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• W1992322558 abstract "Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis. Apoptosis of vascular smooth muscle cells (VSMCs) may lead to atherosclerotic plaque instability and rupture, resulting in myocardial infarction, stroke, and sudden death. However, the molecular mechanisms mediating survival of VSMCs in atherosclerotic plaques remain unknown. Although plaque VSMCs exhibit increased susceptibility to apoptosis and reduced expression of the IGF1 receptor (IGF1R) when compared with normal VSMCs, a causative effect has not been established. Here we show that increased expression of the IGF1R can rescue plaque VSMCs from oxidative stress-induced apoptosis, demonstrating that IGF-1 signaling is a critical regulator of VSMC survival. Akt mediates the majority of the IGF1R survival signaling, and ectopic activation of Akt was sufficient to protect VSMCs in vitro. Both IGF1R and phospho-Akt expression were reduced in human plaque (intimal) VSMCs when compared with medial VSMCs, suggesting that Akt mediates survival signaling in atherosclerosis. Importantly, downstream targets of Akt were identified that mediate its protective effect as inhibition of FoxO3a or GSK3 by Akt-dependent phosphorylation protected VSMCs in vitro. We conclude that Akt and its downstream targets FoxO3a and GSK3 regulate a survival pathway in VSMCs and that their deregulation due to a reduction of IGF1R signaling may promote apoptosis in atherosclerosis. Insulin-like growth factor 1 (IGF1) 2The abbreviations used are: IGF1, insulin-like growth factor 1; IGF1R, IGF1 receptor; IGF1R YF, inactive point mutation of IGF1R; VSMC, vascular smooth muscle cell; pVSMC, atherosclerotic plaque-derived VSMC; HT, 4-hydroxytamoxifen; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; DN, dominant negative. 2The abbreviations used are: IGF1, insulin-like growth factor 1; IGF1R, IGF1 receptor; IGF1R YF, inactive point mutation of IGF1R; VSMC, vascular smooth muscle cell; pVSMC, atherosclerotic plaque-derived VSMC; HT, 4-hydroxytamoxifen; EGFP, enhanced green fluorescent protein; ER, estrogen receptor; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; DN, dominant negative. is a ubiquitous factor exhibiting pleiotropic effects on different cell types. Stimulation of the IGF1 receptor (IGF1R) initiates signaling pathways involved in cell proliferation, differentiation, transformation, and survival. IGF1R-dependent signaling is crucial for the survival of many cell types including vascular smooth muscle cells (VSMCs). VSMCs are the principle source of collagen and extracellular matrix that maintain the tensile strength of atherosclerotic plaques, and VSMC loss induces multiple features of plaque instability (1Clarke M.C. Figg N. Maguire J.J. Davenport A.P. Goddard M. Littlewood T.D. Bennett M.R. Nat. Med. 2006; 12: 1075-1080Crossref PubMed Scopus (523) Google Scholar). In humans, rupture or erosion of the atherosclerotic plaque underlie the majority of myocardial infarctions, stroke, and sudden death (2Burke A.P. Farb A. Malcom G.T. Liang Y.H. Smialek J. Virmani R. N. Engl. J. Med. 1997; 336: 1276-1282Crossref PubMed Scopus (1449) Google Scholar). We have previously shown that VSMCs derived from atherosclerotic plaques (pVSMCs) are more sensitive to apoptosis than cells derived from non-diseased vessels (3Bennett M.R. Evan G.I. Schwartz S.M. J. Clin. Investig. 1995; 95: 2266-2274Crossref PubMed Scopus (610) Google Scholar) and exhibit a defect in IGF1-dependent survival signaling (4Patel V.A. Zhang Q.J. Siddle K. Soos M.A. Goddard M. Weissberg P.L. Bennett M.R. Circ. Res. 2001; 88: 895-902Crossref PubMed Scopus (98) Google Scholar). Oxidative stress is increasingly implicated in the development of atherosclerosis (5Stocker R. Keaney Jr., J.F. J. Thromb. Haemostasis. 2005; 3: 1825-1834Crossref PubMed Scopus (148) Google Scholar) and increased oxidative damage, and elevated levels of DNA strand breaks occur in human atherosclerotic plaques (6Martinet W. Knaapen M.W. De Meyer G.R. Herman A.G. Kockx M.M. Circulation. 2002; 106: 927-932Crossref PubMed Scopus (364) Google Scholar). Oxidative stress reduces IGF1R expression and induces VSMC apoptosis in culture (7Kavurma M.M. Figg N. Bennett M.R. Mercer J. Khachigian L.M. Littlewood T.D. Biochem. J. 2007; 407: 79-87Crossref PubMed Scopus (47) Google Scholar, 8Pedruzzi E. Guichard C. Ollivier V. Driss F. Fay M. Prunet C. Marie J.C. Pouzet C. Samadi M. Elbim C. O'Dowd Y. Bens M. Vandewalle A. Gougerot-Pocidalo M.A. Lizard G. Ogier-Denis E. Mol. Cell. Biol. 2004; 24: 10703-10717Crossref PubMed Scopus (361) Google Scholar, 9Sandberg E.M. Sayeski P.P. J. Biol. Chem. 2004; 279: 34547-34552Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 10Song H.J. Lee T.S. Jeong J.H. Min Y.S. Shin C.Y. Sohn U.D. J. Pharmacol. Exp. Ther. 2005; 312: 391-398Crossref PubMed Scopus (33) Google Scholar). Reduced IGF1R expression is also seen within plaques, suggesting that IGF1R-dependent survival regulates apoptosis in vivo (11Okura Y. Brink M. Zahid A.A. Anwar A. Delafontaine P. J. Mol. Cell. Cardiol. 2001; 33: 1777-1789Abstract Full Text PDF PubMed Scopus (87) Google Scholar, 12Delafontaine P. Song Y.H. Li Y. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 435-444Crossref PubMed Scopus (432) Google Scholar). However, plaque VSMCs also show increased sensitivity to multiple proapoptotic stimuli, including the tumor suppressor gene P53 and death receptor ligation (13Bennett M.R. Littlewood T.D. Schwartz S.M. Weissberg P.L. Circ. Res. 1997; 81: 591-599Crossref PubMed Scopus (118) Google Scholar, 14Chan S.W. Hegyi L. Scott S. Cary N.R. Weissberg P.L. Bennett M.R. Circ. Res. 2000; 86: 1038-1046Crossref PubMed Scopus (55) Google Scholar). It is therefore not known whether defective IGF1R expression alone is an important cause of reduced survival of pVSMCs.A major downstream effector of IGF1R signaling is the serine/threonine kinase Akt (also known as Protein Kinase B (PKB)). Akt phosphorylates a large number of targets involved in glucose metabolism and cell differentiation, proliferation, and survival (reviewed in Ref. 15Lawlor M.A. Alessi D.R. J. Cell Sci. 2001; 114: 2903-2910Crossref PubMed Google Scholar). We have previously shown that pVSMCs also exhibit reduced activation of Akt in response to IGF1 treatment (4Patel V.A. Zhang Q.J. Siddle K. Soos M.A. Goddard M. Weissberg P.L. Bennett M.R. Circ. Res. 2001; 88: 895-902Crossref PubMed Scopus (98) Google Scholar), suggesting that Akt mediates IGF1R-dependent signaling in these cells. However, it is not known whether activation of Akt is necessary and/or sufficient for VSMC survival in response to apoptotic stimuli. Similarly, although Akt targets involved in survival have been identified in many cell types, Akt targets are frequently cell type-specific, and those important for VSMC survival have not been determined. Here we show that activation of Akt alone is sufficient to protect VSMCs from oxidative stress-induced apoptosis. Moreover, we demonstrate that Akt-dependent phosphorylation and subsequent inactivation of FoxO3a and GSK3 is important for VSMC survival.EXPERIMENTAL PROCEDURESCell Culture—Human VSMCs were isolated from aortas of cardiac transplant patients or from atherosclerotic plaques following carotid endarterectomy with informed consent and approval of the Local Ethics Committee. Rat VSMCs were isolated from aortas of Wistar rats. Cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10 units/ml penicillin, 10 μg/ml streptomycin, 5 μg/ml l-glutamine, and 10% fetal calf serum. Cells were treated with 100 ng/ml recombinant human IGF1 (Peprotech), 100 nm 4-hydroxytamoxifen (HT, Sigma), and 50 μm SB415286 (Sigma), as indicated. Since ongoing oxidative stress is a feature of atherosclerosis (5Stocker R. Keaney Jr., J.F. J. Thromb. Haemostasis. 2005; 3: 1825-1834Crossref PubMed Scopus (148) Google Scholar, 6Martinet W. Knaapen M.W. De Meyer G.R. Herman A.G. Kockx M.M. Circulation. 2002; 106: 927-932Crossref PubMed Scopus (364) Google Scholar) and is associated with VSMC apoptosis, we, like others, have used 25-50 μm hydrogen peroxide to induce VSMC apoptosis. Expression plasmids were transfected into rat VSMCs using SuperFect (Qiagen). Alternatively, infectious replication-deficient retrovirus was harvested from Bosc23 packaging cells and used to infect rat VSMCs in the presence of 8 μg/ml Polybrene (hexadimethrine bromide, Sigma). Rat VSMCs were selected and maintained in 5 μg/ml puromycin (Sigma) or 400 μg/ml G418 as appropriate. Human plaque-derived VSMCs were microinjected at 150 hectopascals for 0.1 s with expression plasmids at 1 mg/ml using Eppendorf Femtotips II and an Eppendorf Femtojet/Injectman 2. Cells expressing EGFP were counted using an Olympus IX51 microscope.Plasmids—mAktER and A2 AktER (16Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar) were cloned into the retroviral vector pBMN IRES puro (17Garton K.J. Ferri N. Raines E.W. BioTechniques. 2002; 32 (832, 834 passim): 830Crossref PubMed Scopus (40) Google Scholar). IGF1R wild type and YF mutant (18Li S. Ferber A. Miura M. Baserga R. J. Biol. Chem. 1994; 269: 32558-32564Abstract Full Text PDF PubMed Google Scholar) were cloned into pBMN IRES puro (17Garton K.J. Ferri N. Raines E.W. BioTechniques. 2002; 32 (832, 834 passim): 830Crossref PubMed Scopus (40) Google Scholar). Dominant negative Akt (DN-Akt, T308A, S473A (19Fujio Y. Guo K. Mano T. Mitsuuchi Y. Testa J.R. Walsh K. Mol. Cell. Biol. 1999; 19: 5073-5082Crossref PubMed Scopus (188) Google Scholar)) was cloned into pCDNA3, and the wild type and the A3 mutant of FoxO3a (20Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5366) Google Scholar) were cloned into pCDNA3.1. pCDNA3 GSK3β wild type and S9A have been described (21Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (503) Google Scholar).Antibodies—Antibodies to the following proteins were used: Akt (Cell Signaling antibodies 9272 and 4691), phospho-Akt Ser-473 (Cell Signaling antibodies 9271 and 4060), GSK3α/β (Upstate Biotechnology antibody 05-412), GSK3α (AbCam antibody 28833), phospho-GSK3α/β Ser-21/Ser-9 (Cell Signaling antibody 9331), phospho-GSK3α Ser-21 (Cell Signaling antibody 9316), FoxO1 (Cell Signaling antibody 9462 and AbCam antibody 39670), FoxO3a (Santa Cruz Biotechnology antibody sc-11351 and Cell Signaling antibody 9467), phospho-FoxO1/phospho-FoxO3a Thr-24/Thr-32 (Cell Signaling antibody 9464), Mcl1 (Santa Cruz Biotechnology antibody sc-819), Bad (Cell Signaling antibody 9292), phospho-Bad Ser-136 (Cell Signaling antibody 9295), Bim (Chemicon antibody AB17003), hemagglutinin (Sigma antibody H9658), phospho-Ser (antibody AbCam ab17465), β-actin (Sigma clone AC-15), and α-smooth muscle actin (Dako antibody 1A4).Western Blot—Equal amounts of protein lysates prepared in Laemmli sample buffer were resolved on SDS-polyacrylamide gels, transferred onto polyvinylidene difluoride membranes (Millipore), and incubated with the indicated primary antibody. Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (GE Healthcare).Cell Viability and Apoptosis—Cells were plated at the same density 16 h prior to each experiment. Proliferation and apoptosis were determined by time-lapse digital microscopy using an Olympus IX71 microscope controlled with Openlab software (Improvision). Apoptotic and dividing cells in two or more fields each containing 100 cells at the start of the experiment were counted. Apoptosis was verified by morphological changes, including nuclear condensation in the presence of Hoechst 33258 and membrane blebbing.Immunohistochemistry—Paraffin-embedded sections of human carotid arteries were sectioned at 5-μm intervals. Specimens were dewaxed and microwaved in 120 mm sodium citrate buffer, and endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Sections were incubated in 10% bovine serum albumin for 1 h at room temperature and then with primary antibodies overnight at 4 °C followed by biotinylated secondary antibodies and horseradish peroxidase-conjugated streptavidin using the ABC kit (Vector Laboratories). Peroxidase activity was detected with diaminobenzidene. Sections were counterstained with hematoxylin. Cells expressing Akt, FoxO3a, GSK3α, or their phosphorylated species were counted in three or more random fields, each from three different plaque donors. Only cells whose staining exceeded a 125-pixel density were counted.Statistical Analysis—Statistical analysis was performed using analysis of variance or Student's t test as appropriate. Significance was established when p < 0.05.RESULTSIGF1R Mediates Survival of VSMCs—Previously, we demonstrated that VSMCs derived from human plaques (pVSMCs) express lower levels of IGF1R when compared with VSMCs derived from normal human aortas (4Patel V.A. Zhang Q.J. Siddle K. Soos M.A. Goddard M. Weissberg P.L. Bennett M.R. Circ. Res. 2001; 88: 895-902Crossref PubMed Scopus (98) Google Scholar, 7Kavurma M.M. Figg N. Bennett M.R. Mercer J. Khachigian L.M. Littlewood T.D. Biochem. J. 2007; 407: 79-87Crossref PubMed Scopus (47) Google Scholar). pVSMCs also show reduced proliferation and increased sensitivity to apoptosis (3Bennett M.R. Evan G.I. Schwartz S.M. J. Clin. Investig. 1995; 95: 2266-2274Crossref PubMed Scopus (610) Google Scholar, 4Patel V.A. Zhang Q.J. Siddle K. Soos M.A. Goddard M. Weissberg P.L. Bennett M.R. Circ. Res. 2001; 88: 895-902Crossref PubMed Scopus (98) Google Scholar). Ectopic expression of IGF1R in VSMCs increases the rate of proliferation and markedly reduces apoptosis (7Kavurma M.M. Figg N. Bennett M.R. Mercer J. Khachigian L.M. Littlewood T.D. Biochem. J. 2007; 407: 79-87Crossref PubMed Scopus (47) Google Scholar, 22Li Y. Higashi Y. Itabe H. Song Y.H. Du J. Delafontaine P. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 2178-2184Crossref PubMed Scopus (77) Google Scholar). Although there is a good correlation between reduced IGF1R expression and reduction in proliferation and survival in pVSMCs, a causative effect has not been established. To determine whether increased IGF1R expression could rescue the increased sensitivity of pVSMCs to apoptosis, we co-microinjected expression plasmids for either the wild-type IGF1R or the YF mutant along with EGFP into human pVSMCs. Co-injection with EGFP-expressing plasmids allowed tracking of injected cells within the population. When compared with plaque VSMCs microinjected with empty vector or IGF1R-YF, those injected with wild-type IGF1R showed increased protection against H2O2-induced apoptosis in response to IGF1 (Fig. 1A). This suggests that the level of IGF1R expression regulates the survival of VSMCs in response to oxidative stress.IGF1R-dependent Survival of VSMCs Is Mediated by Akt—The serine/threonine kinase Akt is a major downstream effector of IGF1R signaling. Consistent with this, human pVSMCs demonstrated a transient increase in Akt phosphorylation (Ser-473) peaking at 15 min after IGF1 stimulation when compared with normal human VSMCs that demonstrated a more robust and persistent response (Fig. 1B). The reduction in the active phosphorylated form (Ser-473) of Akt was not due to reduced total Akt expression since this was not affected by IGF1 stimulation. Similar results were obtained from an additional two normal and two plaque-derived primary isolates of human VSMCs, indicating that reduced phospho-Akt was a common feature of plaque-derived cultures when compared with normal VSMCs (data not shown).To examine whether the reduced phospho-Akt expression seen in pVSMCs in vitro was a valid representation of that seen in plaques in vivo, we examined expression of Akt and phospho-Akt in atherosclerotic plaques from three different patients where both the fibrous cap intimal cells and a relatively normal media could be examined in the same section. VSMCs (α-smooth muscle actin-positive, Fig. 1C) within the fibrous cap of the atherosclerotic plaque exhibited reduced phospho-Akt (Ser-473) expression in vivo when compared with medial cells (Fig. 1C). Again, this appeared to be due to a reduction in Akt activation (phosphorylation at Ser-473) rather than a reduction in total Akt expression.To demonstrate directly that Akt mediates the effects of IGF1R activation in VSMCs, we expressed a dominant negative mutant of Akt (DN-Akt, T308A, S473A) (19Fujio Y. Guo K. Mano T. Mitsuuchi Y. Testa J.R. Walsh K. Mol. Cell. Biol. 1999; 19: 5073-5082Crossref PubMed Scopus (188) Google Scholar) in rat VSMCs (Fig. 2A). DN-Akt markedly inhibited IGF1-stimulated survival (Fig. 2A), strongly suggesting that the major downstream effector of IGF1R-mediated survival in VSMCs is Akt. To examine whether Akt was sufficient to protect VSMCs from H2O2-induced apoptosis, we employed an ectopically inducible allele of Akt. Rat VSMCs were infected with retroviruses that express either a constitutively active Akt (myristoylated Akt, mAkt) or an allele containing an inactivating mutation in the myristoylation domain (A2Akt) fused to the ligand-binding domain of the estrogen receptor (mAktER and A2AktER, respectively; Fig. 2B (16Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar)). These proteins are inactive in the absence of ligand (4-hydroxytamoxifen, HT), and the kinase activity of mAktER is only stimulated by the addition of HT to the culture medium (16Kohn A.D. Barthel A. Kovacina K.S. Boge A. Wallach B. Summers S.A. Birnbaum M.J. Scott P.H. Lawrence Jr., J.C. Roth R.A. J. Biol. Chem. 1998; 273: 11937-11943Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). Consistent with this, phosphorylation of mAktER (Ser-473) was only observed following the addition of HT (Fig. 3A). In contrast, A2AktER was not phosphorylated at Ser-473 following the addition of HT and exhibited no kinase activity (see below). Note that a low level of endogenous phospho-Akt is observed in these cells even after 24 h in the absence of serum and that this was markedly increased by IGF1 treatment (Fig. 3A). Rat VSMCs cultured in the absence of IGF1 for 24 h (to suppress endogenous IGF1R signaling) showed ∼35-60% apoptosis 24 h after the addition of 50 μm H2O2 (Fig. 2, A and B). In contrast, ectopic activation of mAktER elicited a protective effect against H2O2-induced apoptosis in these cells and reduced the level of apoptosis to ∼12%, similar to that seen following treatment with IGF1 (Fig. 2, A and B). No protection was seen in HT-treated A2AktER-expressing cells, nor in mAktER-expressing cells treated with vehicle (ethanol). These results demonstrate that IGF1R-dependent survival signaling in rat VSMCs is mediated primarily by Akt and that activation of Akt alone is sufficient to mimic the protective effects of IGF1R treatment.FIGURE 2IGF1-dependent survival of VSMCs is mediated by Akt. A, time-lapse microscopic quantification of apoptosis in control rat VSMCs (□, ♦) or cells expressing a dominant negative Akt protein (•). Cells were cultured in serum-free conditions for 24 h and then treated with 50 μm H2O2 in the absence (□) or presence (♦, •) of 100 ng/ml human IGF1. Results are mean ± S.D. (n = 3 different experiments). ** indicates p < 0.01. The inset shows expression of hemagglutinin-tagged DN-Akt by immunoblot. B, time-lapse microscopic quantification of apoptosis of rat VSMCs expressing mAktER (□, •) or A2AktER (•). Cells were cultured in serum-free conditions for 24 h and then treated with 50 μm H2O2 in the presence of either 100 nm HT (○, •) or an equivalent volume of the carrier, ethanol (□). Results are mean ± S.D. (n = 3 different experiments). ** indicates p < 0.01 versus both mAktER + ethanol and A2AktER + HT. The inset shows expression of both endogenous Akt and the AktER fusion proteins in rat VSMCs by immunoblot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Akt phosphorylates FoxO3a and GSK3 in VSMC. Immunoblots of rat VSMCs expressing mAktER or A2AktER were cultured in serum-free conditions for 24 h and then treated with either 100 nm HT or 100 ng/ml IGF1 as indicated. Expression of β-actin is shown as a protein loading control. Densitometry is shown as the ratio of phosphorylated Akt (p-Akt, white bars) or AktER (p-AktER, black bars) relative to total Akt (A). Densitometry is also shown for the ratio of phosphorylated species of GSK3α (p-GSK3α, B) or FoxO3a (p-FoxO3a, C) relative to the total for each protein, respectively. Results are mean ± S.D. (n = 3 different experiments). * indicates p < 0.05, ** indicates p < 0.01. Immunoblots for FoxO1 (p-FoxO1), Bad (p-Bad), and phospho-Bad are also shown (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Akt-dependent Survival of VSMCs Is Mediated by Inhibition of FoxO3a and GSK3—There are many reported substrates for Akt. For example, Bad, caspase 9, and members of the FoxO family are proapoptotic proteins that have been identified as Akt targets in some cells. Since we have shown that Akt is an important regulator of cell death in VSMCs, we sought to identify which targets of Akt mediate survival in these cells. We compared phosphorylation of putative Akt substrates after activation of mAktER or A2AktER by HT or by the addition of IGF1 to cells deprived of serum for 24 h (Fig. 3). Although phosphorylation of Bad by Akt confers survival in BALB/c 3T3 cells (23Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4914) Google Scholar), we were unable to demonstrate phosphorylation of Bad following activation of Akt in vitro (Fig. 3D). In contrast, ectopic activation of Akt with HT resulted in rapid phosphorylation of FoxO3a and both α and β isoforms of GSK3 (Fig. 3, B and C). This was not observed in A2AktER-expressing control cells. Phosphorylation of FoxO3a and GSK3 α/β was also seen after stimulation with IGF1, consistent with the idea that Akt mediates signaling downstream of IGF1R. Although FoxO3a is phosphorylated by Akt in VSMCs, other members of the FoxO family are not. For example, FoxO1 is also expressed in these cells but was not phosphorylated in response to HT or IGF1 (Fig. 3, C and D).FoxO3a is a transcription factor that activates many genes involved in cell death, including fasL, bim, and puma (reviewed in Ref. 24Burgering B.M. Kops G.J. Trends Biochem. Sci. 2002; 27: 352-360Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). To determine whether inhibition of FoxO3a by Akt is important in the survival of VSMCs, we expressed a mutant FoxO3a allele (A3) in which all three potential Akt phosphorylation sites (Thr-32, Ser-253, and Ser-315) are changed to alanines (Fig. 4A) and which, therefore, cannot be phosphorylated and inhibited by Akt (20Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5366) Google Scholar). The protection afforded by activation of Akt in H2O2-treated control rat VSMCs or those expressing wild-type FoxO3a was severely comprised by expression of FoxO3a-A3 (Fig. 4A). This indicates that inhibition of FoxO3a activity by Akt-dependent phosphorylation is required for survival of these cells.FIGURE 4Akt-dependent inhibition of apoptosis is mediated by phosphorylation of FoxO3a. A, time-lapse microscopic quantification of apoptosis in rat VSMCs expressing mAktER and transfected with a control empty plasmid (C, □) or plasmids encoding either wild type (WT, •) or the A3 mutant (○) of FoxO3a. Cells were cultured in serum-free conditions for 24 h and then treated with 100 nm HT and 50 μm H2O2. Apoptosis was determined by time-lapse microscopy. Results are mean ± S.D. (n = 3 different experiments). * indicates p < 0.05. The inset shows expression of hemagglutinin-tagged FoxO3a (HA-FoxO3a) by immunoblot. B, immunoblot for Bim in rat VSMCs expressing mAktER and either wild type or the A3 mutant of FoxO3a. Cells were cultured in serum-free conditions for 24 h and then treated with 100 nm HT for 8 h. β-actin is shown as a loading control. Densitometry is shown below as the ratio of Bim/β-actin. Results are mean ± S.D. (n = 3). * indicates p < 0.05, ** indicates p < 0.01. p-AktER, phosphorylated AktER. C, immunoblots of primary VSMC cultures from normal human aorta (N) or two different human plagues (P). Densitometry is shown as the ratio of phospho-Akt (p-Akt, white bars), phospho-FoxO3a (p-FoxO3a, gray bars) or phospho-GSK3 (p-GSK3α/β, black bars) relative to total protein for each species, respectively. Results are mean ± S.D. from three replicate immunoblots using the same VSMC isolates. D, representative immunohistochemistry images for total and phospho-FoxO1/3a in sections of a human carotid atherosclerotic plaque demonstrating reduced p-FoxO1/3a in intimal VSMCs when compared with healthy medial VSMCs. The inset shows a higher power magnification of the boxed area. Scale bars = 100 or 50 μm (inset). The proportion of FoxO3a-positive (white bars) or phospho-FoxO1/FoxO3a (p-FoxO1/3a, Thr-32/Thr-24)-positive (black bars) VSMCs in the media or intima is quantified in the same individual plaques from three different patients shown in Fig. 1C. * indicates p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether proapoptotic targets of FoxO3a were regulated after Akt activation, we examined the expression of Bim. Expression of Bim was reduced following activation of Akt in rat VSMCs expressing wild-type FoxO3a but not in cells expressing FoxO3a A3 (Fig. 4B).To determine whether FoxO3a is a physiological target of Akt in human VSMCs as well as rat cells, we examined the expression of p-FoxO3a in lysates derived from normal human aorta or atherosclerotic plaque primary cultures (Fig. 4C). Reduced levels of phosphorylated Akt and FoxO3a were observed in the plaque-derived cultures when compared with normal aorta. Moreover, VSMCs within the fibrous cap of the atherosclerotic plaque exhibited reduced phospho-FoxO1/3a expression in vivo when compared with medial cells despite the presence of FoxO3a in these cells (Fig. 4D). The antibody used recognizes phosphorylated forms of both FoxO1 and FoxO3a, and we cannot be sure which protein is detected. However, we had not previously detected Akt-dependent phosphorylation of FoxO1 in rat VSMCs (Fig. 3), suggesting that the antibody is primarily detecting phosphorylated FoxO3a.It is likely that Akt-dependent survival of VSMCs is mediated by more than one downstream effector. Indeed, GSK3 was also phosphorylated by Akt in VSMCs (Fig. 3), and recent data indicate that GSK3 may have a role in apoptosis regulation as well as glucose metabolism (25Maurer U. Charvet C. Wagman A.S. Dejardin E. Green D.R. Mol. Cell. 2006; 21: 749-760Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar). To determine whether Akt-dependent inhibition of GSK3 is important for survival of VSMCs, we expressed either the wild-type allele or a mutant allele of GSK3β (S9A) in which the primary phosphorylation site targeted by Akt is mutated (Fig. 5A). In cells expressing GSK3β S9A, the ability of Akt to suppress apoptosis in response to H2O2 was delayed and partially impaired (Fig. 5A).FIGURE 5Akt-dependent inhibition of apoptosis is mediated by phosphorylation of GSK3. A, time-lapse microscopic quantification of apoptosis in rat VSMCs expressing mAktER and transfected with control empty plasmid (C, □) or" @default.
• W1992322558 created "2016-06-24" @default.
• W1992322558 creator A5014538781 @default.
• W1992322558 creator A5041459747 @default.
• W1992322558 creator A5043387899 @default.
• W1992322558 creator A5067198585 @default.
• W1992322558 date "2008-07-01" @default.
• W1992322558 modified "2023-10-17" @default.
• W1992322558 title "Akt Regulates the Survival of Vascular Smooth Muscle Cells via Inhibition of FoxO3a and GSK3" @default.
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