Regulation of the Proapoptotic Factor FOXO1 (FKHR) in Cardiomyocytes by Growth Factors and 1-Adrenergic Agonists
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《内分泌学杂志》
Cellular Biochemistry Laboratory, Baker Heart Research Institute, Melbourne 3004, Victoria, Australia
Abstract
Apoptotic responses in cardiomyocytes are opposed by the protein kinase Akt (protein kinase B) and thus can be suppressed by a number of growth factors and cytokines. In some cell types, Akt phosphorylates and inactivates members of the forkhead box (FOXO) family of transcription factors that are active in regulating the expression of proapoptotic cytokines and signaling intermediates. In the current study, we investigated the possibility that FOXO1 (FKHR) was expressed, regulated, and functional in cardiomyocytes. Addition of epidermal growth factor (EGF) (10 nM) to neonatal rat cardiomyocytes caused rapid phosphorylation of Akt and slower FOXO1 phosphorylation. In contrast, the 1-adrenergic receptor agonist phenylephrine (50 μM) did not phosphorylate Akt and caused dephosphorylation of FOXO1 acutely and increased FOXO1 expression with chronic exposure. Phenylephrine, but not EGF, caused nuclear translocation of FOXO1, a response that is associated with dephosphorylation. Overexpression of FOXO1 activated transcription of the proapoptotic cytokine, TNF-related apoptosis-inducing ligand, as indicated by reporter gene activity. This response was enhanced by phenylephrine and inhibited by EGF. FOXO1 is expressed, regulated, and functionally active in cardiomyocytes and thus may contribute to apoptotic responses in heart.
Introduction
BECAUSE CARDIOMYOCYTES HAVE only limited ability to regenerate, cell death by apoptosis or oncosis is especially problematic in heart. As in other cell types, apoptosis of cardiomyocytes can be mediated via a number of different but interacting pathways. These include the death receptor pathways initiated by Fas or TNF receptors (1), whereby the activated cytokine receptors bind and activate death effector proteins that subsequently activate caspases and the downstream effector pathways (2). Another pathway involves activation of the proapoptotic Bcl family member BAD whose activation causes loss of mitochondrial membrane integrity, cytochrome c release, and subsequent caspase activation (3). In addition, activation of inositol-1,4,5-trisphosphate IP3 receptors and subsequent perturbations in Ca2+ may also contribute to overall apoptotic responses (4). Although modest activation of Gq-coupled receptors, such as 1-adrenerigc receptors, causes cardiomyocyte hypertrophy both in vitro and in cardiomyocyte models, substantially heightened activity causes mitochondrial damage and apoptosis. This response is apparently associated with increased expression of the proapoptotic factor Nix (5, 6) and with reductions in phosphatidylinositol (PI) 3-kinase activity (3).
In cardiomyocytes, as in other cell types, apoptotic responses are opposed by the protein kinase Akt (protein kinase B) (7). Akt phosphorylates BAD, caspase 9, and nuclear factor-B (NF-B), and these phosphorylations can account for much of the protective action of Akt against cell destruction (8, 9). However, in a number of cell types, another protective pathway initiated by Akt has been described. Akt has been shown to phosphorylate and inactivate members of the forkhead (FOXO) family of transcription factors (10, 11). In their phosphorylated form, these factors are tethered in the cytoplasm, but activation by dephosphorylation permits nuclear translocation and activation of responsive genes. Included among these FOXO-responsive genes are some that encode apoptotic effectors including TNF and Fas-ligand (12, 13) and more recently the proapoptotic factor Bim (14). Thus, in addition to its effect on immediate apoptotic signaling pathways via suppression of caspase and BAD activities, Akt also has the potential to reduce the generation of proapoptotic cytokines and thus to cause a sustained, secondary inhibition of apoptotic responses.
Expression of the proapoptotic cytokines, TNF and Fas-ligand, increases in cardiomyocytes under pathological conditions such as ischemia and reperfusion (15, 16). Animals in which one or another of these genes has been silenced show reduced apoptosis, smaller infarct, and improved functional recovery after ischemic insult (17, 18). It is also known that activation of Akt, either by overexpressing a constitutively active mutant (7) or by adding known activators of Akt (19, 20), improves functional recovery, reduces infarct size, and decreases apoptosis after ischemia and reperfusion. Akt is regulated by PI 3-kinase and phospholipid-dependent kinase 1 and is thus directly controlled by receptors for many growth factors and cytokines, including epidermal growth factor (EGF) family members, insulin, and IGF-I as well as the GP-130 ligands cardiotropin-1 and leukemia inhibitory factor (21). All of these factors have been shown to offer protection when applied during reperfusion after an acute ischemic episode (19, 20). Much of the benefit provided by Akt may be explained by inhibition of BAD and caspase 9, but the possibility of other Akt substrates protecting from cardiomyocyte apoptosis has not been exhaustively addressed. In the current study, we investigated the possibility that members of the FOXO family of transcription factors are expressed in the myocardium, that their activity is regulated, and that they are transcriptionally active.
Materials and Methods
Culture of neonatal cardiomyocytes
Neonatal rat ventricular myocyte cultures (NRVM) were prepared from 1- to 3-d-old Sprague Dawley rat pups as previously described (22). Cells were preplated twice for 30 min each to remove nonmyocytes and left to attach for 18 h in DMEM, 10% fetal calf serum, 0.1 mM bromodeoxyuridine (BrdU), 50 U/ml penicillin G and 50 μg/ml streptomycin sulfate onto uncoated dishes. Medium was then replaced with a defined serum-free medium consisting of DMEM, 10 μg/ml human insulin, 10 μg/ml bovine apo-transferrin, 0.1 mM BrdU, 50 U/ml penicillin G, 50 μg/ml streptomycin sulfate, and 125 μg/ml fungizone. BrdU was omitted after 3 d. All experiments were approved by the Alfred Monash Research and Education Precinct Animal Ethics Committee, approval no. 0296, and all procedures adhered to the National Health and Medical Research Council code for the care and use of animals for medical research.
FOXO1 phosphorylation
NRVM plated on 9-cm dishes were washed twice with ice-cold PBS and scraped in 100 μl ice-cold lysis buffer (pH 7.7) containing the following constituents (in mM): 50 Tris-HCl, 100 NaCl, 2 EDTA, 2 EGTA, 10 sodium pyrophosphate, 1 sodium orthovanadate, 1 dithiothreitol, 1 phenylmethylsulfonyl fluoride, and 10 NaF, together with 5 μM okadaic acid, 1 μM pepstatin A, 5 μg/ml aprotinin, 10 μg/ml leupeptin, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, and 1% Triton X-100 (23). Lysates were cleared by centrifugation and protein content of the supernatants measured (24). Proteins from whole-cell lysates (150–300 μg protein) were separated by 10% SDS-PAGE (25) and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Membranes were stained with Ponceau-S (Sigma Chemical Co., St. Louis, MO) and probed overnight at 4 C with anti-phospho-Ser256-FOXO1 antibodies (1:2000). Secondary antibodies were diluted in blocking solution (Cell Signaling, Beverly, MA) and blots incubated for 1 h, according to the manufacturer’s instructions. Enhanced chemiluminescence detection was carried out according to the manufacturer’s instructions (Amersham Biosciences, Buckinghamshire, UK). Blots were subsequently stripped and reprobed with antibodies to total FOXO1 (1:2000).
Preparation of nuclear and cytoplasmic fractions from NRVM
NRVM plated on 9-cm dishes were stimulated as indicated, and the medium was removed. Cells were detached from the plates using trypsin-EDTA and the trypsin neutralized by adding 8 ml DMEM-10% fetal calf serum. Detached cells were harvested by low-speed (500 rpm) centrifugation and washed gently with PBS, and 100 ml of nuclear isolation buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40 was then added to the cell pellets. The mixture was vortexed gently for 20 sec, followed by 10 min incubation on ice. Lysed cells were centrifuged at 500 rpm at 4 C to pellet the nuclei, and the supernatants were removed. The nuclear pellets were washed once with nuclear isolation buffer without the Nonidet P-40.
Transient transfection and reporter gene activity
Transfection experiments were performed in triplicate, using cardiomyocytes (400/mm2) on 35-mm wells 1 d after isolation. Transient transfection, using a total of 4.8 μg DNA per well, was performed by the calcium phosphate method (23, 26). Cytosolic fractions were harvested into lysis buffer containing 0.1 M K2HPO4, 1% Triton X-100, and 1 mM dithiothreitol (pH 7.9). Luciferase activity was measured in 100 mM tricine, 10 mM MgSO4, 2 mM EDTA, 2 mM ATP, and 75 μM luciferin (pH 7.8) for 2 min using a Lumat LB9507 luminometer (23, 27) (Berthold Instruments, Melbourne, Australia).
Materials
Antibodies were from the following sources: total FOXO1 from Santa Cruz Biotechnology (Santa Cruz, CA) and P-Ser356-FOXO1, P-Ser473-Akt, total Akt, P-Ser9-GSK3, and total GSK3 from Cell Signaling. The pGL3-TRAIL-luciferase plasmid was provided by Dr. S. Ghaffari (Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY) and has been described previously (27). Plasmids encoding wild-type FOXO1 (FOXO1-WT) and FOXO1–3A were obtained from Dr. E. Tang (Department of Biological Chemistry, University of Pennsylvania, Philadelphia, PA) and were prepared as described elsewhere (10). The myr-Akt plasmid was provided by Dr. Richard Pearson, Peter MacCallum Cancer Research Institute (Melbourne, Australia).
Results
EGF phosphorylates FOXO1 in NRVM, but phenylephrine reduces phosphorylation
EGF reduces apoptotic responses in NRVM via Akt-dependent mechanisms (28). In some cell types, protection from apoptosis involves members of the FOXO family of transcription factors that are phosphorylated and deactivated by Akt (10) (29). In the current studies, we investigated the possibility that FOXO1 was expressed, regulated, and functional in NRVM. NRVM were treated with EGF for 30 min, and cell lysates were prepared, subjected to SDS-PAGE, and blotted with antibodies to phospho-Ser356-FOXO1 and total FOXO1. As shown in Fig. 1 (top left), EGF caused a time-dependent phosphorylation of FOXO1. Phosphorylation was maximal at 30 min, and no further increase was seen after this (data not shown). In marked contrast, activation of 1-adrenergic receptors by addition of 50 μM phenylephrine (together with 1 μM propranolol to block -adrenergic receptors) caused a decrease in FOXO1 phosphorylation over 30 min (Fig. 1, top) even though we have shown previously that phenylephrine transactivates EGF receptors (EGFR) (23).
EGF, but not phenylephrine, activates Akt
We next examined the phosphorylation of Akt in response to EGF and phenylephrine, because Akt has been shown to phosphorylate FOXO1 (10). NRVM were treated with 10 nM EGF or 50 μM phenylephrine (plus 1 μM propranolol) for times up to 30 min. Extracts were prepared, subjected to SDS-PAGE, transferred, and blotted with antibodies to phosphorylated Akt (phospho-Ser473-Akt). As shown in Fig. 1, EGF caused phosphorylation and activation of Akt with a maximal response at 1–3 min. Phenylephrine did not cause Akt phosphorylation over the 30-min period studied. Akt phosphorylation by EGF preceded that of FOXO1, suggesting the possibility that Akt mediated the phosphorylation of FOXO1 by EGF. Blotting with antibodies to phospho-Thr308-Akt produced similar results (data not shown). In contrast to Akt, both EGF and phenylephrine caused phosphorylation of glycogen synthase kinase-3 (GSK3) on Ser9 (Fig. 1).
Phosphorylation of FOXO1 by EGF was inhibited by 10 μM LY294002 and by 5 μM AG-1478 (Fig. 2, bottom), indicating involvement of both PI 3-kinase and the EGFR kinase in this response. The EGF-induced phosphorylation of Akt was similarly inhibited LY294002 and by AG-1478. Thus, EGF phosphorylation of FOXO1 involves both the EGFR kinase activity and PI 3-kinase and very likely is mediated via Akt. The basal level of FOXO1 phosphorylation was reduced by LY294002 but not by AG-1478, implying an involvement of PI 3-kinase but not EGFR in the maintenance of FOXO1 under unstimulated conditions (Fig. 2).
Chronic exposure to phenylephrine increased FOXO1 expression
NRVM were treated with 50 μM phenylephrine (plus 1 μM propranolol) or with 10 nM EGF for 24 h and effects on FOXO1 expression and phosphorylation examined. Chronic exposure to phenylephrine increased the expression of FOXO1, while reducing phosphorylation levels, thereby substantially increasing the content of potentially active dephosphorylated FOXO1 (Fig. 3). Treatment with EGF for 24 h, in contrast, did not cause major changes in FOXO1 expression or phosphorylation (data not shown), possibly because EGF-induced Akt phosphorylation is transient, as shown in Fig. 1. Chronic activation of Akt was achieved by overexpressing a constitutively active (myristylated) Akt (myr-Akt) using an adenoviral vector. After 24 h infected cells expressed heightened levels of active Akt and reduced levels of FOXO1. These findings agree with previous studies (30, 31) demonstrating that degradation of FOXO1 follows phosphorylation.
Phenylephrine causes nuclear translocation of FOXO1, but EGF does not
Activation of FOXO1 by dephosphorylation is associated with increased nuclear retention (32, 33). NRVM were treated with 50 μM phenylephrine (plus 1 μM propranolol) or 10 nM EGF for 2 h. Cells were lysed, nuclear and cytoplasmic fractions prepared, and the content of FOXO1 assessed by Western blotting with anti-FOXO1 antibodies. Phenylephrine caused a reduction in FOXO1 content in the cytoplasm together with an appearance of FOXO1 in the nuclear fraction. EGF did not cause observable changes in FOXO1 distribution (Fig. 4).
FOXO1-WT activates TNF-related apoptosis-inducing ligand (TRAIL) expression, a response that is inhibited by phosphorylation
In some cell types, FOXO1 activation has been shown to increase transcription of proapoptotic effectors, including TRAIL, TNF, and Fas ligand (13, 27, 34). In the current studies, we investigated whether FOXO1 could increase transcription of sensitive promoters in NRVM and whether this response was modified by phosphorylation. NRVM were transfected with pGL3-TRAIL-luciferase together with FOXO1-WT, as described in the Materials and Methods. After 18 h, 10 nM EGF or 50 μM phenylephrine (plus 1 μM propranolol) was added, cells were harvested, and luciferase activity was measured after another 24 h. Overexpression of FOXO1-WT increased transcription from the TRAIL promoter, as indicated by heightened luciferase activity. Phenylephrine enhanced the response to FOXO1-WT, whereas EGF caused a substantial reduction (Fig. 5A). EGF also reduced TRAIL expression in cells expressing blank vector, implying an effect on endogenous FOXO1. To establish whether the TRAIL response was associated with dephosphorylation in NRVM, we transfected NRVM with a FOXO1 mutant in which the three residues phosphorylated by Akt are mutated to alanines, T24A, S253A, and S316A (FOXO1–3A). Expression of FOXO1–3A increased TRAIL-luciferase expression as shown in Fig. 5A. Neither phenylephrine nor EGF altered the response to FOXO1–3A, showing that phosphorylation is involved in the responses to both stimulatory and inhibitory factors.
In similar experiments, we overexpressed a constitutively active mutant of Akt (myr-Akt) together with FOXO1-WT or FOXO1–3A. Overexpression of myr-Akt reduced responses to FOXO1-WT but not to FOXO1–3A. Thus, FOXO1 can activate TRAIL promoter elements, and this is opposed by Akt phosphorylation or by removal of residues susceptible to Akt phosphorylation.
Discussion
Members of the FOXO family of forkhead transcription factors were defined initially as homologues of the Caenorhabditis elegans transcription factor DAF16. The genes for three mammalian homologues of DAF16, FKHR (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4) were first identified as chromosomal breakpoints in human tumors, implying a relationship to cell growth and survival. In various cell types, activation of FOXO family members by dephosphorylation leads to cell cycle arrest and quiescence (35), alterations in energy metabolism (36), and apoptosis (27, 29). In differentiated cells, dephosphorylated forms of FOXO proteins have been shown to directly activate transcription of genes encoding proapoptotic effectors such as TNF, TRAIL, Fas-ligand (27, 13), and more recently the proapoptotic factor Bim (14). FOXO family members are regulated by Akt phosphorylation as well as by other kinases. Phosphorylation reduces DNA binding, interferes with interactions with other transcription factors, and causes nuclear exclusion and tethering in the cytoplasm by binding to 14-3-3 proteins (12, 33, 37). In some cell types, phosphorylation of FOXO proteins has been shown to precede ubiquitinization and degradation (31), pointing to complex overall regulation.
Although there is now a considerable body of knowledge about the functioning of these proteins in various mammalian cells, their possible involvement in the myocardium has only recently been investigated (38). In the current study, EGF phosphorylated FOXO1 in NRVM by a mechanism that apparently involved PI 3-kinase as well as the kinase activity of the EGFR. In contrast, treatment with phenylephrine dephosphorylated FOXO1, even though we have recently shown that phenylephrine transactivates EGFR (Fig. 1) (23). Unlike EGF, phenylephrine does not activate Akt, presumably reflecting a lack of PI 3-kinase activation (Fig. 1) (3). The degree of phosphorylation of EGFR after phenylephrine stimulation is much lower than for EGF, and it is likely that failure to activate PI 3-kinase and Akt reflects this difference in the degree of phosphorylation. It is also possible, however, that there are qualitative differences in signaling from the EGFR depending on whether it is activated by EGF itself or by a transactivating Gq-coupled receptor. In contrast to findings with EGF, phenylephrine caused dephosphorylation of FOXO1 acutely (Fig. 1) and substantially increased FOXO1 expression and the relative amount of dephosphorylated FOXO1 in the longer term (Fig. 3), implying that phenylephrine is an activator of FOXO1 in cardiomyocytes. Enhanced expression might be expected if phosphorylation of FOXO1 leads to degradation. This finding thus implies that FOXO1 is constantly being synthesized and degraded in cardiomyocytes. The mechanism of dephosphorylation of FOXO1 by phenylephrine remains to be established but possibly involves phosphorylation, and thus inactivation, of GSK3. Unlike Akt, phosphorylation of GSK3 at Ser9 is inhibitory rather than stimulatory and so phenylephrine might dephosphorylate FOXO1 by reducing GSK3-induced phosphorylation. EGF also phosphorylates GSK3, but the stimulatory phosphorylation of Akt by EGF possibly overrides any inhibitory effect caused by phosphorylating GSK3. Thus, phosphorylating GSK3 without simultaneous phosphorylation of Akt is a possible mechanism by which Gq-coupled receptors could dephosphorylate FOXO1. However, it is more than possible that other mechanisms are involved. Although acute stimulation of Gq-coupled receptors is not generally associated with apoptotic responses in cardiomyocytes, sustained overactivity of Gq does cause apoptosis both in cell models and in the myocardium in vivo (39). Gq-mediated apoptosis is associated with activation of Nix and loss of mitochondrial integrity (40), but we suggest dephosphorylation of FOXO family members may also make a contribution to the overall cell death response.
Phenylephrine caused nuclear translocation of FOXO1, whereas EGF did not, as expected from the phosphorylation data (Fig. 4). In agreement with this, phenylephrine enhanced transcriptional responses to overexpressed FOXO1, measured as TRAIL promoter activation (Fig. 5). EGF, in contrast, was strongly inhibitory to TRAIL promoter activation by overexpressed FOXO1 and also reduced basal TRAIL promoter activity (Fig. 5). This implies that EGF can effectively phosphorylate and functionally inactivate endogenous or overexpressed FOXO1. Our data also support the notion that Akt regulates FOXO1 action in cardiomyocytes. First, coexpressing Akt reduced the stimulatory activity of overexpressed FOXO1 on TRAIL promoter activity. Second, FOXO1–3A with the Akt phosphorylation sites mutated was fully active and its activity was not sensitive either to EGF or to overexpressed Akt.
Until recently, FOXO members have not been identified in cardiomyocytes, but a recent study has shown that FOXO1, FOXO3a, and FOXO4 are all expressed in NRVM. Furthermore, FOXO3a was shown to inhibit hypertrophic responses in its dephosphorylated, active form via a process involving activation of an atrogene transcriptional program (39). In the current study, we investigated FOXO1 and showed that it too was subject to regulation by phosphorylation/dephosphorylation in NRVM. Factors that activate Akt are known to reduce cardiomyocyte apoptosis and are well-established cardioprotective agents, at least in prevention of ischemic injury (41). Thus, overexpression of Akt or PI 3-kinase reduces ischemic injury, and similar results have been reported after addition of EGF, neuregulin, insulin, IGF-I, or cardiotropin-1, all of which activate Akt (19, 20, 41, 42). Akt has a number of important substrates other than FOXO1, including BAD, caspase 8, and nuclear factor B, all of which may contribute to its antiapoptotic effectiveness (21). However, ischemia and reperfusion cause increased expression of TNF, Fas-ligand, and TRAIL, all of which are regulated by FOXO transcription factors, and diminishing the generation of these cytokines reduces apoptosis and improves functional recovery (15, 43). It therefore seems possible that FOXO1 contributes to apoptosis in the myocardium by increasing expression of proapoptotic effectors and that inhibiting this response is an important part of the protective effect of Akt in the heart. In support of this, there is suggestive evidence for a role for FOXO1 family members in ischemia-induced apoptosis in other cell types. Cerebral ischemia has been shown to be associated with activated, dephosphorylated FOXO1 that paralleled the deactivation of Akt (11, 44). Cell survival after renal ischemia/reperfusion requires phosphorylation and deactivation of both FOXO1 and FOXO3a (45).
Our studies show that, in NRVM, FOXO1 is regulated acutely by phosphorylation and dephosphorylation and chronically at the level of protein expression. The finding that FOXO1 activates transcription from a TRAIL promoter suggests a role in regulating the expression of proapoptotic cytokines in the myocardium. Thus, FOXO1 may be an important downstream effector of Akt in cardiomyocytes.
Acknowledgments
We thank Dr. Saghi Gaffari, Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, for supplying the pGL3TRAIL-luciferase plasmid; Dr. E. Tang, Department of Biological Chemistry, University of Pennsylvania, for supplying the FOXO1 expression plasmids; and Dr. R. Pearson, Peter MacCallum Cancer Institute, Melbourne, for the myr-Akt plasmid.
Footnotes
This work was supported by grants from the Australian National Heart Foundation and the National Health and Medical Research Council of Australia (NHMRC) No. 317801. E.A.W. is an NHMRC Research Fellow. This work was supported by NHMRC Project Grant 317802 and NHMRC Research Fellowship 317803 (to E.A.W.).
Abbreviations: BrdU, Bromodeoxyuridine; EGF, epidermal growth factor; EGFR, EGF receptor; FKHR, forkhead homolog of rhabdosarcoma; FOXO, forkhead box-containing factor; FOXO1-WT, wild-type FOXO1; GSK3, glycogen synthase kinase-3; myr-Akt, myristylated Akt; NRVM, neonatal rat ventricular myocyte cultures; PI, phosphatidylinositol.
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Abstract
Apoptotic responses in cardiomyocytes are opposed by the protein kinase Akt (protein kinase B) and thus can be suppressed by a number of growth factors and cytokines. In some cell types, Akt phosphorylates and inactivates members of the forkhead box (FOXO) family of transcription factors that are active in regulating the expression of proapoptotic cytokines and signaling intermediates. In the current study, we investigated the possibility that FOXO1 (FKHR) was expressed, regulated, and functional in cardiomyocytes. Addition of epidermal growth factor (EGF) (10 nM) to neonatal rat cardiomyocytes caused rapid phosphorylation of Akt and slower FOXO1 phosphorylation. In contrast, the 1-adrenergic receptor agonist phenylephrine (50 μM) did not phosphorylate Akt and caused dephosphorylation of FOXO1 acutely and increased FOXO1 expression with chronic exposure. Phenylephrine, but not EGF, caused nuclear translocation of FOXO1, a response that is associated with dephosphorylation. Overexpression of FOXO1 activated transcription of the proapoptotic cytokine, TNF-related apoptosis-inducing ligand, as indicated by reporter gene activity. This response was enhanced by phenylephrine and inhibited by EGF. FOXO1 is expressed, regulated, and functionally active in cardiomyocytes and thus may contribute to apoptotic responses in heart.
Introduction
BECAUSE CARDIOMYOCYTES HAVE only limited ability to regenerate, cell death by apoptosis or oncosis is especially problematic in heart. As in other cell types, apoptosis of cardiomyocytes can be mediated via a number of different but interacting pathways. These include the death receptor pathways initiated by Fas or TNF receptors (1), whereby the activated cytokine receptors bind and activate death effector proteins that subsequently activate caspases and the downstream effector pathways (2). Another pathway involves activation of the proapoptotic Bcl family member BAD whose activation causes loss of mitochondrial membrane integrity, cytochrome c release, and subsequent caspase activation (3). In addition, activation of inositol-1,4,5-trisphosphate IP3 receptors and subsequent perturbations in Ca2+ may also contribute to overall apoptotic responses (4). Although modest activation of Gq-coupled receptors, such as 1-adrenerigc receptors, causes cardiomyocyte hypertrophy both in vitro and in cardiomyocyte models, substantially heightened activity causes mitochondrial damage and apoptosis. This response is apparently associated with increased expression of the proapoptotic factor Nix (5, 6) and with reductions in phosphatidylinositol (PI) 3-kinase activity (3).
In cardiomyocytes, as in other cell types, apoptotic responses are opposed by the protein kinase Akt (protein kinase B) (7). Akt phosphorylates BAD, caspase 9, and nuclear factor-B (NF-B), and these phosphorylations can account for much of the protective action of Akt against cell destruction (8, 9). However, in a number of cell types, another protective pathway initiated by Akt has been described. Akt has been shown to phosphorylate and inactivate members of the forkhead (FOXO) family of transcription factors (10, 11). In their phosphorylated form, these factors are tethered in the cytoplasm, but activation by dephosphorylation permits nuclear translocation and activation of responsive genes. Included among these FOXO-responsive genes are some that encode apoptotic effectors including TNF and Fas-ligand (12, 13) and more recently the proapoptotic factor Bim (14). Thus, in addition to its effect on immediate apoptotic signaling pathways via suppression of caspase and BAD activities, Akt also has the potential to reduce the generation of proapoptotic cytokines and thus to cause a sustained, secondary inhibition of apoptotic responses.
Expression of the proapoptotic cytokines, TNF and Fas-ligand, increases in cardiomyocytes under pathological conditions such as ischemia and reperfusion (15, 16). Animals in which one or another of these genes has been silenced show reduced apoptosis, smaller infarct, and improved functional recovery after ischemic insult (17, 18). It is also known that activation of Akt, either by overexpressing a constitutively active mutant (7) or by adding known activators of Akt (19, 20), improves functional recovery, reduces infarct size, and decreases apoptosis after ischemia and reperfusion. Akt is regulated by PI 3-kinase and phospholipid-dependent kinase 1 and is thus directly controlled by receptors for many growth factors and cytokines, including epidermal growth factor (EGF) family members, insulin, and IGF-I as well as the GP-130 ligands cardiotropin-1 and leukemia inhibitory factor (21). All of these factors have been shown to offer protection when applied during reperfusion after an acute ischemic episode (19, 20). Much of the benefit provided by Akt may be explained by inhibition of BAD and caspase 9, but the possibility of other Akt substrates protecting from cardiomyocyte apoptosis has not been exhaustively addressed. In the current study, we investigated the possibility that members of the FOXO family of transcription factors are expressed in the myocardium, that their activity is regulated, and that they are transcriptionally active.
Materials and Methods
Culture of neonatal cardiomyocytes
Neonatal rat ventricular myocyte cultures (NRVM) were prepared from 1- to 3-d-old Sprague Dawley rat pups as previously described (22). Cells were preplated twice for 30 min each to remove nonmyocytes and left to attach for 18 h in DMEM, 10% fetal calf serum, 0.1 mM bromodeoxyuridine (BrdU), 50 U/ml penicillin G and 50 μg/ml streptomycin sulfate onto uncoated dishes. Medium was then replaced with a defined serum-free medium consisting of DMEM, 10 μg/ml human insulin, 10 μg/ml bovine apo-transferrin, 0.1 mM BrdU, 50 U/ml penicillin G, 50 μg/ml streptomycin sulfate, and 125 μg/ml fungizone. BrdU was omitted after 3 d. All experiments were approved by the Alfred Monash Research and Education Precinct Animal Ethics Committee, approval no. 0296, and all procedures adhered to the National Health and Medical Research Council code for the care and use of animals for medical research.
FOXO1 phosphorylation
NRVM plated on 9-cm dishes were washed twice with ice-cold PBS and scraped in 100 μl ice-cold lysis buffer (pH 7.7) containing the following constituents (in mM): 50 Tris-HCl, 100 NaCl, 2 EDTA, 2 EGTA, 10 sodium pyrophosphate, 1 sodium orthovanadate, 1 dithiothreitol, 1 phenylmethylsulfonyl fluoride, and 10 NaF, together with 5 μM okadaic acid, 1 μM pepstatin A, 5 μg/ml aprotinin, 10 μg/ml leupeptin, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, and 1% Triton X-100 (23). Lysates were cleared by centrifugation and protein content of the supernatants measured (24). Proteins from whole-cell lysates (150–300 μg protein) were separated by 10% SDS-PAGE (25) and electrophoretically transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Membranes were stained with Ponceau-S (Sigma Chemical Co., St. Louis, MO) and probed overnight at 4 C with anti-phospho-Ser256-FOXO1 antibodies (1:2000). Secondary antibodies were diluted in blocking solution (Cell Signaling, Beverly, MA) and blots incubated for 1 h, according to the manufacturer’s instructions. Enhanced chemiluminescence detection was carried out according to the manufacturer’s instructions (Amersham Biosciences, Buckinghamshire, UK). Blots were subsequently stripped and reprobed with antibodies to total FOXO1 (1:2000).
Preparation of nuclear and cytoplasmic fractions from NRVM
NRVM plated on 9-cm dishes were stimulated as indicated, and the medium was removed. Cells were detached from the plates using trypsin-EDTA and the trypsin neutralized by adding 8 ml DMEM-10% fetal calf serum. Detached cells were harvested by low-speed (500 rpm) centrifugation and washed gently with PBS, and 100 ml of nuclear isolation buffer containing 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40 was then added to the cell pellets. The mixture was vortexed gently for 20 sec, followed by 10 min incubation on ice. Lysed cells were centrifuged at 500 rpm at 4 C to pellet the nuclei, and the supernatants were removed. The nuclear pellets were washed once with nuclear isolation buffer without the Nonidet P-40.
Transient transfection and reporter gene activity
Transfection experiments were performed in triplicate, using cardiomyocytes (400/mm2) on 35-mm wells 1 d after isolation. Transient transfection, using a total of 4.8 μg DNA per well, was performed by the calcium phosphate method (23, 26). Cytosolic fractions were harvested into lysis buffer containing 0.1 M K2HPO4, 1% Triton X-100, and 1 mM dithiothreitol (pH 7.9). Luciferase activity was measured in 100 mM tricine, 10 mM MgSO4, 2 mM EDTA, 2 mM ATP, and 75 μM luciferin (pH 7.8) for 2 min using a Lumat LB9507 luminometer (23, 27) (Berthold Instruments, Melbourne, Australia).
Materials
Antibodies were from the following sources: total FOXO1 from Santa Cruz Biotechnology (Santa Cruz, CA) and P-Ser356-FOXO1, P-Ser473-Akt, total Akt, P-Ser9-GSK3, and total GSK3 from Cell Signaling. The pGL3-TRAIL-luciferase plasmid was provided by Dr. S. Ghaffari (Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY) and has been described previously (27). Plasmids encoding wild-type FOXO1 (FOXO1-WT) and FOXO1–3A were obtained from Dr. E. Tang (Department of Biological Chemistry, University of Pennsylvania, Philadelphia, PA) and were prepared as described elsewhere (10). The myr-Akt plasmid was provided by Dr. Richard Pearson, Peter MacCallum Cancer Research Institute (Melbourne, Australia).
Results
EGF phosphorylates FOXO1 in NRVM, but phenylephrine reduces phosphorylation
EGF reduces apoptotic responses in NRVM via Akt-dependent mechanisms (28). In some cell types, protection from apoptosis involves members of the FOXO family of transcription factors that are phosphorylated and deactivated by Akt (10) (29). In the current studies, we investigated the possibility that FOXO1 was expressed, regulated, and functional in NRVM. NRVM were treated with EGF for 30 min, and cell lysates were prepared, subjected to SDS-PAGE, and blotted with antibodies to phospho-Ser356-FOXO1 and total FOXO1. As shown in Fig. 1 (top left), EGF caused a time-dependent phosphorylation of FOXO1. Phosphorylation was maximal at 30 min, and no further increase was seen after this (data not shown). In marked contrast, activation of 1-adrenergic receptors by addition of 50 μM phenylephrine (together with 1 μM propranolol to block -adrenergic receptors) caused a decrease in FOXO1 phosphorylation over 30 min (Fig. 1, top) even though we have shown previously that phenylephrine transactivates EGF receptors (EGFR) (23).
EGF, but not phenylephrine, activates Akt
We next examined the phosphorylation of Akt in response to EGF and phenylephrine, because Akt has been shown to phosphorylate FOXO1 (10). NRVM were treated with 10 nM EGF or 50 μM phenylephrine (plus 1 μM propranolol) for times up to 30 min. Extracts were prepared, subjected to SDS-PAGE, transferred, and blotted with antibodies to phosphorylated Akt (phospho-Ser473-Akt). As shown in Fig. 1, EGF caused phosphorylation and activation of Akt with a maximal response at 1–3 min. Phenylephrine did not cause Akt phosphorylation over the 30-min period studied. Akt phosphorylation by EGF preceded that of FOXO1, suggesting the possibility that Akt mediated the phosphorylation of FOXO1 by EGF. Blotting with antibodies to phospho-Thr308-Akt produced similar results (data not shown). In contrast to Akt, both EGF and phenylephrine caused phosphorylation of glycogen synthase kinase-3 (GSK3) on Ser9 (Fig. 1).
Phosphorylation of FOXO1 by EGF was inhibited by 10 μM LY294002 and by 5 μM AG-1478 (Fig. 2, bottom), indicating involvement of both PI 3-kinase and the EGFR kinase in this response. The EGF-induced phosphorylation of Akt was similarly inhibited LY294002 and by AG-1478. Thus, EGF phosphorylation of FOXO1 involves both the EGFR kinase activity and PI 3-kinase and very likely is mediated via Akt. The basal level of FOXO1 phosphorylation was reduced by LY294002 but not by AG-1478, implying an involvement of PI 3-kinase but not EGFR in the maintenance of FOXO1 under unstimulated conditions (Fig. 2).
Chronic exposure to phenylephrine increased FOXO1 expression
NRVM were treated with 50 μM phenylephrine (plus 1 μM propranolol) or with 10 nM EGF for 24 h and effects on FOXO1 expression and phosphorylation examined. Chronic exposure to phenylephrine increased the expression of FOXO1, while reducing phosphorylation levels, thereby substantially increasing the content of potentially active dephosphorylated FOXO1 (Fig. 3). Treatment with EGF for 24 h, in contrast, did not cause major changes in FOXO1 expression or phosphorylation (data not shown), possibly because EGF-induced Akt phosphorylation is transient, as shown in Fig. 1. Chronic activation of Akt was achieved by overexpressing a constitutively active (myristylated) Akt (myr-Akt) using an adenoviral vector. After 24 h infected cells expressed heightened levels of active Akt and reduced levels of FOXO1. These findings agree with previous studies (30, 31) demonstrating that degradation of FOXO1 follows phosphorylation.
Phenylephrine causes nuclear translocation of FOXO1, but EGF does not
Activation of FOXO1 by dephosphorylation is associated with increased nuclear retention (32, 33). NRVM were treated with 50 μM phenylephrine (plus 1 μM propranolol) or 10 nM EGF for 2 h. Cells were lysed, nuclear and cytoplasmic fractions prepared, and the content of FOXO1 assessed by Western blotting with anti-FOXO1 antibodies. Phenylephrine caused a reduction in FOXO1 content in the cytoplasm together with an appearance of FOXO1 in the nuclear fraction. EGF did not cause observable changes in FOXO1 distribution (Fig. 4).
FOXO1-WT activates TNF-related apoptosis-inducing ligand (TRAIL) expression, a response that is inhibited by phosphorylation
In some cell types, FOXO1 activation has been shown to increase transcription of proapoptotic effectors, including TRAIL, TNF, and Fas ligand (13, 27, 34). In the current studies, we investigated whether FOXO1 could increase transcription of sensitive promoters in NRVM and whether this response was modified by phosphorylation. NRVM were transfected with pGL3-TRAIL-luciferase together with FOXO1-WT, as described in the Materials and Methods. After 18 h, 10 nM EGF or 50 μM phenylephrine (plus 1 μM propranolol) was added, cells were harvested, and luciferase activity was measured after another 24 h. Overexpression of FOXO1-WT increased transcription from the TRAIL promoter, as indicated by heightened luciferase activity. Phenylephrine enhanced the response to FOXO1-WT, whereas EGF caused a substantial reduction (Fig. 5A). EGF also reduced TRAIL expression in cells expressing blank vector, implying an effect on endogenous FOXO1. To establish whether the TRAIL response was associated with dephosphorylation in NRVM, we transfected NRVM with a FOXO1 mutant in which the three residues phosphorylated by Akt are mutated to alanines, T24A, S253A, and S316A (FOXO1–3A). Expression of FOXO1–3A increased TRAIL-luciferase expression as shown in Fig. 5A. Neither phenylephrine nor EGF altered the response to FOXO1–3A, showing that phosphorylation is involved in the responses to both stimulatory and inhibitory factors.
In similar experiments, we overexpressed a constitutively active mutant of Akt (myr-Akt) together with FOXO1-WT or FOXO1–3A. Overexpression of myr-Akt reduced responses to FOXO1-WT but not to FOXO1–3A. Thus, FOXO1 can activate TRAIL promoter elements, and this is opposed by Akt phosphorylation or by removal of residues susceptible to Akt phosphorylation.
Discussion
Members of the FOXO family of forkhead transcription factors were defined initially as homologues of the Caenorhabditis elegans transcription factor DAF16. The genes for three mammalian homologues of DAF16, FKHR (FOXO1), FKHRL1 (FOXO3a), and AFX (FOXO4) were first identified as chromosomal breakpoints in human tumors, implying a relationship to cell growth and survival. In various cell types, activation of FOXO family members by dephosphorylation leads to cell cycle arrest and quiescence (35), alterations in energy metabolism (36), and apoptosis (27, 29). In differentiated cells, dephosphorylated forms of FOXO proteins have been shown to directly activate transcription of genes encoding proapoptotic effectors such as TNF, TRAIL, Fas-ligand (27, 13), and more recently the proapoptotic factor Bim (14). FOXO family members are regulated by Akt phosphorylation as well as by other kinases. Phosphorylation reduces DNA binding, interferes with interactions with other transcription factors, and causes nuclear exclusion and tethering in the cytoplasm by binding to 14-3-3 proteins (12, 33, 37). In some cell types, phosphorylation of FOXO proteins has been shown to precede ubiquitinization and degradation (31), pointing to complex overall regulation.
Although there is now a considerable body of knowledge about the functioning of these proteins in various mammalian cells, their possible involvement in the myocardium has only recently been investigated (38). In the current study, EGF phosphorylated FOXO1 in NRVM by a mechanism that apparently involved PI 3-kinase as well as the kinase activity of the EGFR. In contrast, treatment with phenylephrine dephosphorylated FOXO1, even though we have recently shown that phenylephrine transactivates EGFR (Fig. 1) (23). Unlike EGF, phenylephrine does not activate Akt, presumably reflecting a lack of PI 3-kinase activation (Fig. 1) (3). The degree of phosphorylation of EGFR after phenylephrine stimulation is much lower than for EGF, and it is likely that failure to activate PI 3-kinase and Akt reflects this difference in the degree of phosphorylation. It is also possible, however, that there are qualitative differences in signaling from the EGFR depending on whether it is activated by EGF itself or by a transactivating Gq-coupled receptor. In contrast to findings with EGF, phenylephrine caused dephosphorylation of FOXO1 acutely (Fig. 1) and substantially increased FOXO1 expression and the relative amount of dephosphorylated FOXO1 in the longer term (Fig. 3), implying that phenylephrine is an activator of FOXO1 in cardiomyocytes. Enhanced expression might be expected if phosphorylation of FOXO1 leads to degradation. This finding thus implies that FOXO1 is constantly being synthesized and degraded in cardiomyocytes. The mechanism of dephosphorylation of FOXO1 by phenylephrine remains to be established but possibly involves phosphorylation, and thus inactivation, of GSK3. Unlike Akt, phosphorylation of GSK3 at Ser9 is inhibitory rather than stimulatory and so phenylephrine might dephosphorylate FOXO1 by reducing GSK3-induced phosphorylation. EGF also phosphorylates GSK3, but the stimulatory phosphorylation of Akt by EGF possibly overrides any inhibitory effect caused by phosphorylating GSK3. Thus, phosphorylating GSK3 without simultaneous phosphorylation of Akt is a possible mechanism by which Gq-coupled receptors could dephosphorylate FOXO1. However, it is more than possible that other mechanisms are involved. Although acute stimulation of Gq-coupled receptors is not generally associated with apoptotic responses in cardiomyocytes, sustained overactivity of Gq does cause apoptosis both in cell models and in the myocardium in vivo (39). Gq-mediated apoptosis is associated with activation of Nix and loss of mitochondrial integrity (40), but we suggest dephosphorylation of FOXO family members may also make a contribution to the overall cell death response.
Phenylephrine caused nuclear translocation of FOXO1, whereas EGF did not, as expected from the phosphorylation data (Fig. 4). In agreement with this, phenylephrine enhanced transcriptional responses to overexpressed FOXO1, measured as TRAIL promoter activation (Fig. 5). EGF, in contrast, was strongly inhibitory to TRAIL promoter activation by overexpressed FOXO1 and also reduced basal TRAIL promoter activity (Fig. 5). This implies that EGF can effectively phosphorylate and functionally inactivate endogenous or overexpressed FOXO1. Our data also support the notion that Akt regulates FOXO1 action in cardiomyocytes. First, coexpressing Akt reduced the stimulatory activity of overexpressed FOXO1 on TRAIL promoter activity. Second, FOXO1–3A with the Akt phosphorylation sites mutated was fully active and its activity was not sensitive either to EGF or to overexpressed Akt.
Until recently, FOXO members have not been identified in cardiomyocytes, but a recent study has shown that FOXO1, FOXO3a, and FOXO4 are all expressed in NRVM. Furthermore, FOXO3a was shown to inhibit hypertrophic responses in its dephosphorylated, active form via a process involving activation of an atrogene transcriptional program (39). In the current study, we investigated FOXO1 and showed that it too was subject to regulation by phosphorylation/dephosphorylation in NRVM. Factors that activate Akt are known to reduce cardiomyocyte apoptosis and are well-established cardioprotective agents, at least in prevention of ischemic injury (41). Thus, overexpression of Akt or PI 3-kinase reduces ischemic injury, and similar results have been reported after addition of EGF, neuregulin, insulin, IGF-I, or cardiotropin-1, all of which activate Akt (19, 20, 41, 42). Akt has a number of important substrates other than FOXO1, including BAD, caspase 8, and nuclear factor B, all of which may contribute to its antiapoptotic effectiveness (21). However, ischemia and reperfusion cause increased expression of TNF, Fas-ligand, and TRAIL, all of which are regulated by FOXO transcription factors, and diminishing the generation of these cytokines reduces apoptosis and improves functional recovery (15, 43). It therefore seems possible that FOXO1 contributes to apoptosis in the myocardium by increasing expression of proapoptotic effectors and that inhibiting this response is an important part of the protective effect of Akt in the heart. In support of this, there is suggestive evidence for a role for FOXO1 family members in ischemia-induced apoptosis in other cell types. Cerebral ischemia has been shown to be associated with activated, dephosphorylated FOXO1 that paralleled the deactivation of Akt (11, 44). Cell survival after renal ischemia/reperfusion requires phosphorylation and deactivation of both FOXO1 and FOXO3a (45).
Our studies show that, in NRVM, FOXO1 is regulated acutely by phosphorylation and dephosphorylation and chronically at the level of protein expression. The finding that FOXO1 activates transcription from a TRAIL promoter suggests a role in regulating the expression of proapoptotic cytokines in the myocardium. Thus, FOXO1 may be an important downstream effector of Akt in cardiomyocytes.
Acknowledgments
We thank Dr. Saghi Gaffari, Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, for supplying the pGL3TRAIL-luciferase plasmid; Dr. E. Tang, Department of Biological Chemistry, University of Pennsylvania, for supplying the FOXO1 expression plasmids; and Dr. R. Pearson, Peter MacCallum Cancer Institute, Melbourne, for the myr-Akt plasmid.
Footnotes
This work was supported by grants from the Australian National Heart Foundation and the National Health and Medical Research Council of Australia (NHMRC) No. 317801. E.A.W. is an NHMRC Research Fellow. This work was supported by NHMRC Project Grant 317802 and NHMRC Research Fellowship 317803 (to E.A.W.).
Abbreviations: BrdU, Bromodeoxyuridine; EGF, epidermal growth factor; EGFR, EGF receptor; FKHR, forkhead homolog of rhabdosarcoma; FOXO, forkhead box-containing factor; FOXO1-WT, wild-type FOXO1; GSK3, glycogen synthase kinase-3; myr-Akt, myristylated Akt; NRVM, neonatal rat ventricular myocyte cultures; PI, phosphatidylinositol.
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