Cross-Talk Between PKA and Akt Protects Endothelial Cells From Apoptosis in the Late Ischemic Preconditioning
Objective— The aim of this study was to explore the molecular mechanisms involved in late preconditioning-induced cell protection in endothelial cells.
Methods and Results— Preconditioning (PC) was induced by exposing bovine aortic endothelial cells (BAECs) to 3 cycles of 15 minutes of hypoxia followed by 15 minutes of reoxygenation. A 12-hour period of hypoxia induced cell death in 60% of BAECs (48±5% apoptosis, 12±4% necrosis). Early and late PC decreased hypoxia-induced apoptotic (25±5% and 28±4%, respectively) and necrotic (6±3%, and 8±2%, respectively) cell death. Consistently, hypoxia-induced caspase-3 cleavage was reduced by PC. Pretreatment with H89 (protein kinase A [PKA] inhibitor), LY294002 (phosphatidyl-inositol-3-kinase [PI3K] inhibitor), and N-acetyl-cysteine (antioxidant) abrogated late PC-induced cell protection, whereas inhibition of protein kinase C by Go6983, and of nitric oxide synthesis by L-NAME,1400W and bovine eNOS siRNA did not. In addition, in early and late PC, PKA physically interacted with the phosphorylated form of Akt, suggesting that PKA is required for Akt phosphorylation. Expression of PKA and Akt dominant negative mutants inhibited ischemic late PC-induced protection, indicating that these kinases play a key role in late PC-mediated cell protection.
Conclusions— Late ischemic PC protects BAECs against hypoxia through PKA- and PI3K-dependent activation of Akt.
Ischemic preconditioning (PC) is a physiological phenomenon in which nonsustained, repetitive, sublethal ischemic stimulation enhances tolerance to a subsequent prolonged ischemic stress. PC has great pathophysiological relevance, because it confers protection against ischemia-induced cell death to those organs that are composed of terminally differentiated cells, like the brain and heart.
The molecular mechanisms and mediators that account for PC have been extensively investigated, and it has emerged that nitric oxide (NO) plays a key role in this phenomenon in both the brain1 and heart.2 Endothelium represents one of the principal sources of NO. Although there is considerable evidence that the protective effects of both early and late PC extend to endothelial cells,3,4 the molecular mechanisms of NO generation in endothelial cells during late PC (LPC) have been poorly investigated. In recent years it has been demonstrated that Akt, a serine/threonine kinase, plays a pivotal role in the genesis of NO in endothelial cells by inducing endothelial nitric oxide synthase (eNOS) phosphorylation at serine1177 (ser1177), leading to an increase in eNOS activity.5 Interestingly, Akt is activated by several ligand-receptor systems previously shown to be protective against cell death, such as insulin-,6 IGF-1–,6 and gp130-dependent cytokines.7 Thus, Akt activation results in a powerful protective effect after transient ischemia in the heart8 and brain,9 which probably reflects its ability to inhibit cell death. Moreover, it has been reported that, in cultured endothelial cells, antiapoptotic effects induced by fluid shear stress10 and ischemic PC11 are mediated by an Akt-dependent mechanism.
Therefore, it is possible to hypothesize that ischemic PC activates Akt. If this is the case, Akt-phosphorylation/activation could directly contribute to PC-induced cytoprotection by promoting cell survival and stimulating NO synthesis.
We planned this study to explore the molecular mechanisms involved in LPC-induced cell protection through a model of ischemic PC in cultured endothelial cells, and, in particular, to investigate the specific roles of Akt and NO.
Materials and Methods
Detailed methods can be found in the supplemental material (available online at http://atvb.ahajournals.org).
Cell Culture and Hypoxia
Bovine aortic endothelial cells (BAECs) were subjected, after overnight starvation, to preconditioning or hypoxia.
Estimation of NO Production
NO production was assessed by both conversion of L-arginine into L-citrulline and GRIESS reaction.
Annexin V Staining
Endothelial cells grown on coverslips were stained with Annexin-V-Fluorescein and Propidium Iodide, according to manufacturer’s instructions. Apoptotic and necrotic cell death were assessed as described in the supplemental material.
Protein Kinase A Activity Assay
The protein kinase A (PKA) activity was assessed by a specific radioactive assay, as described in the supplemental material.
Immunoblotting and Immunoprecipitation
Cells were grown in 100-mm plates. At the end of the stimulation period, the medium was removed and the cells processed, as described in the supplemental material.
Plasmids encoding epitope-tagged dominant negative PKA (generous gift of Drs Michael S. Deal and Susan S. Taylor, University of California, San Diego) and Akt (HA-Akt [K179M]) were expressed in BAECs by transient transfection, as previously reported.12 eNOS expression was silenced by bovine endothelial NOS-short interfering RNA (beNOS-siRNA).
Data are given as mean±SEM. Statistical analyses were performed using analysis of variance. The post test comparison was performed by the method of Tukey. A χ2 test was used for categorical variables. Significance was accepted at P<0.05 levels.
Preconditioning Protects Endothelial Cells From Hypoxia-Induced Cell Death
We performed a pilot experiment\to evaluate the rate of cell death after different periods of hypoxia (supplemental Figure I and Table I). Because 12 hours represented the time point at which hypoxia induced nearly 60% cell death, mainly attributable to apoptosis, we tested whether brief periods of hypoxia were able to reduce hypoxia-induced cell mortality. Three cycles of 15 minutes of hypoxia followed by 15 minutes of reoxygenation (Figure 1A) reduced the cell death induced by 12 hours of hypoxia. A decrease in apoptotic cell death was mainly responsible for this phenomenon (Figure 1B). The observed cytoprotective effect had a biphasic trend, with early and late windows detectable immediately and 24 hours after PC was applied (supplemental Table II). In particular, apoptotic cell death was reduced in “early” and “late” preconditioned cells to 25±5% and 28±4%, respectively, compared to 48±5% in nonpreconditioned cells. Apoptosis was further explored by analysis of caspase-3 cleavage, which plays a key role in regulation of the cellular suicide cascade.13 This analysis confirmed the biphasic trend of PC-induced cytoprotection (Figure 1C), with early and late windows characterized by reduction of hypoxia-induced caspase-3 cleavage.
These results show that in endothelial cells, brief periods of repeated hypoxia followed by reoxygenation reduce hypoxia- induced apoptotic cell death with 2 windows of protection (early and late), indicating that our experimental conditions resemble the PC phenomenon.
Preconditioning Induces NO Synthesis in Endothelial Cells
Because NO is believed to be a key mediator of PC, we measured NO production by citrulline accumulation and by the colorimetric GRIESS method. PC induced eNOS-dependent NO production with 2 peaks, which were detected immediately and 24 hours after preconditioning and were characterized by an increase of 1.5- and 1.9-fold in citrulline accumulation, respectively (Figure 2A). Evaluation of PC-induced NO production by the GRIESS reaction showed the same biphasic trend (Figure 2B). Analysis of enzymes accounting for NO production showed that both eNOS and inducible nitric oxide synthase (iNOS) were involved in preconditioning-induced NO synthesis. In particular, phosphorylation/activation of eNOS at serine1177 (ser1177) and expression of iNOS were detected immediately and 24 hours after PC (Figure 2C and 2D).
These data indicate that, in endothelial cells, PC induces NO production, eNOS activation, and iNOS expression.
PKA and PI3K Mediate NO Synthesis in the Late Preconditioning
Because the LPC confers a longer window of cytoprotection, we focused our interest on the molecular pathways that regulate LPC-induced NO production. Furthermore, in other models, it has been demonstrated that several mechanisms account for NO production in LPC, including activation of protein kinase A (PKA),14 phosphatidyl-inositol-3-kinase (PI3K),5 and protein kinase C (PKC)15 and production of reactive oxygen species (ROS).16 To examine which molecular pathway accounts for LPC-induced NO production in endothelial cells, we measured LPC-induced NO production in the presence of PKA-, PI3K-, PKC-, and iNOS-inhibitors, nonspecific NOS inhibition and an antioxidant (N-acetyl-cysteine, NAC). Inhibition of iNOS by 1400W (20 μmol/L, 15 minutes pretreatment) significantly blunted PC-induced NO production, whereas administration of NO synthesis inhibitor Nω-Nitro-l-arginine methyl ester (L-NAME; 1 mmol/L, 15 minutes pretreatment), alone or in combination with 1400W, abrogated it completely (Figure 3A and 3B). Pretreatment with either H89 (PKA inhibitor; 10 μmol/L, 30 minutes pretreatment) or LY294002 (PI3K inhibitor; 30 μmol/L, 30 minutes pretreatment) also abrogated PC-induced NO production, and NAC exposure (10 mmol/L, 30 minutes pretreatment) reduced it, but inhibition of PKC with Go6983 (1 μmol/L, 60 minutes pretreatment) did not (Figure 3A and 3B). Consistently, LPC-induced phosporylation/activation of eNOS was inhibited by H89, LY294002, and NAC (supplemental Figure IVA). In addition, LPC-induced iNOS expression was inhibited by pretreatment with H89 and NAC, and was significantly blunted by LY294002 (supplemental Figure IVB).
Next, because we found that NAC inhibited LPC-induced NO synthesis, we checked whether PC induced ROS production. ROS generation was assessed by evaluation of protein nitration.17 LPC induced an increase in nitrite protein content (3.2-fold versus control), which was significantly smaller than those detected at 1, 6, and 12 hours after PC application (6.1-fold versus control; Figure 3C). Pretreatment of late preconditioned BAECs with NAC abrogated formation of nitrated proteins (Figure 3D).
Together, these data indicate that PKA, PI3K, and ROS play a pivotal role in NO production during the late phase of PC.
Preconditioning Activates Both PKA and Akt
Because we found that LPC-induced NO production was mediated by PKA- and PI3K-dependent pathways, we asked whether LPC activates these kinases.
PKA activation was assayed by phosphorylation of kemptide (Kp), which is a synthetic substrate of PKA. Both early and late PC induced phosphorylation of Kp. In particular, Kp phosphorylation increased by 3.8-fold in LPC, compared to control (supplemental Figure V).
Next, we explored Akt, the serine/threonine kinase downstream of PI3K that promotes NO synthesis through the activation of eNOS.5 LPC resulted in phosphorylation/activation of Akt at both threonine308 (thr308; 3.2-fold versus control, P<0.001; Figure 4A) and serine473 (ser473; 5.8-fold versus control, P<0.001; Figure 4B).
To confirm that PC-induced PKA and Akt activation account for NO production through phosphorylation/activation of eNOS, we evaluated the physical interaction between PKA, Akt, and phospho-eNOS (ser1177) during PC. For this purpose, cell lysates from different time points after PC induction were subjected to immunoprecipitation with antibodies against the PKA catalytic subunit and Akt. These samples were then blotted with an antiphospho-eNOS (ser1177) antibody. Early PC induced a complex between PKA and phospho-eNOS, whereas LPC induced a complex between Akt and phospho-eNOS (Figure 4C and 4D).
Together these experiments indicate that PC activates PKA and Akt, which, in turn, physically interact with and phosphorylate eNOS. In particular, PKA is mainly involved in eNOS phosphorylation during the early phase of PC, whereas Akt accounts for eNOS phosphorylation in the late phase of PC.
PKA and PI3K/Akt Mediate Cell Protection During Late PC
Next, we explored which mechanism accounts for LPC-induced cytoprotection. For this purpose, we evaluated hypoxia-induced cell death and caspase-3 cleavage in LPC in the presence and absence of PKA-, PI3K-, and PKC-inhibitors, nonspecific inhibition of NO production and ROS scavenger. Hypoxia-induced apoptotic and necrotic cell death (supplemental Figure VIA and supplemental Table IV), as well as caspase-3 cleavage, were reduced by LPC (Figure 5A). This phenomenon was unaffected by inhibition of PKC (Go6983) and of NO production (L-NAME+1400W), but was abolished by inhibition of PKA (H89), PI3K (LY294002), and ROS activity (NAC). Interestingly, hypoxia-induced caspase-3 cleavage detected during PKA and PI3K inhibition was dramatically increased compared to that induced by hypoxia alone. To further rule out the contribution of iNOS and eNOS in LPC-induced cell protection, we evaluated cell death and caspase-3 cleavage in LPC after treatment with 1400W (supplemental Figure VIB) and after silencing eNOS expression by beNOS-siRNA transfection (Figure 5B). The efficacy of beNOS-siRNA transfection in BAECs was assessed by measurement of both eNOS protein expression and by enzymatic activity. Transfected cells showed a reduction in both eNOS expression (Figure 5B) and nitrite production (supplemental Figure XIA). Neither 1400W nor beNOS-siRNA treatment affected the cytoprotective effect of LPC, assessed by measurement of apoptotic and necrotic cell death and by caspase-3 cleavage.
Finally, to explore whether PKA and PI3K are required for LPC-induced cell protection, we assessed the effect of LPC on hypoxia-induced cell death and caspase-3 cleavage in cells with transient expression of dominant negative mutants of PKA (DN PKA) and Akt (DN Akt), which is the serine/threonine kinase that regulates several PI3K-dependent biological activities including cell survival.18 Transfection of plasmid harboring either DN PKA or DN Akt inhibited cell protection conferred by LPC. In fact, both plasmids abolished the inhibition of hypoxia-induced apoptotic and necrotic cell death (supplemental Figure VIIA and supplemental Table V) and caspase-3 cleavage induced by LPC (Figure 5C and 5D). The efficacy of DN PKA and DN Akt transfection in BAECs was assessed by immunoblotting (supplemental Figure VIIB and VIIC). Evaluation of Bad phophorylation, which confers cell protection against apoptosis, showed that LPC induces phosphorylation of serine112 and serine136, consensus sites for PKA and Akt, respectively (supplemental Figure VIC).
Together these data indicate that, in endothelial cells, the LPC-induced cytoprotective effect requires a PKA- and PI3K/Akt-dependent pathway.
PKA and PI3K Mediate PC-Induced Akt Activation
Because both PKA- and PI3K/Akt-dependent pathways account for the LPC-induced cytoprotective effect, we explored the cross-talk between these 2 pathways. First, we analyzed the mechanism of LPC-induced Akt activation. For this purpose, LPC-induced Akt phosphorylation was assessed in the presence of H89, LY294002, Go6983, L-NAME, 1400W, and NAC. LPC-induced Akt phosphorylation, at thr308 and ser473, was abrogated by pretreatment with H89 and NAC, but was only blunted by LY294002 (supplemental Figure XA and XB), suggesting that PKA plays a critical role in Akt activation during LPC. Therefore, we asked whether PKA and Akt physically interact during PC. For this purpose, lysates from cells subjected to PC were immunoprecipitated with antibodies against the PKA catalytic subunit and then blotted with antiphospho-Akt (thr308/ser473) antibodies. Both early and late PC induced an immunocomplex between PKA and phospho-Akt. (Figure 6A and 6B). To verify that Akt is a substrate of PKA during PC, we immunoprecipitated cell lysates using an anti-Akt antibody and blotted with an antibody recognizing PKA substrates. PC increased the recognition of Akt by anti-PKA phospho-substrate antibody, confirming that Akt is phosphorylated by PKA during PC (Figure 6C).
These results indicate that both PKA and PI3K are required for LPC-induced Akt activation and that Akt is a substrate of PKA.
The present study focused on the molecular mechanisms that account for the PC-induced cytoprotective effect in endothelial cells. Our data appear to contrast with the reports showing that NO production is required for PC-induced cytoprotection. Several factors can account for these discrepancies. First, our experimental setting was completely different from that used by other authors, because we used cell culture, whereas Xuan et al19 evaluated infarct size in transgenic mice and Laude et al20 analyzed coronary vasorelaxation in preconditioned rat hearts. Moreover, we exclusively explored the molecular mechanisms involved in cell survival, whereas these authors investigated different functional aspects of PC. These controversial data highlight the need to specifically define the mechanisms that account for the different biological components of PC.
In this study, we found that PC induces activation of Akt, which in turn accounts for the PC-induced cytoprotection. This mechanism allows for speculation that PC-induced NO production, rather than being a mediator of cell survival, is an epiphenomenon of Akt activation. However, we do not exclude the possibility that in a more complex biological system, like in vivo experimental models, NO may play a critical role in PC-induced cytoprotection. In addition, it should be noted that our experimental setting excluded the contribution of inflammation to the endothelial response. This could explain the absence of an NO protective role in PC-induced endothelial protection.
Our data show that, in endothelial cells, the PC-induced cytoprotective effect is mediated by PKA- and PI3K-dependent activation of Akt. A substantial body of evidence indicates that PI3K-dependent Akt activation accounts for a cytoprotective effect in different cell types and in response to several stress stimuli. In contrast, it has been demonstrated that PKA activation, rather than leading to a survival signal, promotes apoptosis in different cell types, including endothelial cells.21 Akt can be activated by distinct mechanisms, including wortmannin-sensitive and -insensitive pathways,22 and it has been reported that Akt can be activated by a PKA-dependent mechanism in endothelial cells.23 In the present study, we found that Akt activation in LPC requires PKA, that PKA forms a physical complex with Akt, and, more interestingly, that Akt acts as a substrate of PKA. This suggests that, in endothelial cells, the PC-induced cytoprotective effect is mediated by the cross-talk between PKA and Akt, which directly interact. Thus, PKA does not exclusively act as a proapoptotic kinase, but can also account for a cytoprotective effect through its downstream target.
We found that LPC-induced phosphorylation of Akt was blunted, but not entirely inhibited, by pretreatment with LY294002, suggesting that, in endothelial cells, PI3K plays a partial role in PC-induced cytoprotection. Although this was an unexpected result, it is reasonable to speculate that the differential relevance of PKA and PI3K in promoting cell protection depends on the characteristics of the survival stimuli. For instance, PI3K-dependent mechanisms play a pivotal role in insulin-24 or IGF-1–25 induced cytoprotection. On the other hand, PKA-dependent mechanisms account for some biological responses evoked by hypoxia.26
We also noted that NAC abrogates LPC-induced protection in BAECs, suggesting an important role of ROS in this phenomenon. Consistently, protein nitration peaked between early and late PC, when the protective effect is abolished. These data highlight the need to clarify the role of ROS production. In particular, low doses of ROS, by reducing thiolic residues in transmembrane receptors, induce activation of downstream protection pathways,27 whereas high doses of ROS, by affecting the tertiary structure of these receptors, inhibit activation of protective mechanisms.27 Finally, at high concentrations, ROS react with NO, inducing generation of peroxynitrite which uncouples eNOS function.28
We found that expression of beNOS-siRNA significantly reduced LPC-induced nitrite production. Although this would appear to be inconsistent with the finding that, during LPC, iNOS mainly accounts for nitrite production, there is, in fact, no contradiction, because it has been demonstrated that nuclear translocation of eNOS is required for iNOS expression.29 Consistent with this observation, we found that LPC induces nuclear translocation of eNOS and that beNOS-siRNA transfection inhibits LPC-induced iNOS expression (supplemental Figure XI).
In conclusion, our data indicate that, in endothelial cells, the protective effect of LPC against hypoxic injury is mediated by PKA-, PI3K-dependent Akt activation and ROS generation.
We thank Daniela Zablocki for critical reading of the manuscript.
Sources of Funding
This study was supported by a grant from the Italian Ministry of University and Research (MIUR), annuity 2006. A.B. was supported by a grant from the Italian Society of Hypertension, annuity 2006.
Received February 28, 2008; revision accepted May 5, 2009.
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