Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis
Kimberly A. Cullen,1
John McCool,1
M. Sawkat Anwer,2 and
Cynthia R. L. Webster1
Departments of 1Clinical and 2Biomedical Science, Tufts University School of Veterinary Medicine, North Grafton, Massachusetts 01536
Submitted 12 December 2003
; accepted in final form 20 March 2004
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ABSTRACT
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cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-
-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-
. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-
-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.
bile acid apoptosis; death receptor apoptosis; phosphatidylinositol 3-kinase; Akt; Rap 1
CAMP TRANSDUCES SURVIVAL SIGNALS in a diverse array of cell types (18, 19, 28, 31, 36, 55, 60). In hepatocytes, cAMP protects against apoptosis induced by hydrophobic bile acids, TNF-
, and Fas ligand (11, 14, 27, 44, 55). The downstream effectors of cAMP's antiapoptotic action have not been fully characterized. In classic signaling cascades, cAMP activates PKA, a serine threonine kinase, which in turn regulates intracellular pathways via phosphorylation of signaling intermediates (25). cAMP activation of PKA alone, however, cannot account for cAMP's survival effect in all cell types. Whereas in neurons and gastric epithelial cells, cAMP's antiapoptotic effect is PKA dependent (19, 28), in neutrophils and pancreatic beta cells the survival effect of cAMP is PKA independent (8, 31). In hepatocytes, cAMP-mediated survival in bile acid and TNF-
-mediated apoptosis is only partially PKA dependent (14, 27, 55). These studies suggest that the antiapoptotic effect of cAMP may be mediated primarily through PKA-independent signaling pathways in hepatocytes.
It is now well established that cAMP controls intracellular signaling by regulating proteins other than PKA (25). cAMP can bind to a novel class of guanine nucleotide exchange factors (GEFs) known as exchange proteins regulated by cAMP (Epacs) or cAMP-GEFs (3, 4). These cAMP-GEFs link cAMP production to activation of the small GTPases, Rap 1 and Rap 2. Rap GTPases, members of the Ras subfamily of GTP binding proteins, exist in an inactive GDP-bound form and an active GTP-bound conformation. GEFs catalyze the release of GDP allowing GTP to bind. In the active GTP-bound state, Rap interacts with target proteins to promote cellular responses.
The biological significance of cAMP-mediated Rap activation is beginning to emerge (3, 4). Recent studies (22, 30) show that Rap activation accounts for cAMP-regulated secretion of insulin and amyloid precursor protein from pancreatic beta cells and neurons, respectively. In addition to its role in exocytosis, Rap activation plays a pivotal role in mediating inside-out integrin signaling and in this context modulates lymphocyte adhesion and migration in response to integrin binding (24, 42). cAMP-mediated Rap activation also potentiates the response to mitogenic stimuli in thyroid follicular cells and promotes neurite outgrowth (6, 52).
Several studies suggest that a cAMP-GEF/Rap pathway may mediate cell survival. Selective activation of cAMP-GEF in pancreatic beta cells protects against free fatty acid-induced apoptosis in a pancreatic beta cell line (26). Genetic deletion of Rap 1 is embryonic lethal in Drosophila and mice and makes it difficult to establish viable mammalian and slime mold cell lines (4, 23). Rap activation is necessary for growth factor-mediated survival in hematopoietic cells (49) and mediates intracellular trafficking of survival receptors in neurons (2, 35, 61). These studies made it logical to hypothesize that the PKA-independent antiapoptotic effects of cAMP may be linked to cAMP-GEF/Rap activation.
Two protein kinases linked to hepatocyte survival, ERK and Akt, are proposed downstream effectors of Rap. Depending on cell types studied, Rap-GTP can activate or inhibit Akt or ERK (35, 29). The role of Rap in modulating these kinases in hepatocytes remains uncharacterized. It is known that cAMP can activate Akt and inhibit ERK in hepatocytes (12, 14, 27, 48, 55, 56, 58). In hepatocytes, ERK inhibition has been reported to be both a PKA-dependent and -independent event (14, 48), although in nonhepatic cells, cAMP modulation of ERK is PKA dependent (9). The role of PKA in Akt activation in hepatocytes has not been well characterized.
Modulation of both the Akt and ERK signaling pathways has been implicated in hepatocyte survival (16, 41, 50, 55, 57). Several studies have demonstrated that cAMP activates phosphatidylinositol 3-kinase (PI3-kinase)/Akt (10, 21, 40), and the results of our work show that this activation is necessary for the survival effect of cAMP in bile acid-induced hepatocyte apoptosis (58). In our previous studies (55, 57), we demonstrated that ERK inhibition had a weak protective effect in bile acid-induced hepatocyte apoptosis; however, other studies (41) demonstrate that growth factor-mediated ERK activation is cytoprotective.
In the present study, the role of cAMP-GEF and PKA activation in cAMP's antiapoptotic action in hepatocytes and in cAMP's modulation of ERK and Akt were investigated. These studies show that cAMP-mediated protection from apoptosis induced by various stimuli is largely independent of PKA and that cAMP-induced activation of Akt and inhibition of ERK are PKA-independent and PKA-dependent events, respectively. Furthermore, a novel cAMP-GEF binding cAMP analog, which activates Rap 1 but not PKA in hepatocytes, promotes hepatocyte survival and PI3-kinase/Akt activation, but does not affect ERK activity.
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MATERIALS AND METHODS
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Reagents.
Collagenase, wortmannin, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), glucagon, Hoechst 33258, glycochenodeoxycholate (GCDC), actinomycin D, and all tissue culture reagents were purchased from Sigma-Aldrich (St. Louis, MO). The cAMP-GEF-specific cAMP analog CPT-2'-O-methyl (CPT-2-Me)-cAMP, the PKA inhibitor Rp-8-(4-chlorophenythio)-cAMP (Rp-cAMP), and human Fas ligand were from Alexis Biochemical (San Diego, CA). Ly-294002 and PKA inhibitor 622 amide peptide were from Calbiochem (San Diego, CA). Murine recombinant TNF-
was from R&D Systems (Minneapolis, MN). Radiochemicals, [3H]taurocholate and [32P]ATP were from Perkin Elmer (Boston, MA). Phospho-specific antibodies to cAMP response element binding protein (CREB)ser133 were from Upstate Biotechnology (Lake Placid, NY) and phospho-specific antibodies for Aktser473 and p42/p44ERKthr202/tyr204 were obtained from Cell Signaling Technology (Beverly, MA). Actin and caspase 3 antibodies were from Calbiochem and Santa Cruz Biotechnology (Santa Cruz, CA), respectively.
Primary cultures of rat hepatocytes.
Rat hepatocytes were isolated from male Wistar rats (200250 g) as previously described (55). Animal studies were conducted in accordance with National Institutes of Health policy on Care and Use of Animals in Research and were granted approval by the university's Institutional Animal Care and Use Committee. Hepatocytes were plated at 5 x 105 cells/cm on 35- mm3 tissue culture dishes or coverslips coated with Type I rat tail collagen in MEM with L-glutamine, 100 nM insulin, and 10% heat-inactivated fetal calf serum and were incubated at 37°C in a humidified atmosphere of 5% CO2. After 1 h was allowed for cell attachment, cultures were washed, and media without insulin or serum was added. After an additional 3 h, experiments were initiated by adding the hydrophobic bile acid GCDC (50 µM), Fas ligand (50 ng/ml), or the combination of TNF-
(25 ng/ml) and actinomycin D (200 ng/ml). Unless otherwise noted, modulators were added at the indicated concentration 30 min before adding apoptotic stimuli.
Assessment of hepatocyte apoptosis.
Morphological evaluation of apoptotic cell death was done at various times after treatment with GCDC or TNF-
/Fas as previously described (55). Briefly, coverslips were stained with Hoechst 33258 and apoptosis was evaluated with fluorescent microscopy. Apoptotic cells were identified as those whose nucleus exhibits brightly stained condensed chromatin or nuclear fragmentation. Five hundred cells were counted by an observer blinded to the treatment conditions, and the number of apoptotic cells were expressed as a %total number of cells counted.
The presence of the p17 kD cleavage product of caspase 3 was used as a biochemical indicator of hepatocyte apoptosis. Cell lysates were prepared from hepatocyte cultures treated with GCDC for 2 h, Fas ligand for 3 h, or TNF-
for 5 h in cell lysis buffer A [in mM: 20 Tris (pH = 7.5), 150 NaCl, 2 EDTA, 2.5 sodium pyrophosphate, 1 glycerolphosphate, 1 PMSF, and 1 sodium orthovanadate plus 1% Triton, 1 mm EGTA, 100 nM okadiac acid, and 10 µg/ml of leupeptin, aprotonin, and pepstatin]. Protein concentrations were determined by the Lowry method as previously described (55). Lysate protein (100 µg) was separated on SDS-PAGE, and the proteins were electrophoretically transferred to polyvinylidenefluoride (PVDF) membranes. Immunoblotting was performed with anti-caspase 3 antibodies and equal protein loading was verified by stripping the blots and probing with actin antibody.
Determination of PKA activation.
PKA activity was directly assayed by using a commercially available kit (SignaTECH cAMP-dependent protein kinase assay system; Promega, Madison, WI) that relies on phosphorylation of a biotinylated Kemtide substrate. Specificity of the assay was assessed by adding a protein kinase inhibitor peptide (Calbiochem) directly to the assay mixture. Because PKA mediates phosphorylation of CREBser133, this phosphorylation event was used as an additional marker of PKA activation. Whole cell lysates prepared from hepatocytes treated with modulators for 15 min were subjected to immunoblotting with phosphoantibodies to CREBser133.
Determination of Rap 1 activation.
Rap activation was determined by using a commercially available Rap activation assay kit (Upstate Biotechnology, Lake Placid, NY) that measures the amount of activated GTP-bound Rap precipitated by a GST tagged protein corresponding to the Ral GDS binding domain of Rap bound to glutathione-agarose. The GTP-bound Rap is detected by immunoblotting with anti-Rap antibodies that recognize Rap 1.
Preparation of whole cell lysates and Western blot analysis techniques.
Hepatocyte cultures were treated with the indicated modulators and cell lysates prepared at predetermined time points in cell lysis buffer A. After being diluted in SDS gel-loading buffer and boiling, 50100 µg of protein were separated by SDS-PAGE, and proteins were transferred to PVDF (for proteins < 25 kD) or nitrocellulose (for proteins > 25 kD) membranes. After being blocked, membranes were incubated with primary antibody for 2 h at room temperature or overnight at 4°C. After being washed, blots were incubated with the appropriate peroxidase-labeled secondary antibodies (Bio-Rad, Hercules, CA) for 1 h at room temperature. Immunoblots were developed with enhanced chemiluminescence (Amersham, Piscataway, NJ), and proteins were detected by autoradiography. Blots were scanned into Adope Photoshop and subjected to computerized densitometric scanning using Sigma Gel.
Bile acid uptake.
The 30-min accumulation of radiolabeled bile acid, [3H]taurocholate (Perkin Elmer), in hepatocyte cultures was determined as previously described (57).
Statistical evaluation.
All results are expressed as the means ± SD and analyzed for statistical significance with Student's t-test or ANOVA. A P value of <0.05 was considered significant.
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RESULTS
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cAMP-mediated protection against bile acid-induced apoptosis is PKA independent.
The specific PKA inhibitor, Rp-cAMP, was used to assess the role of PKA in cAMP cytoprotection (6, 13, 47). In rat hepatocytes, pretreatment with Rp-cAMP minimally reversed cAMP's antiapoptotic effect in GCDC-induced cell apoptosis (
10%) (Fig. 1A). The role of PKA in cAMP-mediated protection in two physiologically relevant models of death receptor-mediated apoptosis mediated by Fas ligand and TNF-
was evaluated. In both cases, pretreatment with Rp-cAMP had only a marginal effect on the protective effect of cAMP (Fig. 1, B and C). Two strategies were used to verify that RP-cAMP inhibited cAMP-mediated activation of PKA: 1) direct assay of PKA and 2) inhibition of PKA-mediated phosphorylation of CREBser133. PKA activity was determined in cytosolic extracts with a commercially available kit (Promega's SignaTECH), which utilizes a biotinylated Kemtide substrate. Specificity of this assay in rat hepatocytes was verified by using a synthetic peptide derived from the 622 amino acid sequence of the endogenous PKA inhibitor PKA-I. When this inhibitor was directly added to cytosolic extracts, no cAMP-stimulated PKA activity could be measured with the assay kit (data not shown). Phosphorylation of CREB was determined in whole cell lysates by immunoblotting with phospho-specific antibodies to CREBser133. Hepatocytes were treated with Rp-cAMP for 30 min before stimulation with cAMP for 15 min. In these studies, Rp-cAMP (500 µM) consistently inhibited both cAMP-stimulated PKA activity and CREBser133 phosphorylation (Fig. 2). Collectively, these results suggest that cAMP's antiapoptotic effect in hepatocytes is largely PKA independent.

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Fig. 2. Effect of Rp-cAMP on PKA activity and PKA-mediated phosphorylation of CREBser133. A: cell lysates were prepared from untreated hepatocytes and hepatocytes treated with 100 µM CPT-cAMP for 15 min. Some cultures were pretreated with 500 µM Rp-cAMP 30 min before incubation with cAMP. Cytosolic PKA activity was determined by using a biotinylated Kemtide substrate according to the manufacturer's instructions. B: whole cell lysates were prepared from hepatocyte cultures treated for 15 min with 100 µM CPT-cAMP with or without 30-min pretreatment with 500 µM Rp-cAMP. The amount of PKA-dependent CREB phosphorylation was determined by Western blot analysis (50 µg) with phosphos-specific CREBser133 antibodies. The relative activity (means ± SD) represents the amount of phosphorylated protein compared with untreated control cultures after normalization for protein loading and is the result of 3 separate experiments. *Value is significantly different from that seen in control cells; #value is statistically different that that seen in the presence of cAMP.
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cAMP activates Rap 1 in hepatocytes.
Because emerging evidence suggests that cAMP-mediated activation of Rap 1 represents a PKA-independent method by which cAMP controls cell signaling (3, 4), the effect of cAMP on the Rap 1 signaling pathway in hepatocytes was investigated. Treatment of rat hepatocytes with 100 µM CPT-cAMP for varying periods of time resulted in sustained activation of Rap 1 (Fig. 3A). Treatment with glucagon (200 nM), which raises cAMP levels by binding to a G protein-coupled receptor, also activated Rap 1 in hepatocytes (Fig. 3, B and C).

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Fig. 3. cAMP activates Rap 1 in rat hepatocytes. Whole cell lysates were prepared from untreated control hepatocytes and hepatocytes treated with 100 µM CPT-cAMP or 200 nM glucagon for 15 min. Some cultures were pretreated with 20 µM Ly-294002 (Ly) for 30 min before adding cAMP. The amount of activated GTP-bound Rap 1 was determined by using a pull-down assay with a glutathione-agarose conjugate of Ral GDS binding domain and detected by immunoblotting with Rap 1 antibodies. A: time course of cAMP-mediated Rap 1 activation. Rat hepatocytes were treated with 100 µM CPT-cAMP, and whole cell lysates were prepared at the indicated times for determination of the amount of GTP-bound Rap. B: representative immunoblot from the experiments is shown. Positive and negative controls represent the amount of GTP-Rap present in cell lysates loaded in vitro with GTP S and GDP, respectively. C: graphical depiction of the amount of Rap 1 immunoreactivity quantified by computer software. All results represent the means ± SD of a least 3 separate experiments. *Statistically different from the value in control cells.
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The effect of a novel cAMP analog, CPT-2-Me-cAMP, in nonhepatic cells on cAMP-mediated events in hepatocytes was investigated. This analog contains a modification in the cAMP/PKA binding domain that substantially decreases the ability of the compound to activate PKA but does not affect the binding of cAMP to cAMP-GEFs that control Rap activation (6, 9, 22, 43). Cultured hepatocytes were treated with CPT-2-Me-cAMP for 15 min, and the amount of active GTP-bound Rap 1 was determined. The CPT-2-Me-cAMP analog activated Rap 1 to a degree comparable to that seen with cAMP (Table 1). The effect of this analog on PKA activity in rat hepatocytes was assessed by directly determining PKA activity in cytosolic extracts and by determination of the amount of CREBser133 phosphorylation. Treatment with cAMP (100 µM) significantly increased PKA activity and CREBser133 phosphorylation, whereas CPT-2-Me-cAMP had no effect on these PKA-mediated events (Table 1). Even the highest concentration of CPT-2-Me-cAMP tested (100 µM) failed to phosphorylate CREBser133 and resulted in only a mild twofold increase in PKA activity (data not shown). Cumulatively, these results demonstrate that CPT-2-Me-cAMP activates Rap 1 in hepatocytes without appreciable effect on PKA activity or PKA-mediated phosphorylation events. Because this selectivity was somewhat diminished at higher concentrations, a 20 µM dose of CPT-2-Me-cAMP was used for further studies.
Because bile acids must enter hepatocytes to induce apoptosis, the effect of CPT-2-Me-cAMP on the uptake of a radiolabeled bile acid was investigated. Taurocholate accumulation in untreated hepatocytes and hepatocytes treated with 20 µM CTP-2-Me-cAMP for 30 min was 22.3 ± 1.2 and 22.2 ± 1.3 pmol·mg1·30 min1, respectively. At higher concentrations (50 µM), CTP-2-Me-cAMP began to inhibit bile acid accumulation (35% inhibition at 30 min, data not shown).
CPT-2-Me cAMP is antiapoptotic in hepatocytes.
Cultured hepatocytes were pretreated with 20 µM CPT-2-Me-cAMP for 30 min before the addition of GCDC, Fas ligand, or TNF-
, and the amount of apoptosis was determined at sequential time points by morphological evaluation of Hoechst-stained cells. CPT-2-Me-cAMP protected hepatocytes from apoptosis mediated by each of the apoptotic stimuli (Fig. 4).

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Fig. 4. Activation of cAMP-guanine exchange factor (cAMP-GEF) is antiapoptotic in rat hepatocytes. Primary cultures of rat hepatocytes were treated with GCDC (50 µM) (A) Fas ligand (100 ng/ml) (B) or a combination of TNF- (25 µg/ml) plus actinomycin D (200 µg/ml) (C) for the indicated times, and the amount of apoptosis was determined by morphological examination of Hoechst-stained cells. Some cultures were pretreated with 20 µM of CPT-2'-O-methyl-cAMP (CPT-2-Me-cAMP) for 30 min before the addition of the apoptotic stimulus. Results are expressed as %apoptosis and represent the means ± SD for 3 separate experiments.
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Because we have previously shown that CPT-cAMP-mediated cytoprotection against GCDC-induced apoptosis is PI3-kinase dependent (55, 58), the effect of PI3-kinase inhibition with Ly-294002 on the antiapoptotic effect of the cAMP-GEF-specific analog was determined. PI3-kinase inhibition prevented the protective effect of CPT-2-Me-cAMP in bile acid-induced apoptosis (Fig. 5A). The protective effect of CPT-2-Me-cAMP was accompanied by inhibition of caspase 3 processing, which was abolished in the presence of PI3-kinase inhibition (Fig. 5B).

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Fig. 5. Role of phosphoinositide-3 kinase (PI3-kinase) in cAMP-GEF-mediated cytoprotection in hepatocytes. Primary rat hepatocytes were treated for 2 h (A and B) with GCDC (50 µM) or for 4 h with Fas ligand (100 ng/ml) (C and D) or a combination of TNF- (25 µg/ml) plus actinomycin D (200 µg/ml) (E and F), and the amount of apoptosis was determined by morphological examination of Hoechst-stained cells (A, C, and E) or by immunoblotting for the active p17 fragment of caspase 3 (B, D, and F). Some cultures were pretreated with 20 µM of CPT-2-Me-cAMP for 30 min or sequentially with 20 µM LY-294002 and then CPT-2-Me-cAMP for 30 min before adding the apoptotic stimulus. Results are expressed as %apoptosis (A, C, and E) and represent the means ± SD for 3 separate experiments. *Statistically different from that seen with the apoptotic stimulus alone; #statistically different from that seen in the absence of CPT-2Me-cAMP. B, D, and F: immunoblots for the p17 cleavage product of caspase 3 and the corresponding actin immunoblot verifying equal protein loading. In these experiments, cells were pretreated with 20 µM CPT-2Me-cAMP for 30 min before adding the apoptotic stimulus. Some cells were pretreated with 20 µM Ly-294002 before adding the cAMP analog. Cell lysates were prepared 2, 3, or 4 h after adding GCDC, Fas, or TNF- , respectively.
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As with GCDC-induced apoptosis, CPT-2-Me-cAMP protection from Fas ligand-induced apoptosis was dependent on PI3-kinase activation (Fig. 5, C and D). Pretreatment with Ly-294002 augmented TNF-
-induced apoptosis but had no effect on the ability of the CPT-2-Me-cAMP to protect against TNF-
-mediated apoptosis (Fig. 5, E and F). Similar to the results seen with the CPT-2-Me-cAMP analog, PI3-kinase inhibition with Ly-2954002 blocked cAMP protection in Fas ligand-mediated apoptosis but had no effect on the protective effect of cAMP in TNF-
-mediated apoptosis (data not shown).
Role of CPT-2-Me-cAMP in modulation of ERK and PI3-kinase/Akt.
Our previous studies (58) have implicated a role for PI3-kinase-dependent activation of Akt in cAMP-mediated survival from bile acid-induced apoptosis. We and others (10, 21, 27, 34, 58) have shown that cAMP can activate PI3-kinase and Akt in hepatocytes. To determine whether this is a PKA-mediated event, hepatocytes were pretreated with Rp-cAMP before exposure to cAMP. In cell lysates pretreated with Rp-cAMP, cAMP still increased Akt phosphorylation (Fig. 6A). In addition, treatment with CPT-2-Me-cAMP resulted in phosphorylation of Akt in hepatocytes and this phosphorylation was prevented by pretreatment with Ly-294002 (Fig. 6B). Treatment with Ly-294002 had no effect on cAMP-mediated Rap activation, indicating that Rap activation by cAMP occurs upstream of PI3-kinase (Fig. 3, B and C). These results show that cAMP-mediated activation of Akt is PKA independent in hepatocytes.

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Fig. 6. Role of PI3-kinase and PKA in cAMP modulation of Akt and ERK. Whole cell lysates were prepared from control hepatocytes and hepatocytes treated for 15 min with CPT-cAMP (100 µM) (A and C) or CPT-2-Me-cAMP (10, 20 or 50 µM) (B and C) alone or after pretreatment for 30 min with Ly-294002 (20 nM) or Rp-cAMP (500 µM). Proteins (100 µg) were separated by SDS-PAGE, transferred to nitrocellulose, and immunblotted with phospho-specific antibodies for Aktser473 (A and B) or p42/p44 ERKthr202/tyr204 (C). Membranes were reprobed with phosphorylation state-independent antibodies to Akt and p42/p44 ERK. The amount of phosphorylated kinase was quantified by using computer software, normalized for equal protein loading, and expressed as the relative activity. Results represent the means ± SD from 3 separate experiments. *Statistically different from that in control cells; #statistically different from that in cells treated with cAMP.
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Because there is conflicting evidence in the literature on the role of cAMP activation of PKA and cAMP-GEFs on ERK modulation, hepatocytes were treated with Rp-cAMP or the CPT-2-Me-cAMP analog, and the effect on ERK phosphorylation was observed. RP-cAMP reversed cAMP-mediated inhibition of ERK, whereas CPT-2Me-cAMP had no effect on ERK phosphorylation (Fig. 6C). These results show that cAMP-mediated inhibition of ERK phosphorylation is a PKA-dependent event in hepatocytes.
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DISCUSSION
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The major aim of the present study was to determine whether the antiapoptotic effect of cAMP is mediated via PKA and/or cAMP-GEF. Results show that activation of cAMP-GEFs confers PKA-independent protection against hepatocyte apoptosis and results in PI3-kinase-dependent activation of Akt. In addition, the antiapoptotic effect associated with cAMP-GEF activation is PI3-kinase dependent for apoptosis induced by hydrophobic bile acids and Fas ligand but PI3-kinase independent for TNF-
-mediated apoptosis.
Results of the present study raise the possibility for the first time that the antiapoptotic effect of cAMP may be due to activation of cAMP-GEFs instead of PKA in hepatocytes. This conclusion is supported by results that show that the activation of cAMP-GEFs results in cytoprotection against apoptosis induced by bile acid, Fas, and TNF-
but is not associated with activation of PKA. Moreover, the cytoprotective effect of CPT-cAMP is largely independent of PKA. Previous studies (27, 55) in primary hepatocytes have shown that cAMP-mediated protection is partially PKA dependent in both TNF-
and bile acid-induced apoptosis. Results of these studies must be evaluated in light of the fact that a fairly nonspecific PKA inhibitor, KT-5720, was used to incriminate a role for PKA (7). Two recent studies (14, 44) implicating a major role for PKA in cAMP survival signaling in Fas ligand-mediated apoptosis likewise used a nonspecific PKA inhibitor, H-89 (7). In the present study, we used a highly specific inhibitor of PKA, Rp-cAMP (13, 47), to show that cAMP's protective effect in bile acid-, TNF-
-, and Fas ligand-induced apoptosis is largely PKA independent. In addition, we show that the cAMP-GEF-specific cAMP analog protects against hepatocyte apoptosis despite being unable to activate PKA. Thus it is very unlikely that the antiapoptotic effect of cAMP is mediated via PKA in hepatocytes.
Results of the present study suggest the presence of the cAMP-GEF/Rap 1 pathway in hepatocytes because CPT-cAMP increases the amount of active GTP-bound Rap 1, the downstream mediator of cAMP-GEF activation. This result raises the possibility that Rap activation may mediate the survival effect of cAMP-GEF activation. Rap GTPases, Rap 1 and Rap 2, are the only known downstream effectors of cAMP-GEF activation described so far. Studies (4, 23, 49) in nonhepatic cells have suggested that Rap 1 activation may be cytoprotective. However, further studies are needed to determine whether Rap 1 is involved in cAMP-mediated survival in hepatocytes.
In previous studies (58), we have shown that cAMP's antiapoptotic action in bile acid-induced apoptosis is linked to PI3-kinase-dependent activation of Akt. Results of the present study clearly indicate that cAMP-mediated activation of Akt is a cAMP-GEF-dependent but PKA-independent event in hepatocytes. Specific PKA inhibition in hepatocytes fails to block cAMP-mediated Akt phosphorylation, and CPT-2-Me-cAMP mediates PI3-kinase-dependent activation of Akt without activating PKA. cAMP Rap 1 activation via cAMP-GEFs modulates Akt activity in nonhepatic cells. In some cells (WRT thyroid cells, C6 glioma cells, HEK-293 cells), Rap activation increases Akt phosphorylation, whereas in other cells (B lymphocytes, PCCL3 thyroid cells) it inhibits Akt (5, 29, 33, 38, 54). Such cell-type-specific response to cAMP is not unusual (25). The biological basis for divergent cellular responses to cAMP are incompletely understood but may be associated with the relative abundance of cAMP binding proteins such as PKA and EPAC, their subcellular distribution, or the presence of distinct populations of downstream effectors in cAMP signaling pathways.
The mechanism by which cAMP-GEF/Rap modulates PI3-kinase/Akt activity is unknown. The related GTPase, Ras, binds to and activates the p110
- and p110
-catalytic subunits of PI3-kinase (39, 46). Because Ras and Rap 1 have identical effector binding regions (3, 4), it has been hypothesized that Rap may bind Ras effectors such as PI3-kinase. In a recent study (5), Rap 1 binding to the regulatory p85
subunit of PI3-kinase was associated with inhibition of PI3-kinase/Akt signaling. Alternatively, Rap could modulate PI3-kinase/Akt activity by facilitating the movement of these kinases to biological membranes or act on a PI3-kinase/Akt lipid or serine/threonine phosphatase. The mechanism by which cAMP-GEFs activate PI3-kinase/Akt is presently the focus of attention in our laboratory.
The PI3-kinase/Akt signaling pathway mediates hepatocyte survival from diverse stimuli (16, 37, 45, 50, 51, 55, 58). In the present study, PI3-kinase inhibition prevents cAMP-GEFs protective effect in bile acid and Fas-mediated apoptosis. Because apoptosis induced by hydrophobic bile acids proceeds at least in part by ligand-independent activation of the Fas receptor (17), it is not surprising that the response to cAMP is similar in these models of apoptosis. These results suggest that a cAMP-GEF/Rap 1/PI3-kinase/Akt signaling cascade may be important in cAMP survival effect in Fas receptor-mediated apoptosis. It is presently unknown how PI3-kinase/Akt signaling pathways protect against Fas/bile acid-mediated apoptosis, but previous studies (51, 53, 58) suggest that the PI3-kinase-dependent survival effect may occur at or above the level of the mitochondrial amplification cascade seen in death receptor-mediated apoptosis.
Unlike the situation in Fas-mediated apoptosis, cAMP-GEF-mediated protection from TNF-
apoptosis appears to be PI3-kinase independent. This may reflect the fact that the biological response to TNF-receptor binding is more complex than that involved in Fas ligand binding and involves parallel activation of survival and cell death pathways (1). The activation of protective signaling pathways must be inhibited by adding transcriptional inhibitors in order for apoptosis to occur in response to TNF-
. Previous studies concur with our observation that cAMP's antiapoptotic effect in TNF-
-mediated apoptosis in hepatocytes is PI3-kinase independent (27). Our results, like that of others, do support a role for PI3-kinase in TNF-
apoptosis, because we see that PI3-kinase inhibition potentiates TNF-
-mediated cell death (37). The finding of divergent cAMP responses in different models of apoptosis is not unprecedented as the PKA and PI3-kinase dependence of cAMP's antiapoptotic action in fibroblast and pancreatic beta cell apoptosis is stimulus dependent (8, 20, 59, 60). Thus it appears that additional PKA-independent cAMP-GEF-mediated events are involved in cAMP's antiapoptotic actions in apoptosis mediated by TNF-
.
There is conflicting information on the role of ERK signaling in hepatocyte survival. In some studies, ERK activation is antiapoptotic (41, 45), whereas other studies suggest ERK modulation has no effect on hepatocyte apoptosis (14, 15). In the present study, cAMP inhibition of ERK is a PKA-dependent event. This observation, coupled with the finding that cAMP's antiapoptotic effect is largely independent of PKA activation, implies that ERK inhibition does not play a large role in cAMP's antiapoptotic effect. Previously, we have shown that ERK inhibition with PD-98059 or U-0126 results in a mild protective effect against bile acid-induced apoptosis (55, 57). It is possible that this modest protection reflects the small amount of apoptosis not reversed by PKA inhibition in this study (
10%).
In summary, the present study shows for the first time that a PKA-independent cAMP/cAMP-GEF/Rap 1 pathway exists in hepatocytes. In addition, activation of this pathway with a novel cAMP analog, CPT-2-Me-cAMP, promotes hepatocyte survival as well as PI3-kinase/Akt activation. cAMP is an important second messenger in hepatic stress hormone signaling, and increases in intracellular cAMP may represent a general means by which hepatocytes upregulate survival mechanisms during times of metabolic stress. Promotion of cell survival appears to be a common property of the cAMP-coupled glucagon receptor family as glucose-dependent insulinotrophic polypeptide and glucagon-like peptide-1 promote pancreatic beta cell survival, whereas vasoactive intestinal polypeptide and pituitary adenylate cyclase activating polypeptide are antiapoptotic in baby hamster kidney cells and neurons, respectively (32). Elucidation of prosurvival cAMP signaling pathways could thus lead to the identification of potential therapeutic targets to ameliorate cell death in a diverse array of disease processes.
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GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-02721 and DK-061950 (to C. R. L. Webster) and DK-333436 (to M. S. Anwer).
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FOOTNOTES
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Address for reprint requests and other correspondence: C. R. L. Webster, Tufts Univ. School of Veterinary Medicine, 200 Westboro Rd., Grafton, MA 01536 (E-mail: cynthia.leveille-webster{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.
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