β-Arrestin-2 Mediates Anti-apoptotic Signaling through Regulation of BAD Phosphorylation
2009; Elsevier BV; Volume: 284; Issue: 13 Linguagem: Inglês
10.1074/jbc.m808463200
ISSN1083-351X
AutoresSeungkirl Ahn, Ji‐Hee Kim, Makoto R. Hara, Xiu-Rong Ren, Robert J. Lefkowitz,
Tópico(s)Adipose Tissue and Metabolism
Resumoβ-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that β-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate β-arrestin but not G protein-dependent signaling, and are abrogated by β-arrestin-2 small interfering RNA. These findings establish a key role for β-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved. β-Arrestins, originally discovered as terminators of G protein-coupled receptor signaling, have more recently been appreciated to also function as signal transducers in their own right, although the consequences for cellular physiology have not been well understood. Here we demonstrate that β-arrestin-2 mediates anti-apoptotic cytoprotective signaling stimulated by a typical 7-transmembrane receptor the angiotensin ATII 1A receptor, expressed endogenously in rat vascular smooth muscle cells or by transfection in HEK-293 cells. Receptor stimulation leads to concerted activation of two pathways, ERK/p90RSK and PI3K/AKT, which converge to phosphorylate and inactivate the pro-apoptotic protein BAD. Anti-apoptotic effects as well as pathway activities can be stimulated by an angiotensin analog (SII), which has been previously shown to activate β-arrestin but not G protein-dependent signaling, and are abrogated by β-arrestin-2 small interfering RNA. These findings establish a key role for β-arrestin-2 in mediating cellular cytoprotective functions by a 7-transmembrane receptor and define the biochemical pathways involved. β-Arrestins 1 and 2 were originally identified as signal terminators for G protein-dependent 7-transmembrane receptor (7TMR) 2The abbreviations used are: 7TMR, 7-transmembrane receptor; AngII, angiotensin II; AT1R, angiotensin II type 1 receptor; ERK, extracellular signal-regulated kinase; HEK, human embryonic kidney; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; RSK, ribosomal S6 kinase; SII, [Sar1,Ile4,Ile8]angiotensin II; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; PBS, phosphate-buffered saline. signaling. Their binding to the receptor sterically inhibits receptor coupling to G protein leading to inactivation of effectors such as second messenger generating enzymes (1Hall R.A. Premont R.T. Lefkowitz R.J. J. Cell Biol. 1999; 145: 927-932Crossref PubMed Scopus (279) Google Scholar). Besides this classical function, recently accumulating evidence has revealed novel functions of β-arrestins as signal transducers in various signaling pathways (2DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1143) Google Scholar). Among other processes, β-arrestins have been suggested to play a role in regulation of cell death, but this area has been controversial and largely devoid of mechanistic insight. There have been reports implicating β-arrestins in both pro- and anti-apoptotic responses as well as in non-apoptotic cell death (3Shi Y. Feng Y. Kang J. Liu C. Li Z. Li D. Cao W. Qiu J. Guo Z. Bi E. Zang L. Lu C. Zhang J.Z. Pei G. Nat. Immunol. 2007; 8: 817-824Crossref PubMed Scopus (122) Google Scholar, 4Zhao M. Wimmer A. Trieu K. Discipio R.G. Schraufstatter I.U. J. Biol. Chem. 2004; 279: 49259-49267Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 5Revankar C.M. Vines C.M. Cimino D.F. Prossnitz E.R. J. Biol. Chem. 2004; 279: 24578-24584Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 6Povsic T.J. Kohout T.A. Lefkowitz R.J. J. Biol. Chem. 2003; 278: 51334-51339Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 7DeFea K.A. Vaughn Z.D. O'Bryan E.M. Nishijima D. Dery O. Bunnett N.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11086-11091Crossref PubMed Scopus (350) Google Scholar, 8Luan B. Zhang Z. Wu Y. Kang J. Pei G. EMBO J. 2005; 24: 4237-4246Crossref PubMed Scopus (66) Google Scholar, 9Wang P. Gao H. Ni Y. Wang B. Wu Y. Ji L. Qin L. Ma L. Pei G. J. Biol. Chem. 2003; 278: 6363-6370Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 10Castro-Obregon S. Rao R.V. del Rio G. Chen S.F. Poksay K.S. Rabizadeh S. Vesce S. Zhang X.K. Swanson R.A. Bredesen D.E. J. Biol. Chem. 2004; 279: 17543-17553Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). For example, it has been shown that stimulation of a number of 7TMRs including the N-formyl peptide receptor induces apoptosis in β-arrestin 1/2 double knock-out mouse embryonic fibroblasts, and that reintroduction of either β-arrestin-1 or -2 completely prevents this apoptosis (5Revankar C.M. Vines C.M. Cimino D.F. Prossnitz E.R. J. Biol. Chem. 2004; 279: 24578-24584Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Conversely, β-arrestins also have been reported to mediate cell death. β-Arrestin-2, particularly a dephosphorylated form, has been shown to facilitate inhibition of NF-κB activation in response to UV, leading to promotion of UV-induced cell death (8Luan B. Zhang Z. Wu Y. Kang J. Pei G. EMBO J. 2005; 24: 4237-4246Crossref PubMed Scopus (66) Google Scholar).Bcl-2 family proteins are known to determine the outcome of an intrinsic apoptotic process initiated by release of cytochrome c and apoptotic factors from the mitochondria (11Adams J.M. Cory S. Oncogene. 2007; 26: 1324-1337Crossref PubMed Scopus (1671) Google Scholar). BAD, a BH3-only protein is one of the "death-promoting" members of the Bcl-2 family and its pro-apoptotic activity is regulated primarily by phosphorylation at several sites (12Datta S.R. Ranger A.M. Lin M.Z. Sturgill J.F. Ma Y.C. Cowan C.W. Dikkes P. Korsmeyer S.J. Greenberg M.E. Dev. Cell. 2002; 3: 631-643Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 13Bergmann A. Dev. Cell. 2002; 3: 607-608Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Survival factors induce BAD phosphorylation via several protein kinase signaling pathways (13Bergmann A. Dev. Cell. 2002; 3: 607-608Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) including activation of mitogen-activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) (14Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1665) Google Scholar, 15Shimamura A. Ballif B.A. Richards S.A. Blenis J. Curr. Biol. 2000; 10: 127-135Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 16Yang X. Liu L. Sternberg D. Tang L. Galinsky I. DeAngelo D. Stone R. Cancer Res. 2005; 65: 7338-7347Crossref PubMed Scopus (44) Google Scholar) and phosphatidylinositol 3-kinase (PI3K)-AKT (17del Peso L. Gonzalez-Garcia M. Page C. Herrera R. Nunez G. Science. 1997; 278: 687-689Crossref PubMed Scopus (1978) Google Scholar, 18Datta 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 (4915) Google Scholar). Phosphorylated BAD associates with 14-3-3 proteins in the cytoplasm, preventing translocation of BAD to the mitochondria (19Datta S.R. Katsov A. Hu L. Petros A. Fesik S.W. Yaffe M.B. Greenberg M.E. Mol. Cell. 2000; 6: 41-51Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar) and its interaction with the anti-apoptotic proteins Bcl-xL and Bcl-2 (13Bergmann A. Dev. Cell. 2002; 3: 607-608Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1883) Google Scholar). These proteins, freed from BAD, in turn associate with two other pro-apoptotic proteins, BAX and BAK. Such association prevents aggregation of these pro-apoptotic proteins on the mitochondrial membrane, stopping cytochrome c release and consequently inhibiting apoptosis (11Adams J.M. Cory S. Oncogene. 2007; 26: 1324-1337Crossref PubMed Scopus (1671) Google Scholar, 20Yang E. Zha J. Jockel J. Boise L.H. Thompson C.B. Korsmeyer S.J. Cell. 1995; 80: 285-291Abstract Full Text PDF PubMed Scopus (1883) Google Scholar).Recently several 7TMRs have been shown to activate the extracellular signal-regulated kinase (ERK) MAPK cascade and PI3K-AKT pathways in a β-arrestin-dependent manner (2DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1143) Google Scholar). In particular, it has been demonstrated that β-arrestin-mediated ERK activation by angiotensin II type 1A (AT1A) (21Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (546) Google Scholar, 22Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar), vasopressin V2 (23Ren X.R. Reiter E. Ahn S. Kim J. Chen W. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1448-1453Crossref PubMed Scopus (251) Google Scholar), parathyroid hormone (24Gesty-Palmer D. Chen M. Reiter E. Ahn S. Nelson C.D. Wang S. Eckhardt A.E. Cowan C.L. Spurney R.F. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2006; 281: 10856-10864Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), and β2-adrenergic receptors (25Shenoy S.K. Drake M.T. Nelson C.D. Houtz D.A. Xiao K. Madabushi S. Reiter E. Premont R.T. Lichtarge O. Lefkowitz R.J. J. Biol. Chem. 2006; 281: 1261-1273Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar) is G protein-independent. Furthermore, we have previously found that β-arrestin-mediated ERK activation is quite distinct in its temporal (22Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 23Ren X.R. Reiter E. Ahn S. Kim J. Chen W. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 1448-1453Crossref PubMed Scopus (251) Google Scholar, 24Gesty-Palmer D. Chen M. Reiter E. Ahn S. Nelson C.D. Wang S. Eckhardt A.E. Cowan C.L. Spurney R.F. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2006; 281: 10856-10864Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 25Shenoy S.K. Drake M.T. Nelson C.D. Houtz D.A. Xiao K. Madabushi S. Reiter E. Premont R.T. Lichtarge O. Lefkowitz R.J. J. Biol. Chem. 2006; 281: 1261-1273Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar) and spatial (22Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 26Tohgo A. Pierce K.L. Choy E.W. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 2002; 277: 9429-9436Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) patterns from ERK-activated via G protein-dependent stimulation. In the case of AT1AR-mediated ERK activation in receptor-transfected human embryonic kidney (HEK)-293 cells, for example, the G protein-dependent activation is rapid, quite transient (peak at 2-5 min), and leads to nuclear translocation of the activated ERK. In contrast, β-arrestin-2-dependent ERK activation is slower, quite persistent, and entirely confined to the cytoplasm, particularly to endosomal vesicles (22Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). Such differences in ERK activation strongly imply that there must be subsets of ERK targets and physiological outcomes differentially regulated through β-arrestin versus G protein-dependent mechanisms. In the case of AKT activation, stimulation of 7TM protease-activated receptors with thrombin has been shown to activate AKT in a β-arrestin-1-dependent manner (27Goel R. Phillips-Mason P.J. Raben D.M. Baldassare J.J. J. Biol. Chem. 2002; 277: 18640-18648Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Stimulation of the AT1 receptor also has been shown to activate AKT in several cell types including vascular smooth muscle cells (VSMCs) (28Mehta P.K. Griendling K.K. Am. J. Physiol. 2007; 292: C82-C97Crossref PubMed Scopus (1443) Google Scholar, 29Nakashima H. Suzuki H. Ohtsu H. Chao J.Y. Utsunomiya H. Frank G.D. Eguchi S. Curr. Vasc. Pharmacol. 2006; 4: 67-78Crossref PubMed Scopus (117) Google Scholar) although it has not been determined whether β-arrestins are involved in this signaling.Accordingly, the studies reported here were undertaken to examine the role of β-arrestin in 7TMR-regulated apoptosis and to determine the downstream pathways mediating this regulation in a physiologically relevant non-transfected cellular receptor system. For this purpose, we chose the AT1 receptor in rat VSMCs.EXPERIMENTAL PROCEDURESMaterials-[Sar1,Ile4,Ile8]AngII (Sar-Arg-Val-Ile-Ile-His-Pro-Ile) (SII) was synthesized as described (21Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (546) Google Scholar, 30Holloway A.C. Qian H. Pipolo L. Ziogas J. Miura S. Karnik S. Southwell B.R. Lew M.J. Thomas W.G. Mol. Pharmacol. 2002; 61: 768-777Crossref PubMed Scopus (197) Google Scholar). GF109203X (GFX), Ro-31-8425, U0126, and Z-VAD-fmk were purchased from Calbiochem. SL0101 was from Toronto Research Chemicals (North York, Ontario, Canada). All other reagents were purchased from Sigma, unless otherwise described. Plasmids expressing hemagglutinin-tagged wild-type, S112A and S136A mutant BAD were a generous gift from Dr. Michael E. Greenberg (Harvard Medical School).Antibodies-Rabbit polyclonal phospho-AKT (Ser473) (used in an 1:1,000 dilution for immunoblotting), AKT (1:2,000), phospho-BAD (Ser112) (1:500-1,000), BAD (1:1,000 for human BAD), Bcl-xL (1:1,000), caspase-3 (1:1,000), cleaved caspase-3 (Asp175) (1:1,000), phospho-ERK1/2 (Thr202/Tyr204) (1:2,000), and phospho-p90RSK (Thr359/Ser363) (1:500-1,000) antibodies were purchased from Cell Signaling (Danvers, MA). Polyclonal 14-3-3β (C-20) (1:1,000), RSK-1 (C-21) (1:1,000), and mouse monoclonal BAD (C-7) (1:1,000 for human BAD) antibodies were from Santa Cruz (Santa Cruz, CA). Monoclonal BAD (1:2,000 for rat BAD) and polyclonal ERK1/2 (1:10,000) antibodies were from BD Transduction Laboratories (Franklin Lakes, NJ) and Millipore (Billerica, MA), respectively. Human and rat β-arrestins were detected by A1CT and A2CT antibodies, respectively (31Attramadal H. Arriza J.L. Aoki C. Dawson T.M. Codina J. Kwatra M.M. Snyder S.H. Caron M.G. Lefkowitz R.J. J. Biol. Chem. 1992; 267: 17882-17890Abstract Full Text PDF PubMed Google Scholar).Synthesis of Small Interfering RNAs (siRNAs)-Chemically synthesized, double-stranded siRNAs, with 19-nucleotide duplex RNA and 2-nucleotide 3′-dTdT overhangs were purchased from Dharmacon (Lafayette, CO) in deprotected and desalted forms. The two different siRNA sequences targeting human (rat) β-arrestin-2 were 5′-GGACCGC(G)AAAGUGUUUGUG-3′ and 5′-CCAACCUCAUUGAAUUU(C)GA-3′, corresponding to positions 150-168 and 1112(1115)-1131(1133), relative to the start codon, respectively (32DeWire S.M. Kim J. Whalen E.J. Ahn S. Chen M. Lefkowitz R.J. J. Biol. Chem. 2008; 283: 10611-10620Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). A non-silencing RNA duplex (5′-UUCUCCGAACGUGUCACGU-3′), as the manufacturer (Xeragon, Germantown, MD) indicated, was used as a control.Cell Culture and Transfection-VSMCs were isolated from aorta of male Sprague-Dawley rats and maintained as described (32DeWire S.M. Kim J. Whalen E.J. Ahn S. Chen M. Lefkowitz R.J. J. Biol. Chem. 2008; 283: 10611-10620Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Eight to 90% confluent early passage (<5) VSMCs were transfected with siRNA using the Lipofectamine 2000 transfection reagent (Invitrogen) according to the modified manufacturer's instructions. Briefly, 60 μl of the Lipofectamine 2000 transfection reagent was added to 300 μl of the medium-199 and 20 μg of siRNA was added to 400 μl of the medium. Both solutions were allowed to stand 5-10 min at room temperature and mixed by inversion. Following a 15-20-min incubation at room temperature, the entire transfection mixture was added to VSMCs in a 100-mm dish containing 5 ml of the fresh, serum-free medium. After cells were incubated for overnight at 37 °C, an additional 5 ml of the medium with 20% fetal bovine serum and 2% penicillin/streptomycin were added to the dish. Following an additional incubation for 24 h, cells were divided into 6-well plates or 35-mm glass bottom dishes (MatTek, Ashland, MA) for further procedures. For DNA transfection into VSMCs in either 35-mm glass bottom dishes or 6-well plates, the Lipofectamine LTX transfection reagent (Invitrogen) was used with the PLUS reagent (Invitrogen) according to the manufacturer's instructions. HEK-293 cells, stably expressing AT1A receptors (∼600 fmol/mg of proteins), were established after transfection with a zeocin-resistant AT1A receptor expression plasmid using FuGENE 6 (Roche) according to the manufacturer's instructions. Stable clones were selected in the presence of zeocin (300 μg/ml) (Invitrogen). Cells were maintained and transfected with siRNAs using GeneSilencer (Genlantis, San Diego, CA) as described (22Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar).Cleaved Caspase-3 and DNA Fragmentation Assays-VSMCs were serum-starved for ∼24 h and then stimulated with 100 nm AngII or 10 μm SII at 37 °C for 5 min prior to treatment with either 80 μm H2O2 or 50 μm etoposide. After a further incubation overnight, cell lysates were prepared. Pro- and cleaved caspase-3 were visualized, and the amounts of cleaved caspase-3 were quantified as described for immunoblotting in this section. The amounts of fragmented DNA were measured by the enzyme-linked immunosorbent assay using the Cell Death Detection ELISAPLUS kit (Roche) according to the manufacturer's instructions.Immunostaining-VSMCs on 35-mm glass bottom dishes were fixed with 5% formaldehyde diluted in phosphate-buffered saline (PBS). Fixed cells were permeabilized by incubation with 0.2% Triton X-100 in PBS for 20 min. After blocking with 2% bovine serum albumin in PBS for 1 h, cells were incubated with the mouse monoclonal anti-cytochrome c (BD Pharmingen, 1:100) antibody at room temperature for 2 h, and repeatedly washed with PBS. Next, incubation of the Bodipy fluorescein-conjugated secondary antibody (Molecular Probes, 1:100) was done for 1 h at room temperature followed by repeated washes with PBS. For subsequent staining of the hemaglutinin epitope-tagged BAD, cells were incubated with a polyclonal hemaglutinin probe (Y11) antibody (Santa Cruz, 1:200) at room temperature overnight. The Texas Red-conjugated secondary antibody (Molecular Probes, 1:250) was then added for 1 h. Confocal images were obtained on a Zeiss LSM510 laser scanning microscope using single line (488 nm) or multitrack sequential excitation (488 and 568 nm) and emission (515-540 nm, Bodipy fluorescein; 585-615 nm, Texas Red) filter sets.Immunoblotting and Immunoprecipitation-In most cases, cellular extracts from VSMCs or HEK-293 cells on 6-well plates were prepared as described previously (22Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). For caspase-3 immunoblotting, VSMC lysates were prepared using the RIPA lysis buffer. Equal amounts (∼10 μg) of cellular extracts/lysates were used for immunoblotting performed as described previously (22Ahn S. Shenoy S.K. Wei H. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 35518-35525Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). For immunoprecipitation, lysates form HEK-293 cells in 100-mm dishes were prepared using the glycerol/Nonidet P-40 lysis buffer (33Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Equal amounts of cell lysates (300-500 μg of total protein in 1 ml of the buffer) were incubated with 1-2 μg of either rabbit polyclonal 14-3-3 β (C-20) or mouse monoclonal BAD (C-7) antibody and protein A- or G-agarose beads, respectively, overnight at 4 °C. Co-immunoprecipitated proteins were detected by immunoblotting. Each band in immunoblots was quantified by densitometry using the GeneTools program (SynGene).Statistical Analysis-Statistical significance in bar graphs was determined by using a one-way analysis of variance (PRISM software) to correct for multiple comparisons (Bonferroni's multiple comparison test) or t test for single comparisons between AngII or SII-stimulated versus non-stimulated in a certain condition, unless otherwise indicated in each figure. Statistical significance in each time point of kinetic graphs was determined by using a two-way analysis of variance (Bonferroni's post-test) between β-arrestin-2 siRNA-transfected and control cells. p value (*, p < 0.05; **, p < 0.01; ***, p < 0.001) of <0.05 was considered statistically significant.RESULTSβ-Arrestin-2 Is Essential for the Anti-apoptotic Effects of AngII on Primary Cultured Vascular Smooth Muscle Cells-It has been previously reported that the octapeptide hormone angiotensin II (AngII) promotes anti-apoptotic effects in VSMCs through AngII type 1 receptors (AT1Rs) (34Mallat Z. Tedgui A. Br. J. Pharmacol. 2000; 130: 947-962Crossref PubMed Scopus (284) Google Scholar, 35Pollman M.J. Yamada T. Horiuchi M. Gibbons G.H. Circ. Res. 1996; 79: 748-756Crossref PubMed Scopus (312) Google Scholar). We and others have previously found that β-arrestin-2 is the isoform that mediates AT1AR-activated ERK signaling (33Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 36Lee M.H. El-Shewy H.M. Luttrell D.K. Luttrell L.M. J. Biol. Chem. 2008; 283: 2088-2097Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), which is potentially an anti-apoptotic pathway. We have also found that β-arrestin-1 functionally opposes this signaling (33Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Consequently, we used VSMCs to explore the role of β-arrestin-2 in regulating apoptosis. We first monitored the effects of AngII stimulation on the activation of caspase-3 (cleavage of procaspase-3) induced by apoptotic challenges in VSMCs transfected with either control or β-arrestin-2 siRNAs. We used 100 nm AngII to obtain maximal responses in this assay. It has been previously reported that AngII-stimulated ERK activation reaches maximal levels with this concentration in HEK-293 cells transiently expressing AT1AR (33Ahn S. Wei H. Garrison T.R. Lefkowitz R.J. J. Biol. Chem. 2004; 279: 7807-7811Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Apoptotic stimulation by either hydrogen peroxide (H2O2) (Fig. 1A) or etoposide (Fig. 1B) dramatically increases the level of cleaved caspase-3 in control VSMCs. The levels of cleaved caspase-3 induced by such apoptotic stimuli were reduced to ∼50% by AngII pretreatment (Fig. 1, C and D). Next, we examined the effects of siRNA-mediated β-arrestin-2 knock-down on AngII-promoted protection from caspase-3 cleavage. Depletion of β-arrestin-2 not only leads to increases (up to ∼150%) in the induction of cleaved caspase-3 levels by treatment with hydrogen peroxide or etoposide, but also eliminates AngII-mediated decreases in these levels (Fig. 1, C and D). On the other hand, knocking-down β-arrestin-1 leads to decreases in apoptotic reagent-induced caspase-3 cleavage even in the absence of stimulation with AngII, which is similar in extent to the protection observed in AngII-treated control cells. Stimulation with AngII results in no significant further protection in cells knocked down for β-arrestin-1 (data not shown). These results suggest an essential role of β-arrestin-2 in AngII-stimulated protection from caspase-dependent apoptotic processes.Because the release of cytochrome c from mitochondria is one of the key upstream events for the coordinated activation of caspases (34Mallat Z. Tedgui A. Br. J. Pharmacol. 2000; 130: 947-962Crossref PubMed Scopus (284) Google Scholar, 37Fabregat I. Roncero C. Fernandez M. Liver Int. 2007; 27: 155-162Crossref PubMed Scopus (194) Google Scholar), we next examined effects of AngII stimulation on the hydrogen peroxide-induced redistribution of cytochrome c in control or β-arrestin-2 siRNA-transfected VSMCs. In the absence of an apoptotic challenge, cytochrome c is well distributed within cytoplasmic, mitochondrial networks in control cells (Fig. 1E, upper left panel). Treatment with hydrogen peroxide leads to the rearrangement of cytochrome c into a cytoplasmic dotted pattern in cells (Fig. 1E, upper middle panel). Hydrogen peroxide-induced redistribution of cytochrome c is largely reversed by prior stimulation with AngII (Fig. 1E, upper right panel). These staining patterns of cytochrome c are, in all cases, superimposed with that of the Mito-Tracker, a mitochondrial marker (supplemental Fig. S1), confirming that the changes in the staining patterns of cytochrome c shown in Fig. 1E indeed represent mitochondrial remodeling. Mitochondrial rearrangement and fragmentation have been observed in apoptotic cells, especially when apoptosis has been induced by pro-apoptotic members of the Bcl-2 family of proteins (38Desagher S. Martinou J.C. Trends Cell Biol. 2000; 10: 369-377Abstract Full Text Full Text PDF PubMed Scopus (1679) Google Scholar, 39Sun M.G. Williams J. Munoz-Pinedo C. Perkins G.A. Brown J.M. Ellisman M.H. Green D.R. Frey T.G. Nat. Cell Biol. 2007; 9: 1057-1065Crossref PubMed Scopus (196) Google Scholar). Depletion of β-arrestin-2 has no effect on mitochondrial network organization (Fig. 1E, lower left panel), and does not alter the pattern of fragmentation observed after hydrogen peroxide treatment (Fig. 1E, lower middle panel). However, unlike control siRNA-transfected cells, pre-stimulation with AngII fails to reverse the hydrogen peroxide-induced mitochondrial fragmentation in β-arrestin-2 siRNA-transfected cells (Fig. 1E, lower right panel). These results further support an important role for β-arrestin-2 in mediating AngII-stimulated anti-apoptotic activity.To further validate these findings, we measured DNA fragmentation, a hallmark of cell death, in VSMCs transfected with either control or β-arrestin-2 siRNAs. Treatment with hydrogen peroxide (Fig. 1F) or etoposide (Fig. 1G) causes a 4-6-fold increase in DNA fragmentation. In control siRNA-transfected cells, pre-stimulation with AngII reduces (by ∼40%) the extent of DNA fragmentation induced by both reagents (Fig. 1, F and G). On the contrary, we did not observe decreases in DNA fragmentation after prestimulation with AngII in β-arrestin-2 siRNA-transfected cells (Fig. 1, F and G), mirroring the situation observed with caspase-3 activation. In the case of β-arrestin-1 knock-down, effects on DNA fragmentation also mirror the effects observed with caspase-3 cleavage (data not shown). Taken together, these results demonstrate that β-arrestin-2 is crucial for AngII-stimulated protection of VSMCs from apoptotic cell death.To establish whether the β-arrestin-2-dependent AT1R-mediated inhibition of apoptotic processes can be accomplished in the absence of G protein activation, we stimulated VSMCs with an AngII analog, [Sar1,Ile4,Ile8]AngII (SII). This mutant form of AngII was previously shown not to activate G proteins (21Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10782-10787Crossref PubMed Scopus (546) Google Scholar, 30Holloway A.C. Qian H. Pipolo L. Ziogas J. Miura S. Karnik S. Southwell B.R. Lew M.J. Thomas W.G. Mol. Pharmacol. 2002; 61: 768-777Crossref PubMed Scopus (197) Google Scholar). As shown in Fig. 1, H and I, stimulation of the cells with SII leads to significant decreases (35-45%) in the levels of cleaved caspase-3 upon hydrogen peroxide or etoposide treatment. The extent of DNA fragmentation induced by these reagents is also reduced to a similar extent upon prestimulation with SII (Fig. 1J). The extent of SII-stimulated decreases (30-45%) in both assays is similar to those evoked by AngII stimulation (40-50%). This result demonstrates that activation of the AT1 receptor elicits cytoprotective effects mainly through G protein-independent signaling.β-Arrestin-2 Mediates Activation of ERK-RSK and AKT Signaling Pathways, Leading to Phosphorylation of BAD in AngII-stimulated VSMCs-Recently a growing list of biochemical pathways has been shown to be stimulated via 7TMRs through β-arrestin-dependent mechanisms (2DeWire S.M. Ahn S. Lefkowitz R.J. Shenoy S.K. Annu. Rev. Physiol. 2007; 69: 483-510Crossref PubMed Scopus (1143) Google Scholar). These include the ERK1/2 MAPKs (21Wei H. Ahn S. Shenoy S.K. Karnik S.S. Hunyady L. Luttrell L.M. Lefkowitz R.J. Proc. Natl.
Referência(s)