS-7 cells express p110
and p110
, whereas WIF-B9 cells only express p110
.
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cAMP-mediated PI3-K activation was sensitive to an inhibitor of
PKA, KT-5720.
KT-5720, an inhibitor of PKA, did not affect basal PI3-K activity but
decreased cAMP-mediated PI3-K activity by 74% in COS-7 and 79% in
WIF-B9 cells (Fig. 5A).
Addition of PKA catalytic subunit to PI3-K in vitro did not change
PI3-K activity (data not shown). Src kinase can be activated by cAMP
and bind to and activate PI3-K (14). We found that cAMP
induces activation of Src in COS-7 cells (data not shown). However,
PP2, a Src inhibitor, had no effect on PI3-K activity (Fig.
5B). Because several reports indicate cross talk between
Gs and Gi signaling (4), we also
investigated the involvement of Gi signaling with PTX. PTX
also failed to block cAMP-mediated PI3-K activation (Fig.
5B), suggesting that neither Src nor Gi
signaling is involved.
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Role of Ras in cAMP-mediated PI3-K activation.
Ras induces PI3-K activation by direct binding to the p110 catalytic
subunits (41) in a phosphotyrosine-independent manner. Addition of cAMP to COS-7 cells increased GTP-bound Ras in 15-30 min [2.2 ± 0.4 (mean ± SE)-fold increase at 30 min;
P < 0.05 compared with basal level; Fig.
6A]. To determine whether Ras
activation in COS-7 cells is upstream of PI3-K, we determined the
effect of wortmannin on cAMP-induced Ras activation. Wortmannin did not affect cAMP-mediated Ras activation (Fig. 6B). In WIF-B9
cells, cAMP did not activate Ras; however, in the presence of cAMP,
wortmannin reduced Ras activation to below control levels (Fig.
6B). These data suggest that, in COS-7 cells, Ras
participates in PI3-K activation and is either upstream or independent.
In contrast, cAMP-mediated increase in PI3-K activity in WIF-B9 cells
does not involve activation of Ras.
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G involvement in cAMP-mediated PI3-K activation.
G
is reported to stimulate PI3-K in many cell types (27,
47, 49, 51). We used an adenoviral vector that expresses
-ARKct to inhibit signaling through G
.
-ARKct specifically binds to and sequesters G
complexes (7). Cells were
infected with the adenovirus, and the effect of cAMP on PI3-K
activation was quantified. The expression of
-ARKct was confirmed by
Western blotting (Fig. 7A,
top). Whereas control
adenovirus infection had no effect,
-ARKct infection attenuated
cAMP-mediated PI3-K activation by 68% and 70% in COS-7 and WIF-B9
cells, respectively (Fig. 7A, bottom), suggesting
involvement of G
in this process. We also determined whether
G
is required for cAMP-induced Ras activation in COS-7 cells
(Fig. 7B). Expression of
-ARKct blocked cAMP-mediated
activation of Ras, whereas infection with control adenovirus did not
affect Ras activation [fold increase: 1.8 ± 0.3 (mean ± SE) for cAMP + empty adenovirus and 0.9 ± 0.2 for cAMP +
-ARKct; P < 0.05]. Similar results were observed
with pERK1/2 [fold increase: 2.5 ± 0.5 (mean ± SE) for
cAMP + empty adenovirus and 1.2 ± 0.4 for cAMP +
-ARKct; P < 0.05; Fig. 7B], which
provides a positive control for attenuation of Ras function in response
to
-ARKct. These observations suggest that G
is upstream of
Ras in response to cAMP in COS-7 cells.
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DISCUSSION |
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cAMP activated class IA PI3-K in a time- and dose-dependent manner in COS-7 and WIF-B9 cells. There have been few studies determining PI3-K activity in response to cAMP. PI3-K activation was reported in FRTL-5, a rat thyroid follicular cell line (33), and PC12, a pheochromocytoma cell line (37). In our study, increased PI3-K activity was detected at 15 min, remained elevated for 2 h, and declined at 4 h. This time course of response differs from effects observed after addition of growth factors such as PDGF, in which PI3-K activity peaks at 10-15 min and returns to baseline within 1 h (6, 48, 50). On stimulation with growth factors, p85 subunit SH2 domains interact with specific phosphotyrosine motifs (46), resulting in activation of PI3-K activity (8). Tyrosine phosphorylation can be removed quickly by tyrosine phosphatases, which leads to dissociation of p85 from target proteins. In contrast to the response induced by growth factors, cAMP activation of p85-associated PI3-K was independent of phosphotyrosine in both cell lines.
Akt is a downstream target of PI3-K and, in some studies, cAMP-induced Akt activation was blocked by a PI3-K inhibitor, wortmannin (13, 37, 54). However, in 293-EBNA cells, forskolin activation of Akt was PI3-K independent (10), suggesting that Akt response to cAMP may differ by cell type. Although cAMP increased PI3-K activity in both COS-7 and WIF-B9 cells, Akt activation was detected only in COS-7 cells and was inhibited by nanomolar concentrations of wortmannin. In WIF-B9 cells, insulin but not cAMP activated Akt, which suggests that different pools of PI3-K are utilized. Because cAMP activates Akt in hepatocytes (28, 54, 56), WIF-B9 cells may lack intermediate molecules required for cAMP-mediated Akt activation in response to PI3-K. Additional observations suggest that PI3-K activation by cAMP may not be directly linked to Akt. In both cell lines, maximal PI3-K activation was observed at 60 min (Fig. 1, C and D) whereas maximal phosphorylation of Akt occurred at 15 min and declined to baseline levels by 60 min (Fig. 3, A and B).
To examine mechanisms of activation based on PI3-K catalytic subunits,
we determined expression of p110 isoforms in COS-7 and WIF-B9 cells. In
COS-7 cells p110 and p110
catalytic subunits were expressed,
whereas only p110
was detectable in WIF-B9 cells. These data suggest
that activation of p110
is insufficient for transduction of some
PI3-K downstream signals, such as Akt. Because p110
was originally
purified and cloned from rat liver and is widely expressed, the lack of
p110
expression in WIF-B9 was unexpected. Expression of p110
may
be lost during fusion or subsequent passages, similar to loss of
p110
expression in colon cancer cell lines despite expression of
this protein in colonic epithelium (43). Lysophosphatidic
acid- and carbachol-induced Akt activation require p110
, which
suggests that p110
expression may be essential for transducing cAMP
signaling to Akt in WIF-B9 cells.
G specifically activates p110
(47) and p110
(19, 27, 30). Blocking downstream signaling from
heterotrimeric G proteins by expression of
-ARKct, which
specifically binds to and sequesters G
complexes
(7), impaired activation of PI3-K by cAMP in both cell
lines. Because WIF-B9 cells express only p110
, it is likely that
G
involvement is indirect. PI3-K activation was also sensitive to
KT-5720, suggesting involvement of PKA. The molecular mechanism whereby
PKA leads to release of G
is not well understood; a signal switch
to Gi from Gs has been proposed in response to
2-adrenergic receptor stimulation (4).
Because PTX did not block cAMP-induced PI3-K activation, other pathways probably exist. For example, reactive oxygen species can induce G
liberation without activating G protein-coupled receptors (34).
An additional mechanism for PI3-K activation is activation of Ras.
GTP-bound Ras can directly bind to PI3-K catalytic subunits, resulting
in conformational changes and increased catalytic activity (40). Ras can also be a downstream target of PI3-K
(17, 55). cAMP induced an increase in GTP-bound Ras in
COS-7 and melanocytes (1) but not in WIF-B9 cells.
Activation of Ras in COS-7 cells was not blocked by wortmannin, and
expression of the dominant-negative Ras construct (RasN17) abolished
PI3-K activation by cAMP. These data suggest that Ras activation is
upstream of PI3-K in COS-7 cells and is required for cAMP-mediated
PI3-K activation. These observations are also consistent with
activation of ERK1/2 by cAMP, which was not sensitive to wortmannin
(data not shown). Several mechanisms have been proposed for Ras
activation in response to cAMP. cAMP-GEFs can directly activate Rap1/2
(5, 22), and a cyclic nucleotide Ras-GEF, CDS-25Mm, which
is phosphorylated by PKA, has also been described (36).
However, expression of these GEFs has been found specifically in brain.
Other, as yet unidentified, molecules may function as GEFs for Ras in
response to cAMP in other cells. Ras and ERK1/2 activation were blocked by expression of -ARKct, which suggests that G
release is
required for Ras activation in COS-7 cells. Although the molecular
mechanisms are not well defined, G
release may activate Ras by
initiating assembly of a multiprotein complex including
-arrestin or
c-Src in clathrin-coated pits (29, 38).
A recent report showed that cAMP had an opposite effect in COS cells that overexpress PI3-K and Akt (23). Under these conditions, cAMP blocked PI3-K and Akt activation. The difference between our data and these results may be due to the use of different clones of COS cells or to the fact that overexpression of these proteins leads to a different response.
In conclusion, cAMP activated PI3-K in COS-7 and WIF-B9 cells and the
pathway leading to PI3-K activation was cell type specific. PKA and
G were required in both cell lines; however, the response in
COS-7 cells required Ras activation whereas the response in WIF-B9
cells did not. Our study indicates that there are different pathways by
which cAMP activates PI3-K. How these individual mechanisms relate to
specific cellular responses, such as bile acid secretion, requires
additional studies, which are in progress.
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ACKNOWLEDGEMENTS |
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This research was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-54785 (to I. M. Arias) and 30-DK-34928 (Digestive Disease Center).
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FOOTNOTES |
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Address for reprint requests and other correspondence: I. M. Arias, Dept. of Physiology, Tufts Univ. School of Medicine, 136 Harrison Ave., Boston, MA 02111 (E-mail: irwin.arias{at}tufts.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.
August 7, 2002;10.1152/ajpcell.00041.2002
Received 28 January 2002; accepted in final form 24 July 2002.
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REFERENCES |
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1.
Busca, R,
Abbe P,
Mantoux F,
Aberdam E,
Peyssonnaux C,
Eychene A,
Ortonne JP,
and
Ballotti R.
Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes.
EMBO J
19:
2900-2910,
2000
2.
Cass, LA,
Summers SA,
Prendergast GV,
Backer JM,
Birnbaum MJ,
and
Meinkoth JL.
Protein kinase A-dependent and -independent signaling pathways contribute to cyclic AMP-stimulated proliferation.
Mol Cell Biol
19:
5882-5891,
1999
3.
Crowder, RJ,
and
Freeman RS.
The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3-kinase and Akt.
J Neurochem
73:
466-475,
1999[ISI][Medline].
4.
Daaka, Y,
Luttrell LM,
and
Lefkowitz RJ.
Switching of the coupling of the beta2-adrenergic receptor to different G proteins by protein kinase A.
Nature
390:
88-91,
1997[ISI][Medline].
5.
De Rooij, J,
Zwartkruis FJ,
Verheijen MH,
Cool RH,
Nijman SM,
Wittinghofer A,
and
Bos JL.
Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.
Nature
396:
474-477,
1998[ISI][Medline].
6.
Domin, J,
Dhand R,
and
Waterfield MD.
Binding to the platelet-derived growth factor receptor transiently activates the p85-p110
phosphoinositide 3-kinase complex in vivo.
J Biol Chem
271:
21614-21621,
1996
7.
Drazner, MH,
Peppel KC,
Dyer S,
Grant AO,
Koch WJ,
and
Lefkowitz RJ.
Potentiation of beta-adrenergic signaling by adenoviral-mediated gene transfer in adult rabbit ventricular myocytes.
J Clin Invest
99:
288-296,
1997
8.
Fantl, WJ,
Escobedo JA,
Martin GA,
Turck CW,
del Rosario M,
McCormick F,
and
Williams LT.
Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.
Cell
69:
413-423,
1992[ISI][Medline].
9.
Faure, M,
Voyno-Yasenetskaya TA,
and
Bourne HR.
cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells.
J Biol Chem
269:
7851-7854,
1994
10.
Filippa, N,
Sable CL,
Filloux C,
Hemmings B,
and
Van Obberghen E.
Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase.
Mol Cell Biol
19:
4989-5000,
1999
11.
Gatmaitan, ZC,
Nies AT,
and
Arias IM.
Regulation and translocation of ATP-dependent apical membrane proteins in rat liver.
Am J Physiol Gastrointest Liver Physiol
272:
G1041-G1049,
1997
12.
Goi, T,
Rusanescu G,
Urano T,
and
Feig LA.
Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth.
Mol Cell Biol
19:
1731-1741,
1999
13.
Gonzalez-Robayna, IJ,
Falender AE,
Ochsner S,
Firestone GL,
and
Richards JS.
Follicle-stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A kinase-independent signaling by FSH in granulosa cells.
Mol Endocrinol
14:
1283-1300,
2000
14.
Haefner, B,
Baxter R,
Fincham VJ,
Downes CP,
and
Frame MC.
Cooperation of Src homology domains in the regulated binding of phosphatidylinositol 3-kinase. A role for the Src homology 2 domain.
J Biol Chem
270:
7937-7943,
1995
15.
Hartwig, JH,
Kung S,
Kovacsovics T,
Janmey PA,
Cantley LC,
Stossel TP,
and
Toker A.
D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate.
J Biol Chem
271:
32986-32993,
1996
16.
Holzinger, F,
Schteingart CD,
Ton-Nu HT,
Cerre C,
Steinbach JH,
Yeh HZ,
and
Hofmann AF.
Transport of fluorescent bile acids by the isolated perfused rat liver: kinetics, sequestration, and mobilization.
Hepatology
28:
510-520,
1998[ISI][Medline].
17.
Hu, Q,
Klippel A,
Muslin AJ,
Fantl WJ,
and
Williams LT.
Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase.
Science
268:
100-102,
1995[ISI][Medline].
18.
Hubner, S,
Couvillon AD,
Kas JA,
Bankaitis VA,
Vegners R,
Carpenter CL,
and
Janmey PA.
Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides.
Eur J Biochem
258:
846-853,
1998[Abstract].
19.
Igarashi, J,
Bernier SG,
and
Michel T.
Sphingosine 1-phosphate and activation of endothelial nitric-oxide synthase. Differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells.
J Biol Chem
276:
12420-12426,
2001
20.
Ihrke, G,
Neufeld EB,
Meads T,
Shanks MR,
Cassio D,
Laurent M,
Schroer TA,
Pagano RE,
and
Hubbard AL.
WIF-B cells: an in vitro model for studies of hepatocyte polarity.
J Cell Biol
123:
1761-1775,
1993[Abstract].
21.
Kagawa, T,
Misra S,
Varticovski L,
and
Arias IM.
The mechanisms whereby cAMP activates PI 3-kinase which regulates canalicular spgp (Abstract).
Hepatology
32:
306A,
2000.
22.
Kawasaki, H,
Springett GM,
Mochizuki N,
Toki S,
Nakaya M,
Matsuda M,
Housman DE,
and
Graybiel AM.
A family of cAMP-binding proteins that directly activate Rap1.
Science
282:
2275-2279,
1998
23.
Kim, S,
Jee K,
Kim D,
Koh H,
and
Chung J.
Cyclic AMP inhibits Akt activity by blocking the membrane localization of PDK1.
J Biol Chem
276:
12864-12870,
2001
24.
Kipp, H,
Pichetshote N,
and
Arias IM.
Transporters on demand: intrahepatic pools of canalicular ATP binding cassette transporters in rat liver.
J Biol Chem
276:
7218-7224,
2001
25.
Kitamura, T,
Gatmaitan Z,
and
Arias IM.
Serial quantitative image analysis and confocal microscopy of hepatic uptake, intracellular distribution and biliary secretion of a fluorescent bile acid analog in rat hepatocyte doublets.
Hepatology
12:
1358-1364,
1990[ISI][Medline].
26.
Koch, WJ,
Hawes BE,
Inglese J,
Luttrell LM,
and
Lefkowitz RJ.
Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling.
J Biol Chem
269:
6193-6197,
1994
27.
Kurosu, H,
Maehama T,
Okada T,
Yamamoto T,
Hoshino S,
Fukui Y,
Ui M,
Hazeki O,
and
Katada T.
Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110 is synergistically activated by the
subunits of G proteins and phosphotyrosyl peptide.
J Biol Chem
272:
24252-24256,
1997
28.
Li, J,
Yang S,
and
Billiar TR.
Cyclic nucleotides suppress tumor necrosis factor -mediated apoptosis by inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation.
J Biol Chem
275:
13026-13034,
2000
29.
Luttrell, LM,
Ferguson SS,
Daaka Y,
Miller WE,
Maudsley S,
Della Rocca GJ,
Lin F,
Kawakatsu H,
Owada K,
Luttrell DK,
Caron MG,
and
Lefkowitz RJ.
Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.
Science
283:
655-661,
1999
30.
Maier, U,
Babich A,
and
Nurnberg B.
Roles of non-catalytic subunits in G-induced activation of class I phosphoinositide 3-kinase isoforms
and
.
J Biol Chem
274:
29311-29317,
1999
31.
Misra, S,
Ujhazy P,
Gatmaitan Z,
Varticovski L,
and
Arias IM.
The role of phosphoinositide 3-kinase in taurocholate-induced trafficking of ATP-dependent canalicular transporters in rat liver.
J Biol Chem
273:
26638-26644,
1998
32.
Misra, S,
Ujhazy P,
Varticovski L,
and
Arias IM.
Phosphoinositide 3-kinase lipid products regulate ATP-dependent transport by sister of P-glycoprotein and multidrug resistance associated protein 2 in bile canalicular membrane vesicles.
Proc Natl Acad Sci USA
96:
5814-5819,
1999
33.
Nedachi, T,
Akahori M,
Ariga M,
Sakamoto H,
Suzuki N,
Umesaki K,
Hakuno F,
and
Takahashi SI.
Tyrosine kinase and phosphatidylinositol 3-kinase activation are required for cyclic adenosine 3',5'-monophosphate-dependent potentiation of deoxyribonucleic acid synthesis induced by insulin-like growth factor-I in FRTL-5 cells.
Endocrinology
141:
2429-2438,
2000
34.
Nishida, M,
Maruyama Y,
Tanaka R,
Kontani K,
Nagao T,
and
Kurose H.
G alpha(i) and G alpha(o) are target proteins of reactive oxygen species.
Nature
408:
492-495,
2000[ISI][Medline].
35.
Perrotti, N,
He RA,
Phillips SA,
Haft CR,
and
Taylor SI.
Activation of serum- and glucocorticoid-induced protein kinase (Sgk) by cyclic AMP and insulin.
J Biol Chem
276:
9406-9412,
2001
36.
Pham, N,
Cheglakov I,
Koch CA,
de Hoog CL,
Moran MF,
and
Rotin D.
The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP.
Curr Biol
10:
555-558,
2000[ISI][Medline].
37.
Powers, JF,
Misra S,
Schelling K,
Varticovski L,
and
Tischler AS.
Mitogenic signaling by cyclic adenosine monophosphate in chromaffin cells involves phosphatidylinositol 3-kinase activation.
J Cell Biochem Suppl
36:
89-98,
2001.
38.
Pumiglia, KM,
LeVine H,
Haske T,
Habib T,
Jove R,
and
Decker SJ.
A direct interaction between G-protein subunits and the Raf-1 protein kinase.
J Biol Chem
270:
14251-14254,
1995
39.
Richards, JS.
New signaling pathways for hormones and cyclic adenosine 3',5'-monophosphate action in endocrine cells.
Mol Endocrinol
15:
209-218,
2001
40.
Rodriguez-Viciana, P,
Warne PH,
Dhand R,
Vanhaesebroeck B,
Gout I,
Fry MJ,
Waterfield MD,
and
Downward J.
Phosphatidylinositol-3-OH kinase as a direct target of Ras.
Nature
370:
527-532,
1994[ISI][Medline].
41.
Rodriguez-Viciana, P,
Warne PH,
Khwaja A,
Marte BM,
Pappin D,
Das P,
Waterfield MD,
Ridley A,
and
Downward J.
Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras.
Cell
89:
457-467,
1997[ISI][Medline].
42.
Sai, Y,
Nies AT,
and
Arias IM.
Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells.
J Cell Sci
112:
4535-4545,
1999
43.
Sasaki, T,
Irie-Sasaki J,
Horie Y,
Bachmaier K,
Fata JE,
Li M,
Suzuki A,
Bouchard D,
Ho A,
Redston M,
Gallinger S,
Khokha R,
Mak TW,
Hawkins PT,
Stephens L,
Scherer SW,
Tsao M,
and
Penninger JM.
Colorectal carcinomas in mice lacking the catalytic subunit of PI(3)Kgamma.
Nature
406:
897-902,
2000[ISI][Medline].
44.
Servillo, G,
Della Fazia MA,
and
Sassone-Corti P.
Cyclic AMP signaling in the liver: coupling transcription to physiology and proliferation.
In: The Liver: Biology and Pathobiology (4th ed.), edited by Arias IM,
Boyer JL,
Chisari FV,
Fausto N,
Schachter D,
and Shafritz DA.. Philadelphia, PA: Lippincott Williams and Wilkins, 2001, p. 525-536.
45.
Shanks, MR,
Cassio D,
Lecoq O,
and
Hubbard AL.
An improved polarized rat hepatoma hybrid cell line. Generation and comparison with its hepatoma relatives and hepatocytes in vivo.
J Cell Sci
107:
813-825,
1994
46.
Songyang, Z,
Shoelson SE,
McGlade J,
Olivier P,
Pawson T,
Bustelo XR,
Barbacid M,
Sabe H,
Hanafusa H,
Yi T,
and
Cantley LC.
Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.
Mol Cell Biol
14:
2777-2785,
1994[Abstract].
47.
Stephens, L,
Smrcka A,
Cooke FT,
Jackson TR,
Sternweis PC,
and
Hawkins PT.
A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits.
Cell
77:
83-93,
1994[ISI][Medline].
48.
Stephens, LR,
Jackson TR,
and
Hawkins PT.
Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system?
Biochim Biophys Acta
1179:
27-75,
1993[ISI][Medline].
49.
Stoyanov, B,
Volinia S,
Hanck T,
Rubio I,
Loubtchenkov M,
Malek D,
Stoyanova S,
Vanhaesebroeck B,
Dhand R,
Nurnberg B,
Gierschik P,
Seedorf K,
Hsuan JJ,
Waterfield MD,
and
Wetzker R.
Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.
Science
269:
690-693,
1995[ISI][Medline].
50.
Susa, M,
Keeler M,
and
Varticovski L.
Platelet-derived growth factor activates membrane-associated phosphatidylinositol 3-kinase and mediates its translocation from the cytosol. Detection of enzyme activity in detergent-solubilized cell extracts.
J Biol Chem
267:
22951-22956,
1992
51.
Thomason, PA,
James SR,
Casey PJ,
and
Downes CP.
A G-protein beta gamma-subunit-responsive phosphoinositide 3-kinase activity in human platelet cytosol.
J Biol Chem
269:
16525-16528,
1994
52.
Vanhaesebroeck, B,
and
Waterfield MD.
Signaling by distinct classes of phosphoinositide 3-kinases.
Exp Cell Res
253:
239-254,
1999[ISI][Medline].
53.
Wakabayashi, Y,
and
Arias IM.
Microtubular-dependent bidirectional trafficking of BSEP in polarized WIF-B9 cells (Abstract).
Hepatology
34:
258A,
2001.
54.
Webster, CR,
and
Anwer MS.
Role of the PI3K/PKB signaling pathway in cAMP-mediated translocation of rat liver Ntcp.
Am J Physiol Gastrointest Liver Physiol
277:
G1165-G1172,
1999
55.
Yart, A,
Laffargue M,
Mayeux P,
Chretien S,
Peres C,
Tonks N,
Roche S,
Payrastre B,
Chap H,
and
Raynal P.
A critical role for phosphoinositide 3-kinase upstream of Gab1 and SHP2 in the activation of ras and mitogen-activated protein kinases by epidermal growth factor.
J Biol Chem
276:
8856-8864,
2001
56.
Zhao, AZ,
Shinohara MM,
Huang D,
Shimizu M,
Eldar-Finkelman H,
Krebs EG,
Beavo JA,
and
Bornfeldt KE.
Leptin induces insulin-like signaling that antagonizes cAMP elevation by glucagon in hepatocytes.
J Biol Chem
275:
11348-11354,
2000