Cyclic nucleotides suppress tumor necrosis factor alpha-mediated apoptosis by inhibiting caspase activation and cytochrome c release in primary hepatocytes via a mechanism independent of Akt activation.

Cyclic nucleotides have been previously shown to modulate cell death processes in many cell types; however, the mechanisms by which cyclic nucleotides regulate apoptosis are unclear. In this study, we demonstrated that cAMP as well as cGMP analogs suppressed tumor necrosis factor alpha (TNFalpha) plus actinomycin D (ActD)-induced apoptosis in a dose-dependent manner in cultured primary hepatocytes. Furthermore, forskolin, which increases intracellular cAMP levels, also effectively suppressed TNFalpha+ActD-induced apoptosis. Activation of multiple caspases was suppressed in cells exposed to TNFalpha+ActD in the presence of cAMP or cGMP analogs. TNFalpha+ActD-induced cytochrome c release from mitochondria was also inhibited by cAMP or cGMP, reinforcing our conclusion that cyclic nucleotides interfere with the early signaling events of TNFalpha-mediated apoptosis. We evaluated the possibility that cAMP and cGMP inhibit apoptosis by activating the serine/threonine kinase Akt, which is known to promote cell survival. Both cAMP- and cGMP-elevating agents led to marked increases in Akt activation that was inhibited by the phosphatidylinositol 3'-kinase inhibitors, LY294002 and wortmannin. However, complete inhibition of cyclic nucleotide-induced Akt activation had little effect on cyclic nucleotide-mediated cell survival, indicating the existence of other survival pathways. Interestingly, the specific inhibitor of protein kinase A (PKA), KT5720, blocked cGMP-mediated protection but only partially prevented the anti-apoptotic effect of cAMP, indicating that both PKA-dependent and -independent mechanisms are involved in cAMP-mediated suppression of apoptosis signaling. Our data suggest that multiple survival signaling pathways coexist in cells and that cyclic nucleotides delay apoptosis by interfering with apoptosis signaling by both PKA-dependent and -independent mechanisms.

Apoptosis plays fundamental roles in the development of multicellular organisms and the maintenance of homeostasis (reviewed in Refs. 1 and 2). Our understanding of the underlying mechanism for apoptosis has advanced greatly. It is now well accepted that activation of a unique family of cysteine proteases named caspases plays a central role in the signaling and execution phase of apoptosis induced by a number of stimuli, including Fas, TNF␣, 1 TRAIL, radiation, and chemotherapeutic drugs (reviewed in Refs. [3][4][5]. Activation of initiator caspases, such as caspase-8 and caspase-9, is known to transduce apoptotic signal and activate executioner caspases, such as caspase-3. Translocation of cytochrome c from mitochondria to cytoplasm has been viewed by many investigators as an irreversible step in committing cells to apoptotic cell death. We (6 -8) and others (9 -13) previously demonstrated that nitric oxide (NO) prevents apoptosis by inhibiting caspase proteolytic activation as well as by directly suppressing caspase activity. NO is known to activate potently soluble guanylyl cyclase and results in production of intracellular cGMP (14). In some cell systems, the anti-apoptotic function of NO is mediated, at least partially, by NO-dependent generation of intracellular cGMP. For example, the protection of NO against apoptosis is found to be dependent on production of the secondary messenger cGMP in B lymphocytes (15), eosinophils (16), embryonic motor neurons (17), PC12 cells (18,19), and ovarian follicles (20). However, it is unknown how cGMP prevents apoptosis signaling and supports survival. Recently, cAMPelevating reagents have also been shown to suppress etoposide-, Fas-, or cycloheximide-induced apoptosis in neutrophils (21,22), promonocytic leukemia cell lines (23,24), hepatocytes (25,26), nerve cells (18), endothelial cells (27), and smooth muscle cells (28). However, the mechanism underlying this cyclic nucleotide-dependent suppression of apoptosis also remains elusive.
Activation of phosphatidylinositol 3Ј-kinase (PI3ЈK) and its downstream effector Akt by growth factors, cytokines, and certain stress such as heat shock has been demonstrated to transduce signals regulating not only protein synthesis, proliferation, and glycogen metabolism but also cell survival (reviewed in Refs. 29 -31). Activation of the serine/threonine kinase Akt (also named protein kinase) has been shown to suppress apo-ptosis and promote cell survival (32)(33)(34)(35). To date, four in vivo substrates of Akt have been identified, including glycogen synthase kinase-3 (GSK3, Ref. 36), procaspase-9 (37), the proapoptotic member of the Bcl-2 family, Bad (33), and most recently the endothelial nitric oxide synthase (38,39). It has been shown that phosphorylation of Bad by Akt results in dissociation of Bad from Bcl-2 permitting Bcl-2 to prevent apoptosis (32,33). Moreover, cAMP-elevating agents have recently been shown to activate Akt independently of PI3ЈK in transfected cells (40); however, the relationship between the anti-apoptotic action of cyclic nucleotides and the Akt pathway remains poorly understood.
In this study, we investigated the anti-apoptotic mechanisms of cyclic nucleotides in preventing TNF␣ plus actinomycin D (TNF␣ϩActD)-induced apoptosis in cultured primary hepatocytes. Here we report that both cGMP-and cAMP-elevating agents suppressed TNF␣ϩActD-induced apoptosis via inhibiting activation and processing of caspases, including caspase-3, caspase-8, and caspase-9. This is consistent with the finding that cytochrome c release and DNA fragmentation, events downstream of caspase-8 and caspase-3, respectively, during TNF␣ϩActD-mediated apoptosis, were significantly inhibited when cells were cotreated with cyclic nucleotide-elevating agents and TNF␣ϩActD. Interestingly, we also found that both cyclic nucleotides rapidly activate endogenous Akt in cultured hepatocytes in a PI3ЈK-dependent manner. Inhibition of this Akt activation did not block the protective effect of cAMP or cGMP; however, inhibition of protein kinase A did. Our results indicate the existence of multiple survival pathways in cells and suggest that protein kinase A is involved in modulating the apoptotic signaling cascade by cyclic nucleotides.
Preparation of Primary Hepatocytes and Cell Culture-Primary rat hepatocytes were isolated and purified from male Harlan Sprague-Dawley rats and cultured as described previously (41). Highly purified hepatocytes (Ͼ98% purity and Ͼ95% viability by trypan blue exclusion) were suspended in Williams medium E supplemented with 10% calf serum, 2 mM L-glutamine, 15 mM HEPES, pH 7.4, 100 units/ml penicillin, and 100 g/ml streptomycin. The cells were plated on collagencoated tissue culture plates at a density of 2ϫ10 5 cells/well in 12-well plates for cell viability analysis, 4 ϫ 10 5 cells/well in 6-well plates for DNA fragmentation ELISA, or 3 ϫ 10 6 cells/60-mm dish for Western blot and enzyme assays. After attachment to the plates for 4 h, cells were rinsed once and cultured overnight with the same culture medium containing no serum. Apoptosis was induced by incubating the hepatocytes with the culture medium containing 2000 units/ml TNF␣ and 0.2 g/ml actinomycin D for 6 h unless specified in the figure legends. Cells were then scraped off the plates and centrifuged, washed twice with cold phosphate-buffered saline (PBS), and resuspended in 5-fold volume of hypotonic buffer A (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mm MgCl 2 , 1 mM EGTA, 1 mM EDTA, 0.5 mM PMSF, 5 g/ml aprotinin, 5 g/ml pepstatin, and 10 g/ml leupeptin). After three cycles of freezing and thawing, cell debris was removed by centrifugation at 13,000 ϫ g at 4°C for 20 min. The supernatant was used as crude cytosol for caspase assays and Western blot for caspases, and the cell debris was used for Western blotting analysis of PARP. Protein concentration was determined with the BCA assay (Pierce) with bovine serum albumin as standard. Cell viability was determined by the crystal violet method as described previously (8).
Preparation of Whole Cell Lysate-Activation of Akt was evaluated by stimulating cells with indicated reagents for various times. Cells were first rinsed twice with ice-cold PBS, and 600 l of whole cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 1 g/ml leupeptin, and 1 mM PMSF) was then added to each plate. After 5 min incubation on ice, cells were scraped off the plates and lysed at 4°C for 30 min. The whole cell lysate was collected by centrifugation at 13,000 ϫ g for 15 min to remove cell debris, frozen in liquid nitrogen, and stored in aliquots at Ϫ80°C until use.
Akt Activity Assay-Akt enzymatic activity was assayed with 1 g per assay of recombinant glycogen synthase kinase (GSK3) fusion protein as substrate in a reaction mixture containing 25 mM Tris, pH 7.5, 1 mM Na 3 VO 4 , 1 mM EDTA, and 0.5 mM PMSF, 10 mM MgCl 2 , 50 M ATP, and 2 Ci of [␥-32 P]ATP/per assay. The phosphorylation reaction was allowed to proceed for 30 min at 30°C and stopped by adding 3ϫ sample buffer. After boiling at 95°C for 5 min, phosphorylated GSK3 was resolved by SDS-PAGE and quantitated with a ImageQuaNT system (Storm 860, Molecular Dynamics Inc., Sunnydale, CA).
Apoptosis Assay-The cell death detection enzyme-linked immunosorbent assay was used to quantitate changes in DNA fragmentation according to the manufacturer's instructions.
Intracellular cAMP Measurement-cAMP levels were measured by using an iodinated assay system from NEN Life Science Products. Following exposure to indicated reagents for 30 min, the cells were lysed in lysis buffer supplemented with 1 mM 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) to inhibit phosphodiesterases as described above. Intracellular cAMP contents were then determined with the cell lysate (10 g of proteins) by radioimmunoassay according to the manufacturer's protocol.
PKA Activity-cAMP-dependent protein kinase activity was determined by in vitro phosphorylation of Kemptide. Cells were lysed as above, and aliquots of 10 g of proteins were mixed with or without 1 M protein kinase A inhibitor (PKI, 6 -22) in 50 mM Tris, pH 7.5, in a volume of 30 l. After incubation for 15 min at 25°C to allow PKI to bind to PKA, 10 l of 4ϫ substrate solution containing 200 M Kemptide, 40 mM MgCl 2 , 400 M ATP and 1 Ci per assay of [␥-32 P]ATP was added to each sample. Samples were incubated for 5 min at 30°C, and the reactions were terminated by adding EDTA to a final concentration of 20 mM. 20 l of the reaction mix was spotted onto P81 phosphocellulose paper disc. The P81 paper sheets were immersed in 1% (v/v) orthophosphoric acid, washed 5-6 times with the acid solution, and then 2 times with distilled water. The incorporated radioactivity was determined by scintillation counting. Background values obtained from a mixture lacking cell lysate were subtracted from all values. All measurements were performed in duplicate.

RESULTS
cGMP and cAMP Analogs Inhibit TNF␣ϩActD-induced Apoptosis-TNF␣ rapidly triggers hepatocyte apoptotic cell death in the presence of the transcriptional inhibitor actinomycin D (8,42). Previously we have shown that the cell-permeable cGMP analog Br-cGMP partially suppresses TNF␣ϩActD-induced hepatocyte apoptosis (7). To investigate the underlying mechanism of the cyclic nucleotide-dependent inhibition of apoptosis, we first established the optimal concentration for the inhibition of apoptosis by Br-cGMP. When incubated with cells in the presence of TNF␣ϩActD, Br-cGMP suppressed apoptotic cell death in a dose-dependent manner (Fig. 1, A and B). The maximal inhibition was observed at 800 M. Interestingly, cAMP analogs such as Br-cAMP also dose-dependently inhibited TNF␣ϩActD-induced cell death but at a much lower concentration when compared with the corresponding cGMP analog ( Fig. 1, A and B). At the highest concentration used, Br-cAMP or Br-cGMP alone had no significant effect on cell viability and morphology in the absence of TNF␣ϩActD. Other cell permeable analogs of cGMP and cAMP, such as 8-pCPT-cGMP, dibutyryl-cGMP (Bt 2 cGMP), and dibutyryl-cAMP (Bt 2 cAMP) showed similar results for suppression of TNF␣ϩActD-induced hepatocyte cell death (data not shown). Treatment of hepatocytes with 10 M forskolin, a reagent that activates adenylyl cyclase and thus increases intracellular cAMP level, also effectively suppressed TNF␣ϩActD-mediated apoptosis (Fig. 1B).
TNF␣ϩActD-induced cell death and DNA fragmentation were completely inhibited by the pan caspase inhibitor, Z-VADfmk ( Fig. 1 and Ref. 8). Addition of cell-permeable Bt 2 cAMP or Br-cGMP at the same time with TNF␣ϩActD delayed the onset of apoptosis but did not completely inhibit cell death (Fig. 1C). Since cell death was completely inhibited at the 6-h time point by cyclic nucleotides, this time point was thus chosen for sub-sequent studies to elucidate the mechanism by which cyclic nucleotides regulate apoptotic signaling events.
cGMP and cAMP Elevating Agents Prevent Caspase-3 Proteolytic Processing and Activation-Previously we have demonstrated that multiple caspases are activated in hepatocytes challenged with TNF␣ϩActD and that caspase activation is required for hepatocyte apoptosis in response to this stimulus (8). To examine whether cGMP and cAMP analogs suppress cell death by inhibiting activation of caspases, we first evaluated caspase-3-like activity of cells exposed to TNF␣ϩActD in the presence and absence of cyclic nucleotides. As shown in Fig. 2A, cyclic nucleotides alone did not have any effect on caspase activity. However, concomitant exposure of cells to cyclic nucleotides with TNF␣ϩActD resulted in significant inhibition of caspase-3-like activity. The effect of cAMP and cGMP analogs on caspase-3-like protease activity closely correlated with their effect on hepatocyte DNA fragmentation (Fig. 2B). Moreover, these cyclic nucleotide analogs suppressed proteolytic activation of procaspase-3 as determined by immunoblotting analysis (Fig. 2C). Consistent with the finding that the caspase-3 activation was inhibited by the cyclic nucleotide analogs, cleavage of PARP, a well established intracellular substrate of caspase-3-like proteases, was also blocked (Fig. 2C). These results demonstrate that cyclic nucleotides suppress TNF␣ϩActD-induced apoptosis at a site upstream of caspase-3 activation.
cGMP and cAMP Elevating Agents Suppress Caspase-8 Proteolytic Processing and Activation-The most proximal caspase activated by TNF␣ is believed to be caspase-8 (3). Activation of caspase-8 can lead to direct activation of other caspases or the release of cytochrome c from mitochondria. We next tested whether activation of procaspase-8 is suppressed by cyclic nucleotide-elevating agents. As shown in Fig. 3A, caspase-8 activity in cytosolic extracts was markedly elevated in cells treated with TNF␣ϩActD. Addition of Br-cGMP or Bt 2 cAMP to cAMP Inhibits Apoptosis Signaling Independent of Akt Activation cells significantly inhibited the caspase-8 activity. As expected, the pan caspase inhibitor Z-VAD-fmk completely prevented caspase-8 activation. Furthermore, forskolin also suppressed TNF␣ϩActD-induced caspase-8 activity. Immunoblotting analysis of procaspase-8 revealed that processing of this caspase was inhibited by cAMP and cGMP analog treatment (Fig. 3B). These results indicate that elevation of cellular cyclic nucleotides interferes with the death signal leading to caspase-8 activation or amplification.
cGMP and cAMP Elevating Agents Partially Prevent TNF␣ϩActD-induced Cytochrome c Release-We previously reported that cytochrome c translocates from mitochondria to cytoplasm in TNF␣ϩActD-treated hepatocytes (8). Release of cytochrome c is known to signal activation of procaspase-9 through interaction with Apaf-1 (43). As shown in Fig. 3C, cytosol from untreated cells contained negligible cytochrome c. However, cytochrome c accumulated in cytosol of cells exposed to TNF␣ϩActD. This release of cytochrome c was significantly inhibited by cotreatment of cells with Bt 2 cAMP or Br-cGMP, demonstrating that cyclic nucleotides act at or above the level of cytochrome c release. Moreover, the activation of caspase-9 was suppressed by cyclic nucleotide-elevating agents (Fig. 3C).
These results are in agreement with our above findings that the cyclic nucleotide agents suppressed TNF␣ϩActD-induced caspase-8 activation.
Activation of Protein Kinase B/Akt by cGMP and cAMP Elevating Agents-The PI3ЈK/Akt pathway plays an important role in inhibiting apoptosis. One mechanism by which Akt signaling suppresses apoptosis is by inhibiting cytochrome c release (44). To explore whether cyclic nucleotide may suppress apoptosis and apoptotic signaling via activating the Akt survival pathway, we then examined the effects of cyclic nucleotides on endogenous Akt activation. Since phosphorylation of Akt at Ser 473 is required for its full activation (45,46), we first examined the phosphorylation status of endogenous Akt using an antibody that specifically recognizes Akt phosphorylated at Ser 473 . We found rapid activation of endogenous Akt when cells were treated with Br-cGMP, and this activation was time-and concentration-dependent (Fig. 4A). Other cGMP analogs such as Bt 2 cGMP and the PKG activator 8-pCPT-cGMP also activated Akt in a dose-and time-dependent manner, revealing a link between cGMP signaling and the Akt signaling pathway. Similar results were observed for cAMP analogs. Treatment of cells with Br-cAMP or Bt 2 cAMP resulted in potent and rapid Cell lysate was then prepared. A, caspase-8 activity was determined with Ac-IETD-pNA as a substrate. B, immunoblotting analysis of procaspase-8 proteolytic activation of cells treated as above for 6 h. C, immunoblots of cytosolic cytochrome c and processed procaspase-9. Cells were treated as indicate for 6 h. Cytosolic extract was obtained by centrifugation at 100,000 ϫ g for 1 h at 4°C after homogenization of cells in buffer A containing 250 mM sucrose. Cleaved caspase-9 large fragment was detected with antibody specific for the p37 fragment. Results are representative of two (B and C) to four (A) independent experiments.

FIG. 2. TNF␣/ActD-induced activation of caspase-3 is inhibited by cyclic nucleotides and caspase inhibitor Z-VAD-fmk.
Primary hepatocytes were treated with TNF␣/ActD (2000 units/ml and 0.2 g/ml, respectively) in the absence and presence of 200 M Bt 2 cAMP, 800 M Br-cGMP, or 100 M Z-VAD-fmk for 6 h. Cells were then collected and washed with ice-cold phosphate-buffered saline, and cell lysate was prepared. A, caspase-3-like protease activity was determined using a colorimetric assay with Ac-DEVD-pNA as substrate. B, apoptosis was evaluated by determining the extent of DNA fragmentation as described above. C, immunoblotting analysis of procaspase-3 activation. Forty g of cellular proteins was loaded into each lane, separated on SDS-PAGE, transferred to nitrocellulose membrane, and probed for caspase-3 and PARP as described under "Experimental Procedures." Results are representative of at least three independent experiments with similar results.
cAMP Inhibits Apoptosis Signaling Independent of Akt Activation phosphorylation of Akt in a concentration-and time-dependent manner (Fig. 4B). Furthermore, incubation of cells with the specific PKA activators, (S p )-Br-cAMPS or Sp-5,6-DCI-IBMPS, resulted in robust activation of Akt, indicating the participation of PKA in cAMP-mediated Akt activation (data not shown). More importantly and consistent with previous reports (46), the extent of phosphorylation of Akt at Ser 473 correlated with Akt enzymatic activity (Fig. 4C).
Akt is a key downstream target of PI3ЈK. To examine whether PI3ЈK is involved in this cyclic nucleotide-mediated Akt activation, we preincubated cells with the specific PI3ЈK inhibitor LY294002 prior to exposing them to cGMP or cAMP agents. It was found that the PI3ЈK inhibitor completely abolished the subsequent Akt activation induced by either cGMP or cAMP analogs (Fig. 5A). Another structurally unrelated PI3ЈK inhibitor, wortmannin, was also able to block cyclic nucleotidemediated Akt activation (Fig. 5A), indicating that this activation is PI3ЈK-dependent.
Activation of Akt Pathway Does Not Account for Cyclic Nucleotide-mediated Protection against Apoptosis-As demonstrated above, cyclic nucleotides dose-dependently protected hepatocytes from TNF␣ϩActD-induced apoptosis via inhibiting caspase activation and in the meanwhile activated the PI3ЈK/ Akt survival signal pathway. As the activation of PI3K/Akt survival pathway has been shown to suppress apoptosis induced by a variety of stimuli in other systems, the relationship between TNF␣ϩActD-mediated apoptosis and PI3ЈK/Akt survival signal pathway was investigated.
Although pretreatment of cells with the PI3ЈK inhibitors LY294002 or wortmannin completely prevented the activation of Akt by cyclic nucleotides, it did not prevent the inhibition of caspase-3-like activity or procaspase cleavage by cyclic nucleotides ( Fig. 5B and data not shown). Furthermore, inhibition of Akt activation did not reverse cyclic nucleotide-mediated inhibition of DNA fragmentation (Fig. 5C). This demonstrates that the PI3ЈK/Akt pathway did not play a major role in the antiapoptotic effects of cyclic nucleotides in primary hepatocytes.
Insulin is known to suppress transforming growth factor ␤-induced hepatocyte apoptosis and activate the PI3ЈK/Akt pathway (47). To ensure that our approach to inhibit the PI3ЈK/ Akt pathway would determine the contribution of this pathway to cell survival, we evaluated the effect of LY294002 and wortmannin on insulin-induced changes. We found that addition of insulin to culture medium partially prevented TNF␣ϩActDinduced DNA fragmentation as well as cell death (Fig. 6, A and  B). However, this partial protection was completely reversed by LY294002 and wortmannin (Fig. 6, A and B). Consistent with a previous report (44), insulin dose-dependently activated Akt, and this activation was blocked by LY294002 or wortmannin (Fig. 6C). To establish whether the differences in the importance of the PI3ЈK/Akt kinase pathway for insulin or cyclic nucleotide-mediated anti-apoptotic actions are due to a difference in the extent of Akt activation, we compared the degrees of Akt activation by these two different type stimuli. It was found that insulin was the most potent stimulus of the PI3ЈK/Akt pathway (about 5-10-fold higher than that of cyclic nucleotides) (Fig. 6D). This difference in the levels of Akt activation may explain the relative contribution of the Akt activation to cell survival. Combined together, these results also indicate that other survival pathways exist for cyclic nucleotide-mediated suppression of apoptosis in hepatocytes.
PKA-dependent and -independent Mechanisms in Cyclic Nucleotide-mediated Protection-Since it has been reported in some cell systems that there exist cross-talks between cAMP and cGMP signaling pathways (48,49), we examined whether a common signaling pathway was used by both cyclic nucleotides. Although higher concentrations of cGMP may increase intracellular cAMP levels by acting on cAMP phosphodiesterase, we observed no increase in hepatocyte intracellular cAMP levels upon exposure to various cGMP analogs (Fig. 7A). As expected forskolin, the phosphodiesterase inhibitor IBMX, and Bt 2 cAMP all markedly increased intracellular cAMP levels (Fig. 7A). Thus, cGMP does not act through increasing intracellular cAMP levels.
Another possibility is that at higher concentrations cGMP may directly activate PKA, a kinase that mediates many of the cAMP effects in cells. Indeed, it has been shown in in vitro experiments that cGMP can directly activate PKA by binding to the regulatory domain of the kinase (49). We therefore performed in vitro kinase assay to examine this possibility. As  (B, lower panel) for various times. After washing cells with ice-cold PBS, cells were lysed with whole cell lysis buffer at 4°C. Phosphorylated Akt was detected by immunoblotting analysis with antibody specific for Ser 473phosphorylated Akt (P-Akt). Protein loading was determined by stripping and reprobing the same nitrocellulose membrane for total Akt protein level. Results are representative of three independent experiments. C, cells were treated with or without 25 M pCPT-cGMP, 100 M Bt 2 cAMP, 10 M (S p )-cAMPS, and 10 M forskolin together with 0.5 mM IBMX for 30 min, and cell lysate was prepared as above. Akt enzymatic activity was determined by kinase assay with recombinant glycogen synthase kinase fusion protein as substrate as detailed under "Experimental Procedures." Results represent two different experiments.
cAMP Inhibits Apoptosis Signaling Independent of Akt Activation expected, PKA enzymatic activity was increased 2-3-fold in cells treated with cAMP analogs and by the PKA specific activator (S p )-cAMPS (Fig. 7B). The specificity of the PKA activity assay was demonstrated by using the highly PKA-specific inhibitor, PKI. Br-cGMP and unexpectedly the potent PKG activator pCPT-cGMP also increased PKA activity in cells, although to a much less extent as compared with cAMP agents (Fig. 7B). Preincubation with the cell-permeable PKA-specific inhibitor KT5720, but not the PKG-specific inhibitor KT5823, completely blocked cGMP-dependent suppression of cell death in TNF␣ϩActD-treated cells. However, KT5720 only partly inhibited cAMP-mediated protection (Fig. 7C), suggesting that both PKA-dependent and -independent pathways contribute to cAMP-mediated suppression of apoptosis.

DISCUSSION
This study was undertaken to characterize the mechanism by which cyclic nucleotides suppress TNF␣ϩActD-mediated apoptosis. Cell-permeable cAMP analogs and other cAMP-elevating agents have been previously shown to suppress or delay apoptosis in several cell types including hepatocytes (25,26); however, the mechanism underlying this suppression remains elusive. In this study, we demonstrated that both cAMP and cGMP inhibited TNF␣ϩActD-induced apoptosis in cultured FIG. 6. Insulin protects hepatocytes via a PI3K/Akt-dependent pathway. Hepatocytes were pretreated with vehicle, LY294002 (10 M) or wortmannin (20 nM), for 45 min. Apoptosis was induced by the addition of TNF␣/ActD in the absence and presence of 1 M insulin. Cell viability (A) and DNA fragmentation (B) were measured in triplicate. C, immunoblots for phosphorylated Akt. Cells were incubated with increasing concentrations of insulin for 30 min. When indicated, cells were pretreated with 10 M LY294002 or 20 nM wortmannin for 45 min prior to being exposed to insulin. Cellular protein was resolved by SDS-PAGE, transferred to nitrocellulose, and probed as described. D, immunoblot of phosphorylated Akt of cells treated with 100 nM insulin, 25 M pCPT-cGMP, 100 M Bt 2 cAMP, 10 M (S p )-cAMPS, or 10 M forskolin plus 0.5 mM IBMX for 30 min. The level of phosphorylated Akt was visualized with ECLϩPlus TM and quantitated with the ImageQuaNT system, and the relative P-Akt level is shown in the lower panel. cAMP Inhibits Apoptosis Signaling Independent of Akt Activation primary hepatocytes by suppressing caspase activation (Fig. 2,  3). This mechanism is further supported by the observations that treatment with cyclic nucleotides inhibited caspase-mediated DNA fragmentation, morphology changes, and PARP cleavage in cells challenged with TNF␣ϩActD ( Figs. 1 and 2) and that TNF␣ϩActD-induced cytochrome c release and activation of procaspase-9 were suppressed by cyclic nucleotides (Fig. 3C). Therefore, we conclude that cyclic nucleotides suppress apoptosis signaling at an early stage by inhibiting caspase proteolytic activation and cytochrome c release.
To explore further the mechanism by which cyclic nucleotides modulate the caspase signaling cascade, we tested the possibility that cyclic nucleotides may modulate or activate cellular survival pathways to antagonize the death signaling. Activation of Akt by growth factors and cytokines promotes cell survival by intervening in the apoptosis cascade via phosphorylation and inactivation of the proapoptotic protein Bad (33) and procaspase-9 (37). In addition, it has also been shown that Akt activation prevents cytochrome c release by a mechanism that is distinct from Bad phosphorylation (44). Therefore, we first examined whether cyclic nucleotide analogs have any effect on Akt activation in hepatocytes and whether this activation accounts for the protective function of cyclic nucleotides. We found that reagents that increase either intracellular cAMP levels or cGMP levels activated endogenous Akt in primary hepatocytes in a time-and dose-dependent manner (Fig. 4). In contrast to recent reports showing that cAMP or PKA stimulates overexpressed and endogenous Akt through a PI3ЈK-independent pathway as determined by the failure of blocking Akt activation with wortmannin in 293 and COS-7 cell lines (40,50), we found that two structurally unrelated PI3ЈK-specific inhibitors, LY294002 and wortmannin, completely inhibited cAMP-and cGMP-induced Akt activation in hepatocytes at low concentrations. This result suggests that PI3ЈK may participate in cyclic nucleotide-stimulated Akt activation in hepatocytes. Others (26) have shown that cAMP reagents do not directly activate PI3ЈK in hepatocytes, but in sympathetic neurons the cAMP analog, CPT-cAMP, increases PI3ЈK activity (51). Whether activation of Akt by cyclic nucleotides described in this study was due to a direct effect of cyclic nucleotides on PI3ЈK or on PI3ЈK regulatory protein(s) remains to be investigated. cAMP-dependent protein kinase (PKA) as well as cGMP-dependent protein kinase (PKG) appear to play a partial role in Akt activation in hepatocytes. This is supported by the observation that inhibition of PKA or PKG with specific inhibitors blocked cyclic nucleotide-mediated Akt activation. 2 Recombinant PKA or PKG directly phosphorylates purified Akt protein in vitro at a site (Ser 422 ) away from the two phosphorylation sites (Thr 308 and Ser 473 ) required for full Akt activation (50). 2 However, cyclic nucleotides induce Akt phosphorylation at Ser 473 in intact hepatocytes, indicating that PKA and PKG activate Akt by affecting other proteins upstream of Akt. Further study is required to elucidate the mechanism for cyclic nucleotide-mediated Akt activation in cells.
Signals transduced from activated Akt have been shown to be required and are sufficient for nerve growth factor and interleukin-3-mediated inhibition of apoptosis and cell survival (33)(34)(35). Unexpectedly, the cytoprotection effects of cyclic nucleotides against TNF␣ϩActD-induced apoptosis reported here were not due to activation of endogenous Akt. This is supported by the findings that complete inhibition of cyclic nucleotidemediated Akt activation had no effect on cyclic nucleotidemediated suppression of TNF␣ϩActD-induced apoptotic cell death nor did it affect cyclic nucleotide-mediated suppression of procaspase-3 activation and DNA fragmentation (Fig. 5). In addition, we were unable to detect phosphorylated Bad under conditions where cyclic nucleotides prevent apoptosis. 2 This dissociation of Akt signaling pathway from TNF␣ϩActD-induced apoptosis is not due to the failure of hepatocytes to respond to Akt survival signaling, since we found that antiapoptotic actions of insulin was completely dependent on PI3ЈK/Akt activation in hepatocytes (Fig. 6). Similarly, others (47) have also shown that insulin protects hepatocytes against transforming growth factor ␤-induced apoptosis via activating the PI3ЈK/Akt-signaling pathway. The dissociation of activation of PI3ЈK/Akt pathway from apoptosis found in our study is further supported by other recent findings in hemopoetic cell lines, fibroblast, and prostate cancer cells where activation of Akt, phosphorylation of the pro-apoptotic protein Bad by cytokines and growth factors did not correlate with cell survivals (52)(53)(54)(55). These studies clearly implicate the existence of a 2 J. Li and T. R. Billiar, unpublished results. cAMP Inhibits Apoptosis Signaling Independent of Akt Activation PI3ЈK/Akt-independent survival signaling pathway. The difference in the magnitude of cyclic nucleotide-and insulin-mediated Akt activation may explain the differential dependence on this pathway for cellular protection.
Many of cellular effects of the cAMP second messenger are mediated by PKA. The anti-apoptotic effect of cAMP also appears to be mediated, at least in part, by activation of PKA. Preincubation of cells with PKA-specific inhibitor partly reversed the cAMP-mediated inhibition of TNF␣ϩActD-induced apoptosis, indicating that both PKA-dependent and -independent pathways account for the anti-apoptotic function of cAMP in hepatocytes. Interestingly, the PKA inhibitor KT5720, but not the PKG inhibitor KT5823, completely reversed the protective effect of cGMP, suggesting that cGMP acts through activation of intracellular PKA. The cross-talk between cGMP and PKA has been previously demonstrated. For example, one study showed that NO/cGMP-induced inhibition of cell growth in vascular smooth muscle cells is completely dependent on activation of PKA (48). Similar observations were found by others (22) where activation of PKA negatively modulates apoptotic cell death. Exactly how PKA intervenes in TNF␣ϩActD-mediated apoptosis signaling is currently unknown. However, a recent study illustrates that one such mechanism is that activated PKA associates with a mitochondria-anchored protein and directly phosphorylates and inactivates Bad at Ser 112 (56).
Increasing cAMP levels are also known to either inhibit or activate mitogen-activated protein kinase (MAPK) in a cell type-and stimulus-specific manner (57)(58)(59). Activation of extracellular signal-regulated kinase has been implicated in a number of systems to contribute as a negative regulator of apoptosis (60 -62). The protective effect of cAMP-elevating agents does not appear to act through the MAPK pathway in our system. This is suggested by the finding that MAPK kinase inhibitor PD98059 and p38 MAPK inhibitor SB203580 had no effect on the anti-apoptotic functions of cyclic nucleotide in hepatocytes. 2 Furthermore, the transcription factor cAMP response element-binding protein promotes cell survival by upregulating pro-survival proteins (63)(64)(65)(66), such as Bcl-2 in cerebellar granule neurons (65) and B-cells (66). Although we cannot completely exclude the possibility that cAMP protects hepatocytes via increased expression of pro-survival proteins, we rendered this unlikely based on the use of the transcription inhibitor actinomycin D in our system. Indeed, immunoblotting analysis of the Bcl-XL level in hepatocytes treated with cyclic nucleotides in the presence of TNF␣ϩActD revealed no differences as compared with control cells. 2 In conclusion, we demonstrated that the critical second messenger cAMP as well as cGMP modulate TNF␣ϩActD-mediated apoptosis signaling pathway via a PKA-dependent and -independent mechanism. Although one of the major functions of Akt is protection of cells against apoptosis, and even though these cyclic nucleotides rapidly activate endogenous Akt, activation of Akt per se does not appear to play a major role in cyclic nucleotide-mediated anti-apoptotic function. Further investigation is needed to unravel the link between apoptosis pathway and other survival pathways activated by the cyclic nucleotides.