Cyclic adenosine monophosphate (cAMP) is a second messenger that activates several intracellular signaling pathways to regulate cell function and cellular gene expression. The mechanisms involved in the cAMP-induced activation of protein kinase A (PKA) and its regulation of regulating hepatocyte metabolism have been extensively studied (1, 2). Cyclic AMP also mediates PKA-independent signaling by binding to guanine nucleotide exchange factors (cAMP-GEFs, also known as exchange protein directly activated by cAMP, Epac) and activating GTPases (3–5). Activation of GTPases such as Rap1 and Raf1 regulates downstream signaling pathways such as ERK, p38, and PI3K in what may be a cell- and tissue-specific manner (3–8). Guanine nucleotide exchange factors also activate calcium-mediated pathways and can regulate downstream signaling through mechanisms other than binding to GTPase that are incompletely understood (9–11).
Two isoforms of Epac, Epac1 and Epac2, are found in mammalian cells (3, 12), and both are expressed in relatively low levels in liver tissue compared with other organs (12). Instead, a specific short form of Epac2 mRNA and protein that uses a translation start site located in exon 10 of the “regular Epac2” is enriched in liver tissue (13). The liver-specific Epac2 lacks the first cAMP-binding domain and the DEP (Dishevelled, Egl, Pleckstrin) domain (13), giving it a structure even shorter than Epac1 (14). Although this liver-specific isoform showed GEF activity toward Rap1, the role of liver-specific Epac in hepatocyte physiology is incompletely understood. Cyclic AMP suppresses hepatocyte apoptosis using both PKA-dependent and PKA-independent mechanisms, and Epac-mediated signaling regulates the PKA-independent effect on apoptosis (15). The role of Epac in regulating the effects of cAMP in other areas of hepatic function is unknown.
We have demonstrated that glucagon and cAMP decrease hepatocyte inducible nitric oxide synthase (iNOS) expression and activity (16, 17) and do so through a mechanism that involves c-Jun N-terminal kinase (JNK) in a manner that is primarily PKA-independent (18). We therefore tested the hypothesis that Epac regulates the PKA-independent effect of cAMP on iNOS. Our data demonstrate that activation of Epac signaling suppresses hepatocyte iNOS expression and activity and does so through effects on both Akt and JNK.
MATERIALS AND METHODS
Williams medium E, penicillin, streptomycin, L-glutamine, Lipofectamine, Human recombinant interleukin 1β (IL-1β), murine recombinant interferon γ (IFN-γ), and HEPES were all from Invitrogen Life Science Inc (Carlsbad, Calif). Insulin was from Lilly (Indianapolis, Ind). 8-(4-Chlorophenylthio)adenosine-3′,5′-cyclic monophosphate (CPTcAMP), 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP), 8-(4-methoxyphenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (OPTmecAMP), and N6-phenyladenosine-3′,5′-cyclic monophosphate (PhecAMP) were purchased from Biolog Life Science Institute (Bremen, Germany) (6). Polyclonal antibodies to iNOS were purchased from BD Bioscience (Billerica, Mass). Antibiodies to Raf1 (phosphorylated at serine 259), Akt, ERK, and actin were purchased from Cell Signaling Technology (Danvers, Mass). LY294002 and farnesyltransferase inhibitor (FTI) were purchased from Calbiochem (San Diego, Calif). The plasmid-expressing dominant-negative Akt was provided by Drs. Burgering and Triest from Utrecht University, Belgium. H-ras cDNA (dominant negative) in pUSEamp vector was purchased from Upstate (Charlottesville, Va). Recombinant adenovirus expressing dominant-negative JNK (DNJNK) was provided by Dr. Hideaki Kaneto from Osaka City University Medical School, Japan. This DNJNK is a kinase-dead mutant (the ATP-binding site is mutated) and can be phosphorylated itself but cannot phosphorylate c-Jun(12). All other reagents were from Sigma (St Louis, Mo).
Hepatocyte isolation and culture
Primary hepatocytes were isolated from male Sprague-Dawley rats (200–250 g) using the modified collagenase perfusion technique as previously described (1). All animal care was in accordance with the University of Louisville’s Animal Care and Use Committee and followed guidelines prescribed by the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Purified hepatocytes (>98% pure with >95% viability by trypan blue exclusion) were cultured onto collagen-coated 100-mm dishes in Williams medium E with L-arginine (0.5 mM), L-glutamine (2 mM), HEPES (15 mM) penicillin, streptomycin, and 10% low-endotoxin calf serum (HyClone Laboratories, Logan, Utah). Once cells attached, the media was changed to insulin-free media with 5% calf serum. The cells were further incubated for 16 h, and the experimental conditions were established. Conditions were performed in duplicate or triplicate, and experiments were repeated three times to ensure reproducibility.
Adenovirus-mediated expression of constitutively active liver-specific Epac2
A unique Xho1 site was introduced at -12 base pairs upstream of the ATG start codon in pSD5-mouse liver cAMP-GEFII (a plasmid containing exon 10-3′UTR of Epac2 provided by Dr. Susumu Seino from Kobe University Graduate School of Medicine, Japan) (13) without interrupting the Kozak cassette. The VLVLE motif in the LID region was then mutated to AAAAA creating LEpac2 (Ala)5, a constitutively activated cDNA sequence (19). After DNA sequencing, the Xho1-EcoR1 fragment was then subcloned into the Xho1-EcoR1 site of a shuttle vector (pAdTrackCMV) from pAdEasy system (Clontech Laboratories, Mountain View, Calif). The linearized shuttle plasmid containing constitutively activated LEpac2 was homogenously recombined with an adenoviral backbone plasmid, pAdEasy-1, in BJ5183 Escherichia coli cells. Successful recombinants were digested with PacI and packaged in the HEK cell line. Viral titers were determined under a florescent microscope (12). Briefly, confluent 293 cells were infected with a 1:10,000 dilution of the final lysate containing AdLEpac2. After 18 h of incubation, the effective titer was determined by the following formula: 107 × the average number of green fluorescent protein–positive cells per field (×100 magnification), which was considered equivalent to plaque-forming units per milliliter. This number was considered to be proportional to the number of infective particles in the original lysate. LEpac2 mRNA and protein expressed by the created adenovirus (AdLEpac2) in hepatocytes were confirmed by Northern blot and Western blot analyses (Fig. 2).
Hepatocytes were washed with ice-cold phosphate-buffered saline and then scraped from the plate in 500 μL of lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM Na2-EDTA; 1 mM EGTA; 1% Triton; 2.5 mM sodium pyrophosphate; 1 mM β-glycerophosphate; 1 mM Na3VO4; 1 μg/mL leupeptin; and 1 mM phenylmethylsulfonyl fluoride). After 30 min at 4°C, the lysates were centrifuged (15,000g for 15 min) and stored at −80°C until use. Proteins were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Nonspecific binding was blocked with Tris buffered saline with Tween 20 (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% Tween 20) containing 5% nonfat milk for 1 h. Primary antibodies were diluted and incubated with membranes for 1 to 2 h at room temperature or overnight at 4°C with agitation. After washing three times with TBS-T, secondary antibodies were incubated at 1:10,000 dilution for 1 h. After five additional washes with TBS-T, the bands were visualized with chemiluminescence according to the manufacturer’s instructions. Membranes were then stripped and reprobed with antibodies for unphosphorylated proteins or actin as described.
Hepatocytes were plated in 6-well plates (106 cells/well) and transfected with plasmids as previously described (18). Briefly, hepatocytes were transfected using Lipofectamine for 6 h and allowed to recover overnight, and the experimental conditions established after washing away unincorporated particles.
Supernatant NO2− was measured as an index of NO production by the Griess reaction as described (20).
Data are shown as the mean (SD). The Student t test or analysis of variance (ANOVA) using SigmaStat (Systat Software Inc, San Jose, Calif) was used to determine statistical significance. P < 0.05 was considered significant.
Cyclic AMP analogs modified in the 2′-ribose position activate Epac signaling to a greater extent than they activate PKA (21). We therefore evaluated the ability of OPTmecAMP to inhibit cytokine-stimulated iNOS activation compared with CPTcAMP and 8-Br-cAMP, cAMP analogs that activate both PKA and Epac. The analog PhecAMP, which does not activate Epac, was used to selectively activate cAMP-dependent protein kinase A (PKA types 1 and 2) (Fig. 1). When cultured hepatocytes were exposed to IL-1β + IFN-γ to induce iNOS, OPTmecAMP inhibited nitrite production to a degree similar to that of CPTcAMP and 8-Br-cAMP, whereas the inhibitory effect of phecAMP was much less and not different from that of vehicle control. OPTmecAMP also effectively suppressed iNOS protein expression as measured by Western Blot (Fig. 1).
To confirm the effect of Epac on cytokine-induced iNOS, we developed a recombinant adenovirus that expressed constitutively active liver-specific Epac2 (AdLEpac2) in cultured hepatocytes. After 24 h of culture, hepatocytes infected with AdLEpac2 had increased expression of Epac2 mRNA and 97-kd Epac2 protein compared with noninfected hepatocytes or hepatocytes infected with a control virus (AdGFP) (Fig. 2A). Notably, basal Epac2 mRNA and protein expression were detected in control hepatocytes and were unchanged by the control virus (Fig. 2A). When hepatocytes infected with either AdLEpac2 or AdGFP were stimulated to express iNOS by IL-1β + IFN-γ, decreased iNOS protein expression was seen in LEpac2-overexpressing cells (Fig. 2B).
The signaling pathways responsible for Epac’s effect on hepatocyte iNOS are not known. Epac regulates mitogen-activated protein kinase (MAPK) through Rap1 and Ras/Raf1 signaling (3–7, 22). We therefore assessed the ability of OPTmecAMP to regulate Raf1 in cultured hepatocytes. OPTmecAMP induced Raf1 phosphorylation at serine 259 (Fig. 3A). Raf-1 phosphorylation at ser259 serves as a binding point for the regulatory adapter protein 14-3-3 and stabilizes the basal inactive Raf-1 conformation, serving as a negative regulatory site (23). Interleukin 1β + IFN-γ phosphorylated Raf1 s259 to a lesser degree than OPTmecAMP. The combination of IL-1β + IFN-γ and OPTmecAMP produced an earlier phosphorylation of Raf1-s259 than either stimulus alone. This result suggests that Epac stabilizes Raf1 and inhibits MEK/ERK signaling in hepatocytes. The phosphorylation of Raf1-s259 induced by OPTmecAMP was mediated in part by PI3K since Raf1 phosphorylation at ser259 was suppressed by the PI3K inhibitor, LY294002 (Fig. 3B). Phosphorylation of Raf1 at serine 259 decreases downstream MEK/ERK activation, and OPTmecAMP produced a corresponding decrease in IL-1β + IFN-γ induced ERK1/2 phosphorylation in hepatocytes (Fig. 4). However, the ERK inhibitor, PD98059, had no effect on IL-1β + IFN-γ–induced nitrite production, and overexpressing a constitutively active ERK2 failed to restore the OPTmecAMP-induced suppression of iNOS expression (data not shown). In addition, OPTmecAMP did not change p38 phosphorylation in hepatocytes stimulated with IL-1β + IFN-γ, and overexpression of a dominant-negative p38 had no effect on the Epac-induced suppression of iNOS (data not shown). These data suggest that although the OPTmecAMP-decreased MAPK activation in hepatocytes, this effect was not responsible for the suppression of iNOS expression produced by Epac.
Cyclic AMP can activate (24) or inhibit (25) PI3K/Akt signaling depending on the cell being studied. In hepatocytes, Epac phosphorylates Akt and prevents apoptosis in a PI3K-dependent manner (15). We therefore evaluated whether Epac activates PI3K/Akt to mediate its effect on iNOS. OPTmecAMP alone induced Akt phosphorylation up to 16 h of culture (Fig. 5, A and B). Overexpression of LEpac2 also increased Akt activation in a dose-dependent manner compared with GFP control (Fig. 5C). Ras FTI and overexpression of dominant-negative HRas decreased OPTmecAMP-induced Akt phos phorylation to an extent similar to that of the specific PI3K inhibitor LY294002 (Fig. 6A) or overexpression of dominant-negative Akt (Fig. 6B). These results suggest that Epac-induced Akt activation is mediated by the Ras/PI3K pathway. We then explored the role of Epac-induced Akt activation on hepatocyte iNOS expression (Fig. 7). Overexpression of dominant-negative Akt increased nitrite production in IL-1β + IFN-γ–stimulated hepatocytes, suggesting that Akt is involved in the endogenous regulation of cytokine-stimulated NO production. Dominant-negative Akt blocked the inhibitory effect of OPTmecAMP on IL-1β + IFN-γ–nitrite production (Fig. 7A). Furthermore, the PI3K inhibitor LY294002 reduced the inhibition of nitrite production and iNOS expression produced by overexpressing LEpac2 in cytokine-stimulated hepatocytes (Fig. 7B). These data support the hypothesis that Epac mediates its effects on iNOS expression through PI3K and Akt.
Cyclic AMP can activate JNK through GEFs (11), and JNK activation mediates the inhibition of IL-1β + IFN-γ–induced iNOS expression by cAMP (18). We therefore evaluated whether JNK was activated by Epac and regulated iNOS production in hepatocytes. OPTmecAMP (0.2 mM) alone induced JNK1/2 phosphorylation, which was detectable early (5 min) and peaked at 30 min (Fig. 8A). We then expressed LEpac2 with or without dominant-negative JNK in hepatocytes and stimulated them with IL-1β + IFN-γ to induce iNOS. Overexpression of LEpac2 decreased IL-1β + IFN-γ–induced nitrite production as previously shown (Fig. 2), whereas dominant-negative JNK enhanced cytokine-stimulated iNOS expression (Fig. 8B). Coexpression of DN-JNK reversed the inhibitory effects of LEpac2 on both nitrite production and iNOS expression (Fig. 8B).
Hepatocyte NO synthesis is an important part of the liver’s response to shock and sepsis, and excessive NO production by iNOS induces tissue injury and organ dysfunction (16, 26). Glucagon and other cAMP-elevating compounds inhibit hepatic NO synthesis in vitro and in vivo (16), but this regulation is complex. Despite PKA signaling being the best characterized pathway mediating cAMP-induced changes in cellular gene expression, we have found that the inhibitory effect of cAMP on cytokine-induced hepatocyte NO synthesis and iNOS expression is predominantly PKA-independent (27). Although cAMP-mediated changes in nuclear factor κB and JNK contribute to this finding, they do not account for all of the PKA-independent effects of cAMP on NO (27). Epac is a guanine nucleotide exchange factor that can activate several distinct intracellular signaling pathways in a PKA-independent manner. Studies have implicated Epac in the regulation of several inflammatory responses including the regulation of endothelial cell–cell junction stability (28), the suppression of cytokine-induced proinflammatory signaling (29), and development of hepatocyte apoptosis (15). We therefore explored the role of Epac in mediating the PKA-independent inhibition of hepatocyte iNOS by cAMP. Our data demonstrate that activation of Epac signaling with OPTmecAMP or overexpression of LEpac2 decreased cytokine-induced iNOS expression.
Epac regulates Ras/Raf/MEK/ERK signaling Epac can inhibit or upregulate MAPK, depending on the cell type being investigated (25, 30, 31). We found that Epac stabilized Raf1 in hepatocytes through phosphorylation of serine 259 associated with the suppression of ERK activation but had no effect on p38 MAPK. However, we could find no role for either p38 MAPK or p44/42 MAPK in regulating hepatocyte iNOS expression. Our data do not exclude the possibility that Epac-induced changes in p38 or p44/42 MAPK can regulate the expression of other hepatocyte genes.
Although cAMP inhibits Akt activation in a number of cell types (25, 32), it activates PI3K/Akt signaling in a PKA-independent manner in hepatocytes (15, 33). We found that pharmacologic activation of Epac signaling with OPTmecAMP or overexpression of liver-specific Epac2 increased Akt phosphorylation. The activation of Akt by OPTmecAMP was inhibited by FTI, an inhibitor of Ras, and LY294002, an inhibitor of PI3K. These data demonstrate that Epac mediated a Ras- and PI3K-dependent Akt activation in cultured rat hepatocytes. Both LY294002 and dominant-negative Akt blocked the inhibitory effect of Epac signaling on iNOS expression in cytokine-stimulated hepatocytes, demonstrating that the effect of Epac on iNOS is mediated through PI3K/Akt.
c-Jun N-terminal kinase inhibits iNOS activation and contributes to the PKA-independent effect of cAMP on iNOS (18). We therefore examined the role of JNK in mediating the effect of Epac activation on iNOS. In hepatocytes, OPTmecAMP increased JNK phosphorylation alone and in combination with cytokines. Expression of a dominant-negative JNK reversed the inhibitory effect of Epac2 on iNOS, suggesting that Epac mediates some of its effects through regulation of JNK. Other studies have shown that Epac activates JNK through Rap1-independent signaling (11) in the HEK cell line, but Rap2 mediates JNK activation in excitatory synapses (34). In hepatocytes, the mechanisms involved in the activation of JNK by Epac are unclear. It is possible that Epac-induced Akt signaling may lead to downstream JNK activation that can subsequently alter c-Jun or other transcription factors important in iNOS expression. These mechanisms will require further investigation to define.
We cannot exclude a role for other PKA-independent signaling pathways in mediating the effects of Epac on hepatocyte iNOS expression seen in these studies. Epac regulates Ca2+ mobilization in pancreatic β cells (5), and Ca2+ regulates the activity of calmodulin-dependent kinases that can alter transcription factor activation and subsequent gene expression (35). Whether Epac-induced changes in Ca2+ or Ca2+-dependent transcription factor activation contribute to the PKA-independent effects of Epac on hepatocyte iNOS will require further study.
In conclusion, we demonstrate that Epac inhibits cytokine-stimulated hepatocyte iNOS expression and does so through effects on PI3K/Akt and JNK. These signaling pathways may represent important regulators of in vivo nitric oxide synthesis during inflammation and sepsis. Additional work will be required to elucidate the specific Akt- and JNK-dependent processes regulating the iNOS gene during shock and sepsis.
The authors thank Drs. Burgering and Triest for providing the plasmids that express dominant-negative Akt and Dr. Kaneto for providing the recombinant adenoviruses that express dominant-negative JNK.
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Epac2; nitric oxide; Akt; JNK; hepatocyte