PTEN Activity Is Modulated During Ischemia and Reperfusion
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循环研究杂志 2005年第12期
the Vascular Biology Program, Institute for Cell Engineering (Z.C., G.L.S.)
the Departments of Pediatrics, Medicine, Oncology
Radiation Oncology (G.L.S.)
the McKusick-Nathans Institute of Genetic Medicine (Z.C., G.L.S.), The Johns Hopkins University School of Medicine, Baltimore, Md.
Abstract
Ischemic preconditioning (IPC), a brief period of ischemia and reperfusion (I/R), generates profound but transient protection against a subsequent prolonged ischemic episode. The serine-threonine kinase Akt has been shown to mediate IPC, and Akt activation is negatively regulated by the phosphatase PTEN, but whether PTEN activity is modulated by IPC has not been investigated. When isolated, perfused rat hearts were subjected to an IPC stimulus consisting of 15-minute ischemia and 30-minute reperfusion (I-15/R-30), PTEN protein levels and activity were decreased, and levels of phospho-AKT were increased, relative to nonischemic hearts. Hearts subjected to IPC demonstrated improved recovery of cardiac function when subsequently subjected to I-30/R-45 as compared with hearts subjected to I-30/R-45 without prior IPC. When hearts were subjected to I-15 followed by R-30, R-60, or R-120, PTEN reaccumulated gradually and its activity was restored. Phospho-Akt levels at R-120 were decreased and these hearts were no longer protected against injury when subjected to I-30/R-45. Wortmannin administration during reperfusion blocked Akt activation and PTEN reaccumulation. In ischemic hearts, PTEN was rapidly degraded. Pretreatment with proteasome inhibitor MG132 blocked ischemia-induced degradation of PTEN and blocked IPC. Reperfusion following I-15 induced oxidation of the remaining PTEN, leading to Akt activation. Perfusion of H2O2 was sufficient to induce Akt activation. Thus, loss of PTEN activity leads to induction of IPC and feedback mechanisms designed to ensure that Akt activation is transient are responsible for decay of IPC.
Key Words: Akt apoptosis ischemia PTEN
Introduction
Coronary artery disease is a leading cause of death in developed countries. Prolonged ischemia and reperfusion (I/R) results in cardiac cell death and ventricular dysfunction. Protecting the heart against I/R injury may reduce disease mortality. Ischemic preconditioning (IPC) was described by Murry et al, who reported that 4 cycles of 5 minutes of ischemia alternating with 5 minutes of reperfusion limited infarct size by 75% in dog hearts.1 IPC was reproduced in other mammalian species, including humans, and other organs, including kidney and gut.2 IPC peaks after &30 minutes of reperfusion and lasts &2 hours. Both ischemia and reperfusion are required to induce IPC.1,2 In most species, 15 minutes of ischemia (I-15) followed by reperfusion is sufficient to generate protection. In rat and pig models, protection afforded by IPC is related to duration of ischemia.3,4 The molecular events following I/R remain poorly understood.
Experimental evidence implicates the serine/threonine kinase Akt as a mediator of IPC.5eC9 Substrates for phosphorylation by Akt include the proapoptotic proteins Bad and Bax,10 endothelial nitric oxide synthase,11 p70 S6 kinase (p70S6K),12 caspase-9,13 and glycogen synthase kinase-3,14 which are all involved in regulating cell survival. IPC activates Akt and overexpression of Akt protects cardiomyocytes against apoptosis.7,9,15,16 Protein kinase C and extracellular signal-regulated kinases 1 and 2 are also involved in cardiac protection induced by IPC and appear to be downstream targets of PI3K and Akt signaling.5,15,17
Akt is activated by phosphatidylinositol (PI)-3,4,5-triphosphate (PIP3). PI3-kinase (PI3K) catalyzes conversion of PI-4,5-diphosphate to PIP3.18 PTEN (Phosphatase and TENsin homologue deleted from chromosome 10) is a PI3-phosphatase that dephosphorylates PIP3 and antagonizes PI3K activity.18 Studies have shown that PIP3 levels are predominantly determined by PTEN activity, thereby controlling Akt activation.19 PI3K is required for IPC15,16 because of its obligatory role in PIP3 production, but the role of PTEN in IPC has not been studied.
PTEN, which was first identified as a tumor suppressor gene, is an essential regulator of cell proliferation, differentiation, growth, and apoptosis.18,20 PTEN activity is inversely related to cell survival: increased apoptosis was observed in neonatal cardiomyocytes that overexpressed PTEN, whereas expression of mutant PTEN activated Akt, increased cell size, and reduced apoptosis.21
PTEN activity is regulated by oxidation and phosphorylation. A reactive cysteine residue (Cys124) is located at the active site of PTEN. H2O2 oxidizes Cys124 to form a disulfide bond with Cys71, leading to PTEN inactivation.22 PTEN oxidation leads to increased PIP3 levels and Akt activation in cell lines after growth factor stimulation.19,23,24 Under basal conditions, PTEN is phosphorylated and inactive. After dephosphorylation and plasma membrane targeting, PTEN is activated but is degraded rapidly.25,26 Phosphorylation of PTEN increases its stability. Amino acid substitutions that block phosphorylation increase PTEN activity and reduce its half-life.27 Thus, activity and half-life are reciprocally regulated. Isoproterenol stimulation or Akt overexpression resulted in PTEN accumulation,21,28 suggesting a feedback loop that autoregulates Akt activity. In this study, we investigated whether changes in PTEN activity contribute to the induction and decay of IPC.
Materials and Methods
Langendorff Preparation
Isolated heart perfusion was performed as described.29
Immunoprecipitation and PTEN Activity
PTEN was immunoprecipitated from 200 e of heart lysates with 20 e蘈 of anti-PTEN antibody overnight, followed incubation with protein GeCagarose beads for 2 hours at 4°C. Precipitates were washed with lysate buffer, and 1 e of PIP3 and 20 e蘈 of assay buffer were added. The enzyme reaction was terminated by adding Malachite Green solution and absorbance was detected at 600 nm. Released phosphate was determined relative to a standard curve.
Analysis of Oxidized and Reduced PTEN
After exposure to H2O2 or I/R, isolated rat hearts were frozen in eC80°C and homogenized in cell lysate buffer containing iodoacetamide (50 mmol/L). The supernatant was fractionated by 10% nonreducing SDS-PAGE, followed by PTEN immunoblot assay.
An expanded Materials and Methods section is provided in the online data supplement available at http://circres.ahajournals.org.
Results
IPC Is Dependent on PI3K-Akt Signaling
We have previously shown that loss of left ventricular (LV) developed pressure (LVDP) closely parallels infarct size in isolated perfused rat hearts subjected to I/R.29 When hearts were subjected to 15-minute ischemia and 30-minute reperfusion (I-15/R-30) as an IPC stimulus followed by prolonged I/R (I-30/R-45), the recovery of LVDP was improved compared with hearts subjected to I-30/R-45 without IPC (LVDPIPC versus LVDPI/R, 77±6 versus 15±5 mm Hg, P<0.001; Figure 1A). I-15/R-30 significantly increased levels of active, phosphorylated Akt (p-AktIPC versus p-AktNIS, 179±18 versus 29±11, P<0.001, where NIS indicates nonischemic hearts; Figure 1B and 1C). The activation of Akt was also demonstrated by the phosphorylation of a known Akt substrate, p70 S6 kinase (p-p70IPC versus p-p70NIS, 178±7 versus 1±0, P<0.001; Figure 1B and 1D). The increased levels of phosphorylated Akt and p70 were not associated with changes in the total amount of Akt or p70 protein (Figure 1B and data not shown). When hearts were subjected to I-15/R-30 in the presence of 1 eol/L wortmannin to inhibit PI3K activity, IPC-induced phosphorylation of Akt and p70 was completely blocked (Figure 1B through 1D) and protective effects of IPC on recovery of LVDP after prolonged ischemia were lost (Figure 1A). These results demonstrate that this model of IPC is dependent on PI3K and Akt signaling as previously described.1eC9
IPC Downregulates PTEN Activity
We determined PTEN activity and protein by phosphatase and immunoblot assays, respectively, with aliquots of the same lysates used to analyze Akt. Compared with nonischemic hearts, I-15/R-30 significantly decreased PTEN activity (PTENNIS versus PTENIPC, 419±56 versus 136±8, P<0.01; Figure 2A) and protein levels (PTENNIS versus PTENIPC, 204±7 versus 37±12, P<0.001; Figure 2B). Wortmannin (WT) decreased PTEN levels (PTENIPC versus PTENIPC+WT, 52±5 versus 24±1 P<0.01) but increased PTEN activity (PTENIPC versus PTENIPC+WT, 135±8 versus 696±53, P<0.01; Figure 1A and 1B). Thus, an IPC stimulus, which increases Akt activity and promotes functional recovery in isolated rat hearts subjected to I/R, downregulates PTEN activity.
PTEN Levels Are Restored During Prolonged Reperfusion
PTEN activity was analyzed in nonischemic hearts and hearts subjected to I-15 followed by R-30, R-60, or R-120. PTEN activity recovered as duration of reperfusion increased (Figure 3A). During extended reperfusion, PTEN levels increased (PTENR-120 versus PTENR-30, 109±24 versus 37±12, P=0.02) and p-Akt levels decreased (p-AktR-120 versus p-AktR-30, 79±2 versus 179±18, P<0.001) without changes in total Akt (Figure 3B and 3C). Thus, extended reperfusion after IPC increases PTEN protein levels and activity, leading to the loss of phosphorylated (activated) Akt.
Decay of Protection During Prolonged Reperfusion
To characterize the effects of extended reperfusion after IPC on heart protection against I/R injury, we exposed hearts to I-30/R-45 directly or following I-15/R-30 or I-15/R-120. Continuously perfused (nonischemic) hearts were used as controls. Recovery of cardiac function was significantly improved in hearts subjected to I-30/R-45 following the IPC stimulus as compared with hearts subjected to I-30/R-45 in the absence of prior IPC (LVDPR-30 versus LVDPI/R, 78±9 versus 15±4 mm Hg, P<0.001; Figure 4A). When reperfusion was extended to 120 minutes, protection was lost (LVDPR-120 versus LVDPR-30, 28±5 versus 78±9 mm Hg, P<0.01; Figure 4A). IPC (I-15/R-30) reduced LV end-diastolic pressure (LVEDP) after I/R (LVEDPR-30 versus LVEDPI/R, 37±3 versus 77±4 mm Hg, P<0.001) but did not affect heart rate or coronary flow rate (supplementary Figure I). When reperfusion during IPC was extended to 120 minutes (I-15/R-120), heart protection was lost (LVEDPR-30 versus LVEDPR-120, 37±3 versus 64±3 mm Hg, P<0.001; supplementary Figure I).
I-30/R-45 resulted in the cleavage of caspase-3 and poly-(ADP-ribose) polymerase (PARP), 2 markers of apoptosis (Figure 4B through 4D). IPC (I-15/R-30) blocked the cleavage of these proteins, an effect that was lost with prolonged reperfusion (I-15/R-120). Thus, prolonged reperfusion during IPC impairs the protective effect of IPC against cardiac injury after I-30/R-45 and allows PTEN reaccumulation.
Recovery of PTEN During Reperfusion Requires PI3K-Akt Signaling
Because phospho-PTEN (p-PTEN) is inactive and stable, we hypothesized that PTEN reaccumulation is attributable to phosphorylation by Akt. Compared with nonischemic hearts, p-PTEN levels were dramatically reduced after I-15/R-30 but recovered to near baseline levels after I-15/R-120 (Figure 5A). When wortmannin was infused at R-15 for 15 minutes to block PI3K activity, Akt phosphorylation was inhibited and p-PTEN levels were decreased (Figure 5B). PTEN reaccumulation at R-30 and R-120 was blocked (PTENR-30 versus PTENR-30+WT, 49±10 versus 12±1, P<0.01; PTENR-120 versus PTENR-120+WT, 98±8 versus 11±5, P<0.01; Figure 5C). Thus, PI3K-Akt signaling is required for phosphorylation and stabilization of PTEN during extended reperfusion. PTEN antibodies coimmunoprecipitated Akt (Figure 5D), suggesting that Akt may directly phosphorylate PTEN.
PTEN Degradation Requires Phosphatase and Proteasome Activity
PTEN levels were markedly decreased after I-15/R-30, indicating that ischemia induces PTEN degradation. To determine the kinetics of degradation, hearts were subjected to ischemia of increasing duration (from 0 to 30 minutes) without reperfusion. PTEN was rapidly degraded in the ischemic hearts, with a half-life of 5.4 minutes, whereas -actin levels were unchanged (Figure 6A). After 30 minutes of ischemia PTEN enzymatic activity was significantly decreased compared with nonischemic hearts (PTENNIS versus PTENISC, 327±36 versus 31±27, P<0.01; Figure 6B). We hypothesized that ischemia-induced dephosphorylation of PTEN targets the protein for degradation. If so, then dephosphorylation should precede degradation. Compared with nonischemic hearts, the levels of phosphorylated PTEN in hearts subjected to 5 minutes of ischemia were reduced by &50%, whereas the levels of total PTEN were reduced by only &10% (Figure 6C).
To determine whether dephosphorylation of PTEN is required for degradation, we pretreated hearts for 15 minutes with the protein phosphatase PP2A inhibitor okadaic acid, then subjected them to I-15. Compared with hearts treated with vehicle only, 40 nmol/L okadaic acid blocked PTEN dephosphorylation and degradation in the ischemic heart, whereas 4 nmol/L okadaic acid was ineffective (Figure 6D). Thus, PTEN degradation requires dephosphorylation that is mediated by PP2A or another okadaic acideCsensitive phosphatase.
To investigate whether rapid degradation of PTEN is mediated by the proteasome, we infused the proteasome inhibitor MG132 at the various concentrations (0 to 4 eol/L) for 15 minutes, followed by I-15. MG132 blocked the ischemia-induced degradation of PTEN in a concentration-dependent manner (Figure 7A). At the lowest concentration tested (1 eol/L), MG132 partially inhibited PTEN degradation (PTENISC+MG versus PTENISC, 47±2 versus 28±2, P<0.01; Figure 7B) but did not block the reduction in p-PTEN levels (p-PTENISC+MG versus p-PTENISC, 20±1 versus 44±3, P<0.01; Figure 7C).
To determine whether PTEN degradation is required for IPC-induced protection, hearts were pretreated with MG132 (0, 2, 4 eol/L) and then subjected to IPC (I-15/R-30). Hearts pretreated with vehicle only and subjected to I-15/R-30 (MG0) showed almost complete recovery of cardiac function (Figure 7D). In contrast, pretreatment with MG132 at 2 eol/L (MG2) significantly inhibited the recovery of cardiac function (LVDPMG0 versus LVDPMG2, 101±7 versus 32±14 mm Hg, P=0.01) and completely blocked recovery at 4 eol/L.
Ischemia Reperfusion Induces PTEN Oxidation
Although ischemia induced the degradation of PTEN, the kinetics of degradation indicated that not all PTEN protein was degraded after I-15. We hypothesized that residual PTEN protein was inactivated by oxidation during reperfusion. To analyze PTEN oxidation, hearts were subjected to I-15 followed by R-0, 1, 5, 10, 15, or 30 and homogenates were exposed to 50 mmol/L iodoacetamide, an alkylating agent that attacks free thiols. The reduced and oxidized forms of PTEN were fractionated by nonreducing SDS-PAGE and identified by immunoblot assay. Reperfusion after I-15 resulted in decreased levels of reduced (active) PTEN, compared with the nonischemic heart (Figure 8A). The proportion of oxidized PTEN increased rapidly after reperfusion (Figure 8B). Comparison of the kinetics of PTEN oxidation, Akt phosphorylation, and PTEN dephosphorylation revealed 2 important findings. First, PTEN oxidation was already maximal at R-1 (31 minutes in Figure 8B) and thus preceded the increase in Akt phosphorylation at R-5 (35 minutes in Figure 8B). Second, although levels of p-PTEN decreased during ischemia, the increase in Akt phosphorylation from R-1 to R-5 was not associated with any further loss of PTEN phosphorylation. These data are consistent with a model in which oxidation of residual PTEN during early reperfusion leads to Akt phosphorylation.
The data presented in Figure 8B indicate that a brief period of reperfusion is sufficient to inactivate PTEN and lead to Akt activation. To determine whether Akt phosphorylation is determined by duration of ischemia, hearts were subjected to ischemia of increasing duration (5 to 20 minutes), followed by 5-minute reperfusion. The decrease in PTEN levels was correlated with the duration of ischemia (Figure 8C). Akt phosphorylation at R-5 was dependent on the duration of ischemia and inversely correlated with PTEN levels.
The oxidation of PTEN during early reperfusion is consistent with the increase in reactive oxygen species (ROS) that occurs during this process. To determine whether ROS are sufficient to induce PTEN oxidation and Akt phosphorylation, hearts were perfused with buffer containing H2O2 (0 to 10 eol/L) for 15 minutes. H2O2 treatment increased the levels of oxidized PTEN and decreased the levels of reduced PTEN in a concentration-dependant manner (Figure 8D). Most importantly, Akt phosphorylation was also increased by H2O2 treatment in a concentration-dependant manner, whereas the levels of total Akt, p-PTEN, and total PTEN were unchanged (Figure 8D). Finally, the induction of p-AKT by I-15/R-5 or I-15/R-30 was markedly reduced when hearts were perfused with 2-mercaptoethanol to block ROS signaling (Figure 8E).
Discussion
Two major conclusions can be drawn from the data presented above. First, IPC induces downregulation of PTEN activity through the combined effects of dephosphorylation/proteasomal degradation during ischemia and oxidative inactivation of residual PTEN during reperfusion, leading to Akt activation and cardiac protection. Second, extended reperfusion induces PTEN reaccumulation and reactivation by a PI3K/Akt-mediated feedback mechanism, resulting in Akt inactivation and loss of protection, thus providing for the first time a molecular basis for the transient nature of IPC.
IPC Stimulus Induces PTEN Inactivation by Dual Mechanisms
Our data indicate that I-15/R-30 induces loss of PTEN activity, which leads to reduced dephosphorylation of PIP3, increased Akt activation, and protection against injury following subsequent prolonged I/R. PIP3 levels are determined by the opposing activities of PI3K and PTEN. We focused on the inactivation of PTEN in this study; it is possible that both PI3K activation and PTEN inactivation increase PIP3 levels during IPC. The absence of active PTEN during early reperfusion allows unopposed PI3K activity and PIP3 accumulation, which is necessary for Akt activation. Additional studies are required to determine whether changes in the activity of other signaling proteins occur as a result of ischemia-reperfusion and are necessary for Akt activation. However, the striking modulation of PTEN activity we have demonstrated represents a paradigm shift in a field where attention has been focused almost entirely on Akt activation by PI3K signaling. The results presented above demonstrate that PTEN activity is modulated by rapid dephosphorylation and proteasomal degradation during ischemia followed by oxidation and inactivation of residual PTEN protein during reperfusion.
Several studies using cultured cells have provided evidence that PTEN plays a critical role in controlling PIP3 levels and Akt activity. In PTEN null cells, Akt could not be activated by H2O2 treatment, but when PTEN was reintroduced into the cells, H2O2 treatment resulted in increased PIP3 levels and Akt activation.23 Insulin-mediated Akt activation required a decrease in PTEN activity, and increased PI3K activity alone was not sufficient to activate Akt in human neuroblastoma cells.19 Exposure of cultured cells to insulin, epidermal growth factor, and platelet-derived growth factor was associated with inactivation of PTEN by reversible oxidation of cysteine residues in its catalytic site.24 A major experimental limitation at present is that, unlike PI3K, specific small molecule inhibitors of PTEN are not available, and it is not possible to determine whether acute PTEN inactivation can mimic IPC. Genetic manipulations that chronically reduce PTEN activity in the heart result in compensatory changes in signal transduction18 and are not useful for delineating mechanisms of IPC.
Ischemia Induces PTEN Dephosphorylation and Degradation
We have shown that dephosphorylation of PTEN during ischemia precedes its degradation and that the protein phosphatase inhibitor okadaic acid blocks PTEN degradation. These results are consistent with a model in which ischemia-induced dephosphorylation of PTEN is a signal for its degradation. Establishing the identity of the PTEN phosphatase and the molecular mechanisms by which ischemia induces its activity, or inhibits the activity of the PTEN kinase, are important goals for future studies.
MG132 also blocked PTEN degradation in the ischemic heart, demonstrating involvement of the proteasome in this process. The effect of MG132 was so severe that it blocked the recovery of cardiac function after brief ischemia and reperfusion (I-15/R-30). In addition to PTEN, other proapoptotic proteins are degraded by the proteasome, including Bax,30 Bim,31 and oxidized proteins,32 which may affect recovery of cardiac function after I/R. Additional studies are needed to investigate the effects of proteasome inhibitors on cardiac function, especially in light of the fact that clinical trials of the proteasome inhibitor bortezomib in cancer patients are currently underway.33
Reperfusion Induces Oxidation of PTEN
We have demonstrated for the first time that PTEN is oxidized in isolated rat hearts subjected to I-15/R-30 or exposed to perfusate containing H2O2 and that either treatment leads to Akt activation. Studies in cultured cells have shown that PTEN is oxidized and inactivated by H2O2.22,34 ROS generation has been demonstrated in cardiomyocytes and intact hearts during I/R.35eC37 Under physiological conditions, H2O2 is an important signaling molecule that regulates the activity of protein tyrosine phosphatases and PTEN.34 Endogenous ROS signals are sufficient to oxidize a cysteine residue at the active site of PTEN, resulting in PTEN inactivation.19,23,24 It has been reported that ROS mediates heart protection in ischemic and pharmacological preconditioning.38eC40 Our data suggest that ROS play an important role by oxidizing PTEN during IPC. Although oxidation has been shown to inactivate PTEN,22 we do not know whether oxidation is also a signal for PTEN degradation in the postischemic heart, but the dramatic increase in the ratio of oxidized:reduced PTEN without a corresponding increase in the absolute levels of oxidized PTEN suggests that this may be so.
Ischemic Duration Determines PTEN Degradation and IPC Protection
We used I-15/R-30 as an IPC stimulus. Fifteen minutes is the maximal duration of ischemia from which cardiac function can be completely recovered after reperfusion.40 The degree of protection afforded by IPC is related to the duration of ischemia in pig and rat models.3,4 Indeed, one cycle of I-5/R-10 as an IPC is sufficient to induce Akt phosphorylation and reduced infarct size, although 2 cycles induce higher levels of p-Akt and increased protection because infarct size reduction depends on the levels of p-Akt in preconditioned rats.41 Our data demonstrate that the duration of ischemia determines the extent of PTEN degradation, which, in turn determines the extent of Akt activation during reperfusion. As an IPC stimulus, I-15/R-30 has a greater effect on Akt phosphorylation than 3 cycles of I-5/R-10 (Z.C., unpublished data, 2005). IPC reduces cardiac infarct size and cell apoptosis as demonstrated in this and previous studies. Caspase-3 activation leads to cardiac contractile dysfunction and cell death after I/R. Activation of caspase-3 is a critical event linking signals from the endoplasmic reticulum, mitochondria, or plasma membrane with downstream effectors of apoptosis. Akt phosphorylates and inhibits caspase-9, which is the activator of caspase-3. PARP plays an important role in maintaining cellular viability and was the first identified substrate of active caspase-3.42 Thus, our studies establish connections between PTEN, Akt, and caspase-3 and myocardial dysfunction/infarction.
Activated AKT Induces PTEN Reaccumulation During Reperfusion
The existence of an important negative feedback loop is supported by the observation that expression of a constitutively active form of Akt increased the phosphorylation of wild-type PTEN, resulting in PTEN accumulation.21 In U2-OS osteosarcoma cells, mutation of PTEN residues serine 380, threonine 382, and threonine 383 resulted in increased activity and decreased stability of the protein.26 Wortmannin treatment of cultured Jurkat cells decreased the phosphorylation and increased the degradation of PTEN.20,21 In the present study, wortmannin blocked both Akt activation induced by IPC and the subsequent reaccumulation of PTEN after I-15/R-120. Taken together, these data suggest that PI3K, Akt, or a downstream target of these kinases is responsible for the phosphorylation of PTEN. While our manuscript was in preparation, chronic administration of atorvastatin, a stimulator of Akt, was reported to increase PTEN levels in rat hearts and result in the loss of heart protection, leading the investigators to conclude that "detailed studies investigating the importance of PTEN in I/R injury need to be undertaken."43 We have performed a detailed analysis of the regulation of PTEN activity in response to acute I/R that has provided new insight into the rapid and transient nature of IPC. The requirement for only transient activation of Akt in response to physiological stimuli is dramatically demonstrated by the increased proliferation and resistance to apoptosis manifested by human cancers in which Akt is constitutively activated as a result of loss-of-function mutations in the PTEN gene.44
IPC in Other Organs
IPC phenomena have been demonstrated in several other organs in addition to the heart, including the kidney.2 In preliminary studies, we have found that 15 minutes of unilateral renal artery occlusion results in a dramatic reduction in PTEN levels in the ipsilateral (ischemic) as compared with the contralateral (nonischemic) kidney, whereas renal artery occlusion for 15 minutes, followed by 30 minutes of reperfusion, results in a marked increase in p-Akt levels in the ipsilateral kidney (Z.C., unpublished data, 2005). Thus, the same regulatory mechanisms that we have delineated in the heart are likely to be involved in the transient protection of other organs against injury caused by I/R.
Conclusion
PTEN is inactivated by IPC, which contributes to Akt activation and cardiac protection. Extended reperfusion leads to reaccumulation of active PTEN by an Akt-mediated feedback loop, resulting in Akt inactivation and loss of protection. Thus, changes in PTEN activity in response to ischemia and reperfusion may play previously unrecognized but critical roles in the induction and decay of IPC. Additional studies are required to determine whether other preconditioning stimuli also modulate PTEN activity.
Acknowledgments
This work was supported in part by Public Health Service grant P01-HL65608 from the NIH.
This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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Sulis ML, Parsons R. PTEN: from pathology to biology. Trends Cell Biol. 2003; 13: 478eC483.(Zheqing Cai, Gregg L. Sem)
the Departments of Pediatrics, Medicine, Oncology
Radiation Oncology (G.L.S.)
the McKusick-Nathans Institute of Genetic Medicine (Z.C., G.L.S.), The Johns Hopkins University School of Medicine, Baltimore, Md.
Abstract
Ischemic preconditioning (IPC), a brief period of ischemia and reperfusion (I/R), generates profound but transient protection against a subsequent prolonged ischemic episode. The serine-threonine kinase Akt has been shown to mediate IPC, and Akt activation is negatively regulated by the phosphatase PTEN, but whether PTEN activity is modulated by IPC has not been investigated. When isolated, perfused rat hearts were subjected to an IPC stimulus consisting of 15-minute ischemia and 30-minute reperfusion (I-15/R-30), PTEN protein levels and activity were decreased, and levels of phospho-AKT were increased, relative to nonischemic hearts. Hearts subjected to IPC demonstrated improved recovery of cardiac function when subsequently subjected to I-30/R-45 as compared with hearts subjected to I-30/R-45 without prior IPC. When hearts were subjected to I-15 followed by R-30, R-60, or R-120, PTEN reaccumulated gradually and its activity was restored. Phospho-Akt levels at R-120 were decreased and these hearts were no longer protected against injury when subjected to I-30/R-45. Wortmannin administration during reperfusion blocked Akt activation and PTEN reaccumulation. In ischemic hearts, PTEN was rapidly degraded. Pretreatment with proteasome inhibitor MG132 blocked ischemia-induced degradation of PTEN and blocked IPC. Reperfusion following I-15 induced oxidation of the remaining PTEN, leading to Akt activation. Perfusion of H2O2 was sufficient to induce Akt activation. Thus, loss of PTEN activity leads to induction of IPC and feedback mechanisms designed to ensure that Akt activation is transient are responsible for decay of IPC.
Key Words: Akt apoptosis ischemia PTEN
Introduction
Coronary artery disease is a leading cause of death in developed countries. Prolonged ischemia and reperfusion (I/R) results in cardiac cell death and ventricular dysfunction. Protecting the heart against I/R injury may reduce disease mortality. Ischemic preconditioning (IPC) was described by Murry et al, who reported that 4 cycles of 5 minutes of ischemia alternating with 5 minutes of reperfusion limited infarct size by 75% in dog hearts.1 IPC was reproduced in other mammalian species, including humans, and other organs, including kidney and gut.2 IPC peaks after &30 minutes of reperfusion and lasts &2 hours. Both ischemia and reperfusion are required to induce IPC.1,2 In most species, 15 minutes of ischemia (I-15) followed by reperfusion is sufficient to generate protection. In rat and pig models, protection afforded by IPC is related to duration of ischemia.3,4 The molecular events following I/R remain poorly understood.
Experimental evidence implicates the serine/threonine kinase Akt as a mediator of IPC.5eC9 Substrates for phosphorylation by Akt include the proapoptotic proteins Bad and Bax,10 endothelial nitric oxide synthase,11 p70 S6 kinase (p70S6K),12 caspase-9,13 and glycogen synthase kinase-3,14 which are all involved in regulating cell survival. IPC activates Akt and overexpression of Akt protects cardiomyocytes against apoptosis.7,9,15,16 Protein kinase C and extracellular signal-regulated kinases 1 and 2 are also involved in cardiac protection induced by IPC and appear to be downstream targets of PI3K and Akt signaling.5,15,17
Akt is activated by phosphatidylinositol (PI)-3,4,5-triphosphate (PIP3). PI3-kinase (PI3K) catalyzes conversion of PI-4,5-diphosphate to PIP3.18 PTEN (Phosphatase and TENsin homologue deleted from chromosome 10) is a PI3-phosphatase that dephosphorylates PIP3 and antagonizes PI3K activity.18 Studies have shown that PIP3 levels are predominantly determined by PTEN activity, thereby controlling Akt activation.19 PI3K is required for IPC15,16 because of its obligatory role in PIP3 production, but the role of PTEN in IPC has not been studied.
PTEN, which was first identified as a tumor suppressor gene, is an essential regulator of cell proliferation, differentiation, growth, and apoptosis.18,20 PTEN activity is inversely related to cell survival: increased apoptosis was observed in neonatal cardiomyocytes that overexpressed PTEN, whereas expression of mutant PTEN activated Akt, increased cell size, and reduced apoptosis.21
PTEN activity is regulated by oxidation and phosphorylation. A reactive cysteine residue (Cys124) is located at the active site of PTEN. H2O2 oxidizes Cys124 to form a disulfide bond with Cys71, leading to PTEN inactivation.22 PTEN oxidation leads to increased PIP3 levels and Akt activation in cell lines after growth factor stimulation.19,23,24 Under basal conditions, PTEN is phosphorylated and inactive. After dephosphorylation and plasma membrane targeting, PTEN is activated but is degraded rapidly.25,26 Phosphorylation of PTEN increases its stability. Amino acid substitutions that block phosphorylation increase PTEN activity and reduce its half-life.27 Thus, activity and half-life are reciprocally regulated. Isoproterenol stimulation or Akt overexpression resulted in PTEN accumulation,21,28 suggesting a feedback loop that autoregulates Akt activity. In this study, we investigated whether changes in PTEN activity contribute to the induction and decay of IPC.
Materials and Methods
Langendorff Preparation
Isolated heart perfusion was performed as described.29
Immunoprecipitation and PTEN Activity
PTEN was immunoprecipitated from 200 e of heart lysates with 20 e蘈 of anti-PTEN antibody overnight, followed incubation with protein GeCagarose beads for 2 hours at 4°C. Precipitates were washed with lysate buffer, and 1 e of PIP3 and 20 e蘈 of assay buffer were added. The enzyme reaction was terminated by adding Malachite Green solution and absorbance was detected at 600 nm. Released phosphate was determined relative to a standard curve.
Analysis of Oxidized and Reduced PTEN
After exposure to H2O2 or I/R, isolated rat hearts were frozen in eC80°C and homogenized in cell lysate buffer containing iodoacetamide (50 mmol/L). The supernatant was fractionated by 10% nonreducing SDS-PAGE, followed by PTEN immunoblot assay.
An expanded Materials and Methods section is provided in the online data supplement available at http://circres.ahajournals.org.
Results
IPC Is Dependent on PI3K-Akt Signaling
We have previously shown that loss of left ventricular (LV) developed pressure (LVDP) closely parallels infarct size in isolated perfused rat hearts subjected to I/R.29 When hearts were subjected to 15-minute ischemia and 30-minute reperfusion (I-15/R-30) as an IPC stimulus followed by prolonged I/R (I-30/R-45), the recovery of LVDP was improved compared with hearts subjected to I-30/R-45 without IPC (LVDPIPC versus LVDPI/R, 77±6 versus 15±5 mm Hg, P<0.001; Figure 1A). I-15/R-30 significantly increased levels of active, phosphorylated Akt (p-AktIPC versus p-AktNIS, 179±18 versus 29±11, P<0.001, where NIS indicates nonischemic hearts; Figure 1B and 1C). The activation of Akt was also demonstrated by the phosphorylation of a known Akt substrate, p70 S6 kinase (p-p70IPC versus p-p70NIS, 178±7 versus 1±0, P<0.001; Figure 1B and 1D). The increased levels of phosphorylated Akt and p70 were not associated with changes in the total amount of Akt or p70 protein (Figure 1B and data not shown). When hearts were subjected to I-15/R-30 in the presence of 1 eol/L wortmannin to inhibit PI3K activity, IPC-induced phosphorylation of Akt and p70 was completely blocked (Figure 1B through 1D) and protective effects of IPC on recovery of LVDP after prolonged ischemia were lost (Figure 1A). These results demonstrate that this model of IPC is dependent on PI3K and Akt signaling as previously described.1eC9
IPC Downregulates PTEN Activity
We determined PTEN activity and protein by phosphatase and immunoblot assays, respectively, with aliquots of the same lysates used to analyze Akt. Compared with nonischemic hearts, I-15/R-30 significantly decreased PTEN activity (PTENNIS versus PTENIPC, 419±56 versus 136±8, P<0.01; Figure 2A) and protein levels (PTENNIS versus PTENIPC, 204±7 versus 37±12, P<0.001; Figure 2B). Wortmannin (WT) decreased PTEN levels (PTENIPC versus PTENIPC+WT, 52±5 versus 24±1 P<0.01) but increased PTEN activity (PTENIPC versus PTENIPC+WT, 135±8 versus 696±53, P<0.01; Figure 1A and 1B). Thus, an IPC stimulus, which increases Akt activity and promotes functional recovery in isolated rat hearts subjected to I/R, downregulates PTEN activity.
PTEN Levels Are Restored During Prolonged Reperfusion
PTEN activity was analyzed in nonischemic hearts and hearts subjected to I-15 followed by R-30, R-60, or R-120. PTEN activity recovered as duration of reperfusion increased (Figure 3A). During extended reperfusion, PTEN levels increased (PTENR-120 versus PTENR-30, 109±24 versus 37±12, P=0.02) and p-Akt levels decreased (p-AktR-120 versus p-AktR-30, 79±2 versus 179±18, P<0.001) without changes in total Akt (Figure 3B and 3C). Thus, extended reperfusion after IPC increases PTEN protein levels and activity, leading to the loss of phosphorylated (activated) Akt.
Decay of Protection During Prolonged Reperfusion
To characterize the effects of extended reperfusion after IPC on heart protection against I/R injury, we exposed hearts to I-30/R-45 directly or following I-15/R-30 or I-15/R-120. Continuously perfused (nonischemic) hearts were used as controls. Recovery of cardiac function was significantly improved in hearts subjected to I-30/R-45 following the IPC stimulus as compared with hearts subjected to I-30/R-45 in the absence of prior IPC (LVDPR-30 versus LVDPI/R, 78±9 versus 15±4 mm Hg, P<0.001; Figure 4A). When reperfusion was extended to 120 minutes, protection was lost (LVDPR-120 versus LVDPR-30, 28±5 versus 78±9 mm Hg, P<0.01; Figure 4A). IPC (I-15/R-30) reduced LV end-diastolic pressure (LVEDP) after I/R (LVEDPR-30 versus LVEDPI/R, 37±3 versus 77±4 mm Hg, P<0.001) but did not affect heart rate or coronary flow rate (supplementary Figure I). When reperfusion during IPC was extended to 120 minutes (I-15/R-120), heart protection was lost (LVEDPR-30 versus LVEDPR-120, 37±3 versus 64±3 mm Hg, P<0.001; supplementary Figure I).
I-30/R-45 resulted in the cleavage of caspase-3 and poly-(ADP-ribose) polymerase (PARP), 2 markers of apoptosis (Figure 4B through 4D). IPC (I-15/R-30) blocked the cleavage of these proteins, an effect that was lost with prolonged reperfusion (I-15/R-120). Thus, prolonged reperfusion during IPC impairs the protective effect of IPC against cardiac injury after I-30/R-45 and allows PTEN reaccumulation.
Recovery of PTEN During Reperfusion Requires PI3K-Akt Signaling
Because phospho-PTEN (p-PTEN) is inactive and stable, we hypothesized that PTEN reaccumulation is attributable to phosphorylation by Akt. Compared with nonischemic hearts, p-PTEN levels were dramatically reduced after I-15/R-30 but recovered to near baseline levels after I-15/R-120 (Figure 5A). When wortmannin was infused at R-15 for 15 minutes to block PI3K activity, Akt phosphorylation was inhibited and p-PTEN levels were decreased (Figure 5B). PTEN reaccumulation at R-30 and R-120 was blocked (PTENR-30 versus PTENR-30+WT, 49±10 versus 12±1, P<0.01; PTENR-120 versus PTENR-120+WT, 98±8 versus 11±5, P<0.01; Figure 5C). Thus, PI3K-Akt signaling is required for phosphorylation and stabilization of PTEN during extended reperfusion. PTEN antibodies coimmunoprecipitated Akt (Figure 5D), suggesting that Akt may directly phosphorylate PTEN.
PTEN Degradation Requires Phosphatase and Proteasome Activity
PTEN levels were markedly decreased after I-15/R-30, indicating that ischemia induces PTEN degradation. To determine the kinetics of degradation, hearts were subjected to ischemia of increasing duration (from 0 to 30 minutes) without reperfusion. PTEN was rapidly degraded in the ischemic hearts, with a half-life of 5.4 minutes, whereas -actin levels were unchanged (Figure 6A). After 30 minutes of ischemia PTEN enzymatic activity was significantly decreased compared with nonischemic hearts (PTENNIS versus PTENISC, 327±36 versus 31±27, P<0.01; Figure 6B). We hypothesized that ischemia-induced dephosphorylation of PTEN targets the protein for degradation. If so, then dephosphorylation should precede degradation. Compared with nonischemic hearts, the levels of phosphorylated PTEN in hearts subjected to 5 minutes of ischemia were reduced by &50%, whereas the levels of total PTEN were reduced by only &10% (Figure 6C).
To determine whether dephosphorylation of PTEN is required for degradation, we pretreated hearts for 15 minutes with the protein phosphatase PP2A inhibitor okadaic acid, then subjected them to I-15. Compared with hearts treated with vehicle only, 40 nmol/L okadaic acid blocked PTEN dephosphorylation and degradation in the ischemic heart, whereas 4 nmol/L okadaic acid was ineffective (Figure 6D). Thus, PTEN degradation requires dephosphorylation that is mediated by PP2A or another okadaic acideCsensitive phosphatase.
To investigate whether rapid degradation of PTEN is mediated by the proteasome, we infused the proteasome inhibitor MG132 at the various concentrations (0 to 4 eol/L) for 15 minutes, followed by I-15. MG132 blocked the ischemia-induced degradation of PTEN in a concentration-dependent manner (Figure 7A). At the lowest concentration tested (1 eol/L), MG132 partially inhibited PTEN degradation (PTENISC+MG versus PTENISC, 47±2 versus 28±2, P<0.01; Figure 7B) but did not block the reduction in p-PTEN levels (p-PTENISC+MG versus p-PTENISC, 20±1 versus 44±3, P<0.01; Figure 7C).
To determine whether PTEN degradation is required for IPC-induced protection, hearts were pretreated with MG132 (0, 2, 4 eol/L) and then subjected to IPC (I-15/R-30). Hearts pretreated with vehicle only and subjected to I-15/R-30 (MG0) showed almost complete recovery of cardiac function (Figure 7D). In contrast, pretreatment with MG132 at 2 eol/L (MG2) significantly inhibited the recovery of cardiac function (LVDPMG0 versus LVDPMG2, 101±7 versus 32±14 mm Hg, P=0.01) and completely blocked recovery at 4 eol/L.
Ischemia Reperfusion Induces PTEN Oxidation
Although ischemia induced the degradation of PTEN, the kinetics of degradation indicated that not all PTEN protein was degraded after I-15. We hypothesized that residual PTEN protein was inactivated by oxidation during reperfusion. To analyze PTEN oxidation, hearts were subjected to I-15 followed by R-0, 1, 5, 10, 15, or 30 and homogenates were exposed to 50 mmol/L iodoacetamide, an alkylating agent that attacks free thiols. The reduced and oxidized forms of PTEN were fractionated by nonreducing SDS-PAGE and identified by immunoblot assay. Reperfusion after I-15 resulted in decreased levels of reduced (active) PTEN, compared with the nonischemic heart (Figure 8A). The proportion of oxidized PTEN increased rapidly after reperfusion (Figure 8B). Comparison of the kinetics of PTEN oxidation, Akt phosphorylation, and PTEN dephosphorylation revealed 2 important findings. First, PTEN oxidation was already maximal at R-1 (31 minutes in Figure 8B) and thus preceded the increase in Akt phosphorylation at R-5 (35 minutes in Figure 8B). Second, although levels of p-PTEN decreased during ischemia, the increase in Akt phosphorylation from R-1 to R-5 was not associated with any further loss of PTEN phosphorylation. These data are consistent with a model in which oxidation of residual PTEN during early reperfusion leads to Akt phosphorylation.
The data presented in Figure 8B indicate that a brief period of reperfusion is sufficient to inactivate PTEN and lead to Akt activation. To determine whether Akt phosphorylation is determined by duration of ischemia, hearts were subjected to ischemia of increasing duration (5 to 20 minutes), followed by 5-minute reperfusion. The decrease in PTEN levels was correlated with the duration of ischemia (Figure 8C). Akt phosphorylation at R-5 was dependent on the duration of ischemia and inversely correlated with PTEN levels.
The oxidation of PTEN during early reperfusion is consistent with the increase in reactive oxygen species (ROS) that occurs during this process. To determine whether ROS are sufficient to induce PTEN oxidation and Akt phosphorylation, hearts were perfused with buffer containing H2O2 (0 to 10 eol/L) for 15 minutes. H2O2 treatment increased the levels of oxidized PTEN and decreased the levels of reduced PTEN in a concentration-dependant manner (Figure 8D). Most importantly, Akt phosphorylation was also increased by H2O2 treatment in a concentration-dependant manner, whereas the levels of total Akt, p-PTEN, and total PTEN were unchanged (Figure 8D). Finally, the induction of p-AKT by I-15/R-5 or I-15/R-30 was markedly reduced when hearts were perfused with 2-mercaptoethanol to block ROS signaling (Figure 8E).
Discussion
Two major conclusions can be drawn from the data presented above. First, IPC induces downregulation of PTEN activity through the combined effects of dephosphorylation/proteasomal degradation during ischemia and oxidative inactivation of residual PTEN during reperfusion, leading to Akt activation and cardiac protection. Second, extended reperfusion induces PTEN reaccumulation and reactivation by a PI3K/Akt-mediated feedback mechanism, resulting in Akt inactivation and loss of protection, thus providing for the first time a molecular basis for the transient nature of IPC.
IPC Stimulus Induces PTEN Inactivation by Dual Mechanisms
Our data indicate that I-15/R-30 induces loss of PTEN activity, which leads to reduced dephosphorylation of PIP3, increased Akt activation, and protection against injury following subsequent prolonged I/R. PIP3 levels are determined by the opposing activities of PI3K and PTEN. We focused on the inactivation of PTEN in this study; it is possible that both PI3K activation and PTEN inactivation increase PIP3 levels during IPC. The absence of active PTEN during early reperfusion allows unopposed PI3K activity and PIP3 accumulation, which is necessary for Akt activation. Additional studies are required to determine whether changes in the activity of other signaling proteins occur as a result of ischemia-reperfusion and are necessary for Akt activation. However, the striking modulation of PTEN activity we have demonstrated represents a paradigm shift in a field where attention has been focused almost entirely on Akt activation by PI3K signaling. The results presented above demonstrate that PTEN activity is modulated by rapid dephosphorylation and proteasomal degradation during ischemia followed by oxidation and inactivation of residual PTEN protein during reperfusion.
Several studies using cultured cells have provided evidence that PTEN plays a critical role in controlling PIP3 levels and Akt activity. In PTEN null cells, Akt could not be activated by H2O2 treatment, but when PTEN was reintroduced into the cells, H2O2 treatment resulted in increased PIP3 levels and Akt activation.23 Insulin-mediated Akt activation required a decrease in PTEN activity, and increased PI3K activity alone was not sufficient to activate Akt in human neuroblastoma cells.19 Exposure of cultured cells to insulin, epidermal growth factor, and platelet-derived growth factor was associated with inactivation of PTEN by reversible oxidation of cysteine residues in its catalytic site.24 A major experimental limitation at present is that, unlike PI3K, specific small molecule inhibitors of PTEN are not available, and it is not possible to determine whether acute PTEN inactivation can mimic IPC. Genetic manipulations that chronically reduce PTEN activity in the heart result in compensatory changes in signal transduction18 and are not useful for delineating mechanisms of IPC.
Ischemia Induces PTEN Dephosphorylation and Degradation
We have shown that dephosphorylation of PTEN during ischemia precedes its degradation and that the protein phosphatase inhibitor okadaic acid blocks PTEN degradation. These results are consistent with a model in which ischemia-induced dephosphorylation of PTEN is a signal for its degradation. Establishing the identity of the PTEN phosphatase and the molecular mechanisms by which ischemia induces its activity, or inhibits the activity of the PTEN kinase, are important goals for future studies.
MG132 also blocked PTEN degradation in the ischemic heart, demonstrating involvement of the proteasome in this process. The effect of MG132 was so severe that it blocked the recovery of cardiac function after brief ischemia and reperfusion (I-15/R-30). In addition to PTEN, other proapoptotic proteins are degraded by the proteasome, including Bax,30 Bim,31 and oxidized proteins,32 which may affect recovery of cardiac function after I/R. Additional studies are needed to investigate the effects of proteasome inhibitors on cardiac function, especially in light of the fact that clinical trials of the proteasome inhibitor bortezomib in cancer patients are currently underway.33
Reperfusion Induces Oxidation of PTEN
We have demonstrated for the first time that PTEN is oxidized in isolated rat hearts subjected to I-15/R-30 or exposed to perfusate containing H2O2 and that either treatment leads to Akt activation. Studies in cultured cells have shown that PTEN is oxidized and inactivated by H2O2.22,34 ROS generation has been demonstrated in cardiomyocytes and intact hearts during I/R.35eC37 Under physiological conditions, H2O2 is an important signaling molecule that regulates the activity of protein tyrosine phosphatases and PTEN.34 Endogenous ROS signals are sufficient to oxidize a cysteine residue at the active site of PTEN, resulting in PTEN inactivation.19,23,24 It has been reported that ROS mediates heart protection in ischemic and pharmacological preconditioning.38eC40 Our data suggest that ROS play an important role by oxidizing PTEN during IPC. Although oxidation has been shown to inactivate PTEN,22 we do not know whether oxidation is also a signal for PTEN degradation in the postischemic heart, but the dramatic increase in the ratio of oxidized:reduced PTEN without a corresponding increase in the absolute levels of oxidized PTEN suggests that this may be so.
Ischemic Duration Determines PTEN Degradation and IPC Protection
We used I-15/R-30 as an IPC stimulus. Fifteen minutes is the maximal duration of ischemia from which cardiac function can be completely recovered after reperfusion.40 The degree of protection afforded by IPC is related to the duration of ischemia in pig and rat models.3,4 Indeed, one cycle of I-5/R-10 as an IPC is sufficient to induce Akt phosphorylation and reduced infarct size, although 2 cycles induce higher levels of p-Akt and increased protection because infarct size reduction depends on the levels of p-Akt in preconditioned rats.41 Our data demonstrate that the duration of ischemia determines the extent of PTEN degradation, which, in turn determines the extent of Akt activation during reperfusion. As an IPC stimulus, I-15/R-30 has a greater effect on Akt phosphorylation than 3 cycles of I-5/R-10 (Z.C., unpublished data, 2005). IPC reduces cardiac infarct size and cell apoptosis as demonstrated in this and previous studies. Caspase-3 activation leads to cardiac contractile dysfunction and cell death after I/R. Activation of caspase-3 is a critical event linking signals from the endoplasmic reticulum, mitochondria, or plasma membrane with downstream effectors of apoptosis. Akt phosphorylates and inhibits caspase-9, which is the activator of caspase-3. PARP plays an important role in maintaining cellular viability and was the first identified substrate of active caspase-3.42 Thus, our studies establish connections between PTEN, Akt, and caspase-3 and myocardial dysfunction/infarction.
Activated AKT Induces PTEN Reaccumulation During Reperfusion
The existence of an important negative feedback loop is supported by the observation that expression of a constitutively active form of Akt increased the phosphorylation of wild-type PTEN, resulting in PTEN accumulation.21 In U2-OS osteosarcoma cells, mutation of PTEN residues serine 380, threonine 382, and threonine 383 resulted in increased activity and decreased stability of the protein.26 Wortmannin treatment of cultured Jurkat cells decreased the phosphorylation and increased the degradation of PTEN.20,21 In the present study, wortmannin blocked both Akt activation induced by IPC and the subsequent reaccumulation of PTEN after I-15/R-120. Taken together, these data suggest that PI3K, Akt, or a downstream target of these kinases is responsible for the phosphorylation of PTEN. While our manuscript was in preparation, chronic administration of atorvastatin, a stimulator of Akt, was reported to increase PTEN levels in rat hearts and result in the loss of heart protection, leading the investigators to conclude that "detailed studies investigating the importance of PTEN in I/R injury need to be undertaken."43 We have performed a detailed analysis of the regulation of PTEN activity in response to acute I/R that has provided new insight into the rapid and transient nature of IPC. The requirement for only transient activation of Akt in response to physiological stimuli is dramatically demonstrated by the increased proliferation and resistance to apoptosis manifested by human cancers in which Akt is constitutively activated as a result of loss-of-function mutations in the PTEN gene.44
IPC in Other Organs
IPC phenomena have been demonstrated in several other organs in addition to the heart, including the kidney.2 In preliminary studies, we have found that 15 minutes of unilateral renal artery occlusion results in a dramatic reduction in PTEN levels in the ipsilateral (ischemic) as compared with the contralateral (nonischemic) kidney, whereas renal artery occlusion for 15 minutes, followed by 30 minutes of reperfusion, results in a marked increase in p-Akt levels in the ipsilateral kidney (Z.C., unpublished data, 2005). Thus, the same regulatory mechanisms that we have delineated in the heart are likely to be involved in the transient protection of other organs against injury caused by I/R.
Conclusion
PTEN is inactivated by IPC, which contributes to Akt activation and cardiac protection. Extended reperfusion leads to reaccumulation of active PTEN by an Akt-mediated feedback loop, resulting in Akt inactivation and loss of protection. Thus, changes in PTEN activity in response to ischemia and reperfusion may play previously unrecognized but critical roles in the induction and decay of IPC. Additional studies are required to determine whether other preconditioning stimuli also modulate PTEN activity.
Acknowledgments
This work was supported in part by Public Health Service grant P01-HL65608 from the NIH.
This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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