Phosphorylation of GATA-6 is required for vascular smooth ... · VASCULAR BIOLOGY Phosphorylation...

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Phosphorylation of GATA-6 is required forvascular smooth muscle cell differentiation aftermTORC1 inhibitionYi Xie,1* Yu Jin,1* Bethany L. Merenick,2,3* Min Ding,1,2,3 Kristina M. Fetalvero,2,3

Robert J. Wagner,3 Alice Mai,3 Scott Gleim,1 David F. Tucker,4† Morris J. Birnbaum,4

Bryan A. Ballif,5 Amelia K. Luciano,1 William C. Sessa,1 Eva M. Rzucidlo,3 Richard J. Powell,3

Lin Hou,6 Hongyu Zhao,6 John Hwa,1,2 Jun Yu,1 Kathleen A. Martin1,2,3‡

Vascular smooth muscle cells (VSMCs) undergo transcriptionally regulated reversible differentiation ingrowing and injured blood vessels. This dedifferentiation also contributes to VSMC hyperplasia aftervascular injury, including that caused by angioplasty and stenting. Stents provide mechanical supportand can contain and release rapamycin, an inhibitor of the mechanistic target of rapamycin complex1 (mTORC1). Rapamycin suppresses VSMC hyperplasia and promotes VSMC differentiation. We reportthat rapamycin-induced differentiation of VSMCs required the transcription factor GATA-6. Inhibition ofmTORC1stabilizedGATA-6andpromoted thenuclear accumulationofGATA-6, itsbinding toDNA, its trans-activation of promoters encoding contractile proteins, and its inhibition of proliferation. These effects weremediated by phosphorylation of GATA-6 at Ser290, potentially by Akt2, a kinase that is activated in VSMCswhen mTORC1 is inhibited. Rapamycin induced phosphorylation of GATA-6 in wild-type mice, but not inAkt2−/−mice. Intimal hyperplasia after arterial injury was greater inAkt2−/−mice than inwild-typemice, andthe exacerbated response in Akt2−/− mice was rescued to a greater extent by local overexpression of thewild-type or phosphomimetic (S290D) mutant GATA-6 than by that of the phosphorylation-deficient(S290A) mutant. Our data indicated that GATA-6 and Akt2 are involved in the mTORC1-mediated regulationof VSMC proliferation and differentiation. Identifying the downstream transcriptional targets of mTORC1may provide cell type–specific drug targets to combat cardiovascular diseases associated with excessiveproliferation of VSMCs.

INTRODUCTION

Mature vascular smooth muscle cells (VSMCs) retain plasticity to un-dergo phenotypic modulation in response to growth factor stimuli or inju-ry. VSMCs in the vessel wall normally exhibit a differentiated contractilephenotype, but can undergo phenotypic switching to a dedifferentiated,proliferative, and migratory phenotype with enhanced protein synthesisin response to extracellular cues (1, 2). This dedifferentiated or “synthetic”phenotype not only contributes to physiological processes such as vascularremodeling and angiogenesis, but it can also contribute to the pathogenesisof both atherosclerosis and intimal hyperplasia. Stents eluting rapamycin orrapamycin analogs have revolutionized coronary artery revascularization,reducing rates of restenosis compared to bare metal stents (3). Exploringthe molecular basis for the actions of mechanistic target of rapamycin

1Yale Cardiovascular Research Center, Vascular Biology and TherapeuticsProgram, and Departments of Medicine and Pharmacology, Yale UniversitySchool of Medicine, NewHaven, CT 06511, USA. 2Department of Pharmacologyand Toxicology, TheGeisel School ofMedicine at Dartmouth, Hanover, NH03755,USA. 3Department of Surgery, Section of Vascular Surgery, TheGeisel Schoolof Medicine at Dartmouth, Lebanon, NH 03756, USA. 4Institute for Diabetes,Obesity and Metabolism, Perelman School of Medicine, University of Pennsyl-vania, Philadelphia, PA19104,USA. 5Department ofBiology,University of Vermont,Burlington, VT 05405, USA. 6Department of Biostatistics, Yale School of PublicHealth, New Haven, CT 06510, USA.*These authors contributed equally to this work.†Present address: Integral Molecular, 3711 Market Street, Suite 900, Phila-delphia, PA 19104, USA.‡Corresponding author. E-mail: [email protected]

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(mTOR) complex 1 (mTORC1) inhibitors has important implications forfuture vascular therapeutics.

mTOR is a ubiquitously distributed serine/threonine protein kinase.When associated with other proteins in mTORC1, it serves an importantcheckpoint function in regulating specific protein synthesis in response tomitogens, stress, energy, and nutritional signals (4). mTORC1 coordinatesanabolic processes including cell growth, proliferation, and metabolism(5). mTORC1 activity can be inhibited by nutrient starvation or pharma-cologically by the inhibitor rapamycin (4).

The mTORC1 pathway is activated in VSMCs in response to vascularinjury in vivo. Rapamycin inhibits VSMC proliferation and migrationin vitro (6–8). Moreover, we have demonstrated that rapamycin treat-ment induces VSMC differentiation through increasing the expressionof contractile protein-encoding mRNAs (9). This is mediated by reliefof the classical feedback loop inwhichmTORC1 and its substrate S6 kinase1 (S6K1) promote insulin receptor substrate 1 (IRS-1) degradation todampen signaling through insulin and insulin-like growth factors (10).We have shown that in VSMCs, Akt2 is specifically activated in responseto mTORC1 inhibition, and that this induction of the activity of Akt2, butnot Akt1, is required for the VSMC differentiation response (10). The keydownstream transcriptional targets of Akt2 in vitro and in vivo are not yetknown.

Whereas mTORC1 was initially appreciated for its role in regulatingprotein synthesis inmammalian cells, little is known regarding mTORC1-mediated regulation of cell type–specific transcription. Here, we demon-strate that rapamycin promotes VSMC differentiation through activationof GATA-binding protein 6 (GATA-6), and that this signaling may be

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mediated by Akt2-mediated phosphorylation of GATA-6. We identify afunction of mTORC1 in regulation of cell type–specific transcription, afinding that has important implications for vascular therapeutics.

RESULTS

GATA-6 mediates the mTORC1-regulated modulation ofSMC differentiation and proliferationWehave previously shown that themTORC1 inhibitor rapamycin promotesVSMCdifferentiation through the classic feedback activation of the IRS-1–PI3K (phosphatidylinositol 3-kinase)–Akt pathway (10). mTORC1inhibition induces expression of VSMC-specific markers including smoothmuscle myosin heavy chain (SM-MHC), h-caldesmon, SM-a-actin, andcalponin at the mRNA and protein levels (9), which requires activation ofthe Akt2 isoform (10). Because smooth muscle contractile proteins aretranscriptionally regulated, we next sought to identify transcription factorsdownstream of Akt2 signaling. GATA-6 is present in mature, differenti-ated smooth muscle, but its abundance is rapidly decreased after vascularinjury and growth factor stimulation (11, 12). Because GATA-6 plays apotent antiproliferative, prodifferentiation role in VSMC invitro and in vivo(11, 12), we investigatedwhether GATA-6 couldmediate rapamycin-induceddifferentiation in human coronary artery SMCs (hCASMCs). Consistentwith our previous studies, rapamycin treatment inducedMYH11mRNAby more than fourfold in control transfected hCASMCs (Fig. 1A). Notably,GATA-6 knockdown significantly reduced the basal amount of MYH11mRNA and prevented rapamycin induction of this gene, which is the mostdefinitive marker of VSMC differentiation (13) (Fig. 1A). The same patternwas also observed at the protein level with multiple contractile markers(SM-MHC, calponin, and SM-a-actin) in hCASMCs from three differentdonors (Fig. 1, B and C, and fig. S1, A and B).

Rapamycin inhibits VSMC proliferation in vitro and in vivo (6–8). Wehave shown that rapamycin induces the cyclin-dependent kinase (CDK) in-hibitors p21cip and p27kip inVSMCs (9). The antiproliferative effect ofGATA-6inVSMCs depends on induction of p21cip (12), suggesting that p21cipmaybe a common antiproliferative effector of rapamycin andGATA-6. Consistentwith this notion,GATA-6knockdown reduced the basal amount of p21cip andprevented its induction by rapamycin (Fig. 1, B andC, and fig. S1,A andB).GATA-6 knockdown also significantly increased proliferation in hCASMCsand attenuated the inhibitory effects of rapamycin on proliferation (Fig. 1, Dand E, and fig. S1, C andD). These data indicate that GATA-6 plays a role inrapamycin-induced differentiation and proliferation, regulating genes encod-ing both SMC contractile proteins and cell cycle progression factors.

mTORC1 inhibition promotes GATA-6 transactivation ofan MHC-luciferase promoter reporterBecause GATA-6 was required for rapamycin-induced VSMC differenti-ation, we next assessed whether rapamycin alters GATA-6 transactivationof a smooth muscle–specific promoter. We used a truncated (836 basepairs) MYH11 promoter-luciferase reporter because it has few other reg-ulatory elements and its activation is GATA-dependent (14). Rapamycininduced a dose-dependent activation of thisMYH11 promoter-reporter intransfected hCASMCs (fig. S2A), suggesting that rapamycin could in-duce MYH11 transcription by activating endogenous GATA-6. Similarto rapamycin-treated cells, those overexpressing GATA-6 showed atwofold increase in reporter activity, an effect that was not significantlyenhanced by rapamycin treatment, suggesting that GATA-6 overexpres-sion had saturated reporter activity (fig. S2B). Notably, a GATA-6 mutantlacking the two DNA binding zinc finger domains (Dzf GATA-6) (12) notonly failed to activate the luciferase reporter but also prevented rapamycin-

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induced promoter-reporter activity (fig. S2B). We further demonstratedthat knockdown of endogenous GATA-6 in hCASMCs reduced basal ac-tivity and prevented rapamycin-induced expression from this reporter (fig.S2C). A reporter with a mutation of the GATA element showed reducedbasal activity and was not responsive to GATA-6 overexpression, thusconfirming the GATA dependence of this reporter (fig. S2D).

mTORC1 inhibition stabilizes GATA-6 protein, promotesits nuclear accumulation, and increases its DNAbinding activityGATA-6 protein abundance increased after rapamycin treatment (Fig. 1, Band C, and fig. S1, A and B). Rapamycin gradually increased endogenous

Fig. 1. GATA-6 is required for rapamycin-induced VSMC differentiation.
(A andB) hCASMCswere transfected with control or GATA-6 siRNAs, thentreated with ethanol (vehicle) or rapamycin for 48 hours, and analyzed for(A) GATA-6 or MYH11 mRNA by quantitative reverse transcription PCR(RT-PCR) (n = 3 biological replicates; *P = 0.05; n.s., not significant) or(B) abundance of SMC contractile proteins and p21cip (Western blot). (C)Quantitation of three replicate experiments from each of three different do-nors as in (B) (**P<0.01, ***P<0.001, ****P< 0.0001). (D) hCASMCsweretransfected with siRNAs as above and treated with either ethanol or rapa-mycin. Proliferation was monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 72 hours (n = 8 biologicalreplicates; ***P < 0.001, ****P < 0.0001). Data were presented as the foldchange of cell indexcompared to input. (E) hCASMCswere transfectedwithsiRNAs as above and treated with either ethanol or rapamycin. Cells weretrypsinized and counted (n = 3 biological replicates; *P = 0.05).


GATA-6 protein abundance over 2 to 24 hours (Fig. 2A)without significant-ly altering GATA-6mRNA abundance (Fig. 1A and fig. S3) in hCASMCs.GATA-6 protein abundance was also increased by rapamycin treatmentin vivo, as intraperitoneal injection of rapamycin increased Gata-6 and SM-MHC abundance in mouse arteries in a time-dependent manner (Fig. 2B).Immunofluorescence staining for endogenous GATA-6 in rapamycin-treatedhCASMCs similarly revealed an increase in GATA-6 abundance over time(Fig. 2C). Whereas the increase in GATA-6 abundance was apparent inboth the cytosol and nucleus, we noted strong nuclear accumulation afterrapamycin treatment (Fig. 2C and fig. S4). Rapamycin-induced nuclearaccumulation of GATA-6 was confirmed by biochemical subcellular frac-tionation of hCASMCs (fig. S5).

We next examinedwhether rapamycin affects GATA-6 protein half-lifeby measuring the rate of protein decay after inhibition of new protein syn-thesiswith cycloheximide. IRS-1 is stabilized after rapamycin treatment inVSMCs (10) and served as a positive control. Rapamycin pretreatmentbefore cycloheximide treatment increased IRS-1 protein abundance andhalf-life in hCASMCs (Fig. 2D and fig. S6). Similarly, rapamycin pre-treatment increased GATA-6 protein abundance and increased its half-lifefrom 4 to 7 hours (Fig. 2D and fig. S6), indicating that mTORC1 inhibi-tion stabilizes GATA-6 protein.

We next assessed the effect of mTORC1 inhibition on GATA-6 DNAbinding activity with chromatin immunoprecipitation (ChIP)–quantitativepolymerase chain reaction (qPCR) assays performed on rapamycin-treatedhCASMCs. In immunoprecipitates of endogenous GATA-6, the relativeabundance of coprecipitated DNAwas analyzed by qPCR with primer setsthat amplified the GATA element–containing regions within the humanSM-a-actin (ACTA2) and SM-MHC (MYH11) gene promoters. There was atime-dependent increase in GATA-6 binding at these GATA elements, indi-cating that rapamycin could increase GATA-6 DNA binding activity (Fig. 2E).

mTORC1 inhibition induces phosphorylation of GATA-6in an Akt2-dependent mannerTo elucidate the mechanism of how mTORC1 inhibition modulates theprotein abundance and DNA binding activity of GATA-6, we next inves-

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tigated posttranslational modifications ofGATA-6 triggered by rapamycintreatment. We have previously found that the inhibition of mTORC1 andS6K1 by rapamycin or adiponectin relieves the feedback inhibition ofIRS-1, resulting in increased PI3K and Akt2 activity (10, 15). VSMC dif-ferentiation induced by mTORC1 inhibition requires activation of Akt2(10, 15). GATA-6 contains an evolutionarily conserved basophilic se-quence motif (KPQKRVPS*), suggesting that Ser290 within this motif

Fig. 2. Rapamycin stabilizes GATA-6 protein and promotes its binding toDNA. (A) hCASMCs were treated with rapamycin for the indicated timesand immunoblotted for endogenousGATA-6 protein. The bar graph showsthe quantitation of GATA-6 protein abundance from three individual expe-riments [*P=0.05comparedwith vehicle (Veh) control]. p, phosphorylated. t,total. (B) Mice were intraperitoneally injected with vehicle or rapamycin andeuthanized at the indicated time points. Aortas and femoral arteries werepooled, and the lysates were immunoblotted for endogenous Gata-6 andSM-MHC proteins. The bar graph shows the quantitation of Gata-6 andSM-MHC protein abundance from three individual experiments (*P = 0.05compared with vehicle control). (C) hCASMCs plated on cover slides weretreated with ethanol or rapamycin, fixed at the indicated time points, and im-munostained forGATA-6.Z-stack imagesare shown. Scalebars,50 mm.Rep-resentative of two independent experiments. (D) hCASMCs were treatedwith ethanol or rapamycin for 4 hours and then with cycloheximide (CHX)for times indicated. Cells were harvested at the indicated time points andimmunoblotted for the indicated proteins. A representative Western blotanalysis is shown in fig. S6. The plot is the quantitation of three individualexperiments (P < 0.05, paired t test). (E) hCASMCs were treated with ve-hicle or rapamycin and subjected to ChIP assay with primer sets flankingtheGATAelements or control regions (lackingGATAelements) in the humanACTA2 andMYH11 gene promoters (n = 3 independent experiments; *P =0.05 compared with time zero).



could potentially be targeted byAkt. To determinewhether themTORC1pathway regulates GATA-6 through phosphorylation at this motif, GATA-6immunoprecipitates from rapamycin-treated hCASMCs were immuno-blotted with an antibody that detects the phosphorylatedAkt substrate motif(RXRXXS*/T*). GATA-6 phosphorylation was detected after 0.5 hour andpeaked between 2 and 4 hours of rapamycin treatment (Fig. 3A).

Because we have previously found that VSMC differentiation in re-sponse to mTORC1 inhibition depends specifically on activation of theAkt2 isoform (10, 15), we tested whether there was a similar Akt isoformspecificity for phosphorylation of GATA-6. Transfection of small inter-

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fering RNAs (siRNAs) directed against Akt2, but not against Akt1, pre-vented the rapamycin-induced phosphorylation of GATA-6 in hCASMCs(Fig. 3B). Consistent with this finding, transfection of Akt2 or Myr-Akt2,but not of Akt1, induced phosphorylation of GATA-6 in human embryonickidney (HEK) 293 cells (fig. S7). Furthermore, intraperitoneal injection ofrapamycin induced phosphorylation of Gata-6 as detected by the Aktphospho-substrate motif antibody in the arteries of wild-type mice but notin Akt2−/−mice (Fig. 3C). Although this evidence supports a role for Akt2in phosphorylation of GATA-6, the motif recognized by the antibody isbasophilic and could potentially be phosphorylated by protein kinase A(PKA) (16). However, treatment of hCASMCs with the PKA inhibitor ef-ficiently inhibited phosphorylation of the PKA substrate cyclic adenosinemonophosphate response element–binding protein (CREB) at Ser133, but didnot diminish rapamycin-induced immunoreactivity with the Akt phospho-substrate motif antibody (fig. S8).

To determine whether Akt2 can directly phosphorylate GATA-6, weperformed invitro kinase assays using recombinant Akt1 orAkt2 proteins.Both recombinant Akt1 and Akt2 phosphorylated a glycogen synthase ki-nase 3b (GSK3b substrate peptide invitro, but only Akt2 phosphorylatedfull-length GATA-6 immunoprecipitated from HEK293 cells, as detectedby the Akt phospho-substrate antibody (Fig. 3D). To determine whetherSer290 was directly phosphorylated by Akt2, we used GST-fusion proteinsencoding 20 amino acids of GATA-6, centered around either wild-typeSer290 or a S290A mutation, as substrates for in vitro kinase assays. Thewild-type, but not the S290A sequence, was phosphorylated specificallybyAkt2; therewasminimal incorporation of 33PwithAkt1, althoughAkt1had greater activity toward the GSK3b control substrate in this assay (Fig.3E). Mass spectrometry (MS) analysis of an Akt2 in vitro kinase assayusing a synthetic substrate peptide encoding this region of GATA-6 con-firmed phosphorylation of Ser290 because this generated a spectrumidentical to that of a corresponding synthetic peptide containing phospho-serine at position 290 (fig. S9). We additionally assessed the Akt isoformspecificity for Ser290 in intact cells by coexpressing Akt1 or Akt2 withGATA-6 wild type or S290A in HEK293 cells. GATA-6 was phospho-rylated in cells coexpressing Akt2, but not Akt1, and this depended onSer290 (fig. S10).

Akt1 and Akt2 are highly homologous (82% identity between mouseAkt1 and Akt2) (17) with a conserved four-domain structure that includesPH, linker, catalytic, and regulatory domains, with the highest degree ofhomology (91%) in the catalytic domain. To identify regions that mediatethe Akt2-specific phosphorylation of GATA-6, we cotransfected GATA-6into HEK293 cells with epitope-tagged chimeric Akt constructs in whichone ormore domains are swapped betweenAkt1 andAkt2 (17) (fig. S11A).Of the eight chimeras tested, only those containing the Akt2 linker domain(Akt1222, Akt2211, Akt2221, and Akt2212), but not those containing theAkt1 linker domain, induced phosphorylation ofGATA-6 (fig. S11,A andB). These data suggest that the linker domain, which has the lowest iden-tity (46%) between (mouse) Akt1 and Akt2 (17), confers GATA-6 sub-strate specificity to Akt2.

Phosphorylation of Ser290 promotes GATA-6 DNAbinding and functionTo investigate the effect of phosphorylation on GATA-6 activity, we usedphospho-mimetic (S290D) and nonphosphorylatable (S290A) mutants.To assess the effects of this phosphorylation on DNA binding, we gen-erated stable hCASMC clones that overexpressed Myc epitope–taggedwild-type GATA-6, S290D, or S290A. We verified Myc–GATA-6 over-expression in ~100%of the cells by immunostaining (fig. S12). ChIP-qPCRanalysis using anti-Myc antibody revealed that the S290Dmutant boundmore target DNA per protein input than did the wild type or S290A,

Fig. 3. Rapamycin induces phosphorylation of GATA-6 in an Akt2-dependent
manner. (A) GATA-6 immunoprecipitates (IP) from hCASMCs treated withrapamycin for the indicated times were Western-blotted with Akt phospho-substrate motif antibody (RXRXXS*/T*). (B) hCASMCs were transfectedwith control or siRNAs against Akt1 or Akt2 and treated with ethanol or ra-pamycin. GATA-6 immunoprecipitates from lysates were Western-blottedwith Akt phospho-substrate motif antibody (RXRXXS*/T*). (C) Wild-type(WT) or Akt2 knockout (KO) mice were injected intraperitoneally with vehi-cle or rapamycin. Aortas and femoral arteries were harvested and lysed.Gata-6 immunoprecipitates from these lysates were Western-blotted withAkt phospho-substrate motif antibody (RXRXXS*/T*). (D) GATA-6 immuno-precipitates fromHEK293cells (upper panel) or recombinant GSK3protein(bottom panel) were used as substrates in in vitro kinase assays with re-combinant Akt1 or Akt2 and immunoblotted with Akt phospho-substratemotif antibody (RXRXXS*/T*). (E) Bacterial glutathione S-transferase(GST), GST-GATA-6_20aa_WT, or GST-GATA-6_20aa_S290A protein wasused as substrate in in vitro kinase assays with recombinant Akt1 or Akt2and [33P]adenosine triphosphate (ATP). The samples were subjected toSDS-PAGE and autoradiography. (A) to (D) are representative of at leastthree individual experiments. (E) is representative of two individual experiments.


indicating that phosphorylation at Ser290 enhanced the DNA binding af-finity. The binding of the S290D mutant to the MYH11 or ACTA2 pro-moters was two- to threefold greater than that of wild-type GATA-6. Thebinding of the S290A mutant was modestly but significantly reducedcompared to that of wild-type GATA-6. Amplification with negativecontrol primers confirmed the specificity of this assay (Fig. 4A).

To determine whether the increased DNA binding of the GATA-6S290D mutant leads to an increase in functional regulation of transcrip-tion, we transfected hCASMCs with plasmids encoding Myc–GATA-6wild type, S290D, or S290A. Expression of the GATA-6 S290D mutantin hCASMCs increased the abundance of smooth muscle differentiation–specific contractile proteins, including SM-MHC, SM-a-actin, and SM-calponin by 1.5- to 2.0-fold compared to untransfected hCASMCs (Fig. 4,B and C). The S290D mutant was about twice as potent as GATA-6 wildtype and nearly four times as potent as S290A (Fig. 4C). Expression of theS290Dmutant also increased the abundance of the CDK inhibitor proteinsp21cip and p27kip (Fig. 4, B and C). Lentiviral overexpression of GATA-6S290D inhibited hCASMC proliferation to a greater extent than did wild-type or S290A GATA-6 (Fig. 4D), consistent with the greater increase inthe abundance of p21cip and p27kip in S290D-expressing cells (Fig. 4, Band C). Collectively, these data support that phosphorylation of GATA-6at Ser290 is an activating modification that increases GATA-6 DNA bind-ing, prodifferentiation, and antiproliferative activities in VSMCs.

To determine whether phosphorylation at Ser290 might mediate therapamycin-induced GATA-6 protein stabilization (as in Fig. 2D), we de-termined the protein half-life of wild-type or mutant GATA-6 expressedat low amounts in cycloheximide-treated hCASMCs. The S290Dmutanthad a longer protein half-life than wild-type or S290AGATA-6 (Fig. 4, Eand F), suggesting that phosphorylation of GATA-6 at Ser290 also en-hances the protein stability. It is likely that this posttranslational modifi-cation enhances GATA-6 functional activity by promoting both its proteinstabilization and DNA binding.

GATA-6 expression attenuates neointima formation inthe mouse femoral artery wire injury modelWe have previously shown that Akt2 is a key effector in rapamycin-inducedVSMC differentiation (10). To support that Akt2 mediates this effectthrough phosphorylation of GATA-6 in vivo, we determined the effectsof Akt2 loss of function and GATA-6 phosphorylation site mutants inthe mouse femoral artery wire injury model. Inserting a guide wire intothe mouse femoral artery models the endothelial denudation and mechan-ical injury that leads to intimal hyperplasia in humans after angioplasty(18) (fig. S13). Wild-type C57BL/6 mice exhibited only modest neointimaformation after this procedure (Fig. 5A). Consistent with a prodifferen-tiation function for Akt2 in VSMCs (10), Akt2 knockout mice exhibitedmore severe intimal hyperplasia than did wild-type mice as indicated by an~80% increase in the intima/media area ratio (Fig. 5, A and B). BecauseGATA-6 overexpression rescues neointimal formation in rats (11), we nextdetermined the effects of local overexpression of wild-type or mutantforms of GATA-6 in Akt2−/− mice. Control lentivirus or virus encodingMyc-taggedGATA-6wild type, S290D, or S290Awas applied in a pluronicgel to the adventitial side of the femoral artery at the time of wire injury.Overexpression of wild-type or mutant forms of GATA-6 significantlyattenuated intimal hyperplasia in the Akt2−/−mouse compared to controlvirus; however, the mutants differed in potency (Fig. 5, C and D). TheS290D mutant more potently inhibited intimal hyperplasia than did wild-type GATA-6, and the S290Amutant conferred only a partial rescue com-pared to control virus (Fig. 5, C and D). The trends in the effects of theGATA-6 constructs on intimal hyperplasia (Fig. 5, C and D) and prolifera-tion (as measured by Ki67 staining in medial and neointimal cells) (Fig. 5E

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and fig. S14) were similar. Proliferation was increased to a greater extentin Akt2−/− arteries compared to the wild-type arteries, an effect that wasinhibited by overexpression of wild-type GATA-6. The S290D mutant

Fig. 4. Phosphorylation of Ser290 promotes GATA-6 DNA binding and
function. (A) hCASMCs transduced to express Myc-tagged WT GATA-6 orthe S290D or S290Amutants were subjected to ChIP-qPCR assay with Mycantibody and primer sets flanking the GATA elements or control regions(lacking GATA elements) in the promoters of human ACTA2 and MYH11genes. Data were normalized to the expression of each mutant (n = 3independent experiments; P = 0.05, indicated by brackets). (B) hCASMCstransfected with vector or WT, S290D, or S290A Myc–GATA-6 were immu-noblotted for the indicated proteins. (C) Quantitation of (B) (n = 6 biologicalreplicates; P < 0.05, indicated by brackets). (D) Proliferation in hCASMCstransduced as in (A) was monitored by MTT assay for 60 hours (n = 7biological replicates; P < 0.01, indicated by brackets). Data were presentedas the fold change of cell index compared to input. (E) hCASMCs trans-fected as in (B) were treated with cycloheximide for the indicated timesbefore immunoblotting with Myc antibody to determine GATA-6 proteinhalf-life. (F) Quantitation of (E) (n = 3 independent experiments; paired t test,P = 0.0625 between the WTGATA-6 and GATA-6 S290D groups, as well asbetween the GATA-6 S290D and S290A groups).


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was significantly more potent than wild-type GATA-6 in inhibiting pro-liferation in Akt2−/− arteries, whereas the S290A mutant only partiallyrescued this phenotype (Fig. 5E and fig. S14). These findings wereconsistent with the in vitro efficacy of these mutants in their regulationof DNA binding, expression of contractile proteins, and proliferation inVSMCs (Fig. 4, A to D).

Immunostaining for the Myc epitope tag indicates that the over-expressed proteinswere expressed at similar amounts at 6 days after injury,and that whereas some adventitial expression was detected, the over-expressed GATA-6 proteins were abundant in the medial layer (fig. S15).The abundance of contractile proteins decreases after vascular injury (2).Notably, immunostaining for SM-a-actin colocalized with the over-expressed wild-type and mutant GATA-6 at 6 days after injury in Akt2−/−

mice (fig. S15). Quantitation of these images indicates that the intensityof SM-a-actin staining per cell in the medial layer after injury correlateswith the efficacy of the GATA-6 constructs in increasing contractile pro-tein abundance in vitro (S290D>wild type > S290A) (fig. S15 and Fig. 4,B and C). These studies confirm the role of Akt2 in the modulation ofVSMC phenotype in vivo as well as the effects of the Ser290 phosphoryl-ation site on GATA-6 function in an in vivo model of intimal hyperplasia.

DISCUSSION

Akt2 is specifically activated in response to mTORC1 inhibition and pro-motes VSMC differentiation in vitro (10, 15). We now report that loss ofAkt2 markedly exacerbates intimal hyperplasia, and that GATA-6 may bethe substrate through which Akt2 promotes VSMC differentiation in vitroand in vivo (fig. S16). Furthermore, local vascular overexpression ofGATA-6, particularly the S290D phospho-mimetic mutant, rescues theAkt2−/− injury phenotype. These findings provide insights into the thera-peutic mechanisms of mTORC1 inhibitors aswell as an important role ofAkt2 in the regulation of VSMC phenotype.

Akt isoform–specific regulation of GATA-6Although the highly homologous Akt isoforms share many common sub-strates (19), the differing phenotypes of the Akt1 and Akt2 knockout micesuggest that unique substrates exist. We have identified GATA-6 as apotential Akt2-specific substrate in VSMCs. In vivo, Akt1−/−mice displayreduced cell and organism size,whereasAkt2−/−mice are prone to diabetes(20, 21). However, the doubleAkt1/Akt2 knockout dies shortly after birth,suggesting that there is also considerable functional redundancy betweenthese isoforms (22). We now report that loss of Akt2 led to severe intimalhyperplasia after injury, and that overexpression of GATA-6 could rescuethis phenotype. This is consistent with our previous in vitro findings thatAkt2, but not Akt1, is necessary for rapamycin-induced VSMC differ-entiation, and that overexpression of Akt2, but not Akt1, is sufficient topromote VSMCdifferentiation (10). Although it is possible that Akt2 reg-ulation of GATA-6 could be indirect, we demonstrate that Akt2 is requiredfor rapamycin-induced phosphorylation of GATA-6 in vitro and in vivo,and contributes to the prodifferentiation and antiproliferative effects of ra-pamycin on SMCs. Akt1 stimulates different responses in VSMCs, namely,migration and proliferation (23). Thus, Akt1 and Akt2 appear not only tobe nonredundant but also to serve opposing functions in VSMCs. Thereis precedence for opposing roles for Akt isoforms in a cell type–specificmanner. For example, Akt1 inhibits but Akt2 promotes migration in breastcancer cells (24–26), whereas Akt2 inhibits and Akt1 promotes migrationin mouse embryonic fibroblasts (MEFs) (27). It is likely that these celltype differences arise because of differences in the abundance and local-ization of both Akt isoforms and substrates, some of which may be cell

Fig. 5. GATA-6 expression attenuates neointima formation in the mousefemoral artery wire injury model. (A) Mice were subjected to wire injury inthe left femoral artery to denudate the endothelial layer and induce neo-intima formation. Neointima formation was analyzed 21 days after injuryby light microscopy. (B) The neointima area and intima/media ratio in (A)were quantitated (n = 11 mice per group; *P < 0.05, **P < 0.01). (C) WT orAkt2 knockout mice received femoral artery injury as in (A). Immediatelyafter injury, lentivirusencodingWT, S290D, or S290AmutantGATA-6cDNAor control virus was loadedonto the injured artery. Neointima formationwasanalyzed as above. (D) Quantitation of intima/media ratio in (C) (n = 4micein eachgroup; *P< 0.05). (E) Cryosections from (A) were subjected to Ki67immunohistochemistry. The data are presented as the percentage of Ki67-positive cells of the total cells in the media and intima areas. Five differentsections from each of two mice per treatment condition were quantitated(*P < 0.05, **P < 0.01). Scale bar,50 mm. Arrowheads denote maximalneointimal thickness in each section.



type–specific. This has important implications for the therapeuticpotential of Akt isoform–specific inhibitors.

The Akt linker domain has been implicated in the isoform-specificregulation of migration in MEFs (17, 27). We determined that phospho-rylation of GATA-6 may require the linker domain of Akt2, the region ofgreatest divergence between Akt isoforms that could potentially mediatea specific interaction between Akt2 and GATA-6. Whereas many Aktsubstrates are phosphorylated by all Akt isoforms in kinase assays in vitro,Akt2 appeared to be specific for GATA-6 in vitro and in intact cells. Simi-larly, Akt2, but not Akt1, regulates FoxO4 in response to mTORC1 inhibi-tion in VSMCs (15).

Mechanisms and consequences of mTORC1 regulationof GATA-6The important role of GATA-6 in intimal hyperplasia (11), together withintriguing data indicating that the TOR pathway could regulate GATA-family transcription factors in yeast (28), prompted us to investigatemTORC1-mediated regulation of GATA-6 in VSMCs. We found thatwhereasmTORC1 inhibition similarly activatesGATA-6, themechanismsin humanVSMCand yeast appear to be distinct. In yeast, the GATA factorGln3 is activated rapidly, translocating to the nucleus within 10 min ofTOR inhibition (28), which requires dephosphorylation of Gln3 by thephosphatase Tap42. We found that GATA-6 activation in human VSMCsis slower, possibly due to the requirement for feedback activation of Akt2.Whereas it is highly probable that GATA-6 contains additional phospho-rylation sites, we found that mutation of Ser290 has marked effects on pro-tein function, especially in vivo. We note that trace 33P incorporation wasdetected in the in vitro kinase reaction with Akt2 and the GST-S290A sub-strate, suggesting that Ser291, the only other proximal serine within thisGATA-6 sequence, could also be phosphorylated, butmost likely onlywhenthe primary site, Ser290, is mutated. This scenario can indeed occur whenthere aremultiple serine residues neighboring a basophilic site (29). OurMSanalysis did not detect phosphorylation of Ser291 in the wild-type GATA-6peptide. We detected a modest immunoreactivity with the RXRXXS*/T*phospho-substrate antibody in cells overexpressing GATA-6 S290A andAkt (fig. S10). This immunoreactivity could represent detection of phos-phorylation at Ser291, or perhaps another basophilic site elsewhere in theprotein. Although the contribution of other potential phosphorylation sitesis not yet known, our data suggest that phosphorylation of Ser290 affectsGATA-6 protein function and that Akt2 could be the kinase that phospho-rylates this site.

We found that phosphorylation of GATA-6 at Ser290 affected multipleparameters that collectively led to increased GATA-6 activity: increasedDNA binding, perhaps due to the location of this phosphorylation site be-tween the two zinc finger domains, and increased stability. The S290Dmutant also increased the expression of GATA-6 target genes, includingthose encoding contractile proteins and inhibitors of proliferation, in cul-tured cells. When locally overexpressed at the site of vascular injury, thewild type, S290D, and S290A forms of GATA-6 all diminished intimalhyperplasia and proliferation in Akt2−/− vessels. However, the S290Dmutant was more effective than wild type, and both forms were substan-tially more effective than the S290Amutant. These effects are consistentwith the differences in protein half-life observed in vitro. Whereas differ-ences in antiproliferative activity between themutantswere not significantin vitro, likely due to the relatively short time point assessed, the phospho-mimetic mutant was the most potent inhibitor of proliferation in vivo,followed by wild type, then the S290A mutant. Our results suggest thatinhibition of proliferation plays a major role in the inhibitory effect ofGATA-6 on intimal hyperplasia. However, the increase in contractile proteinabundance colocalized with the overexpressed GATA-6 proteins and also

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correlated with phosphorylation status, suggesting that promotion ofVSMC differentiation may also contribute to this effect.

Role of GATA-6 in VSMC phenotypeStudy of GATA-6 in vivo has been hindered by the very early (E6.5) em-bryonic lethality of the global knockout (30) and severe developmentaldefects in the smooth muscle– or neural crest–specific knockouts (31).GATA-6 exerts potent antiproliferative effects in VSMCs (12) and mesangialcells (32). Its abundance is decreased upon vascular injury, and localGATA-6 overexpression can rescue injury-induced intimal hyperplasiain vivo (11). Consistent with these findings, our study also supports a pro-differentiation, antiproliferative, and antihyperplastic role for GATA-6.Others have reported that the transcription factor CHF1/Hey2, implicatedin intimal hyperplasia, directly interacts with and represses GATA-6 (33).In contrast, a microarray using an engineered dominant negative GATA-6fusion protein suggested that GATA-6 regulates genes associated with thededifferentiated VSMC phenotype (34).

The ability of VSMCs to reversibly differentiate has been intenselyinvestigated. The current paradigm has focused on the interaction ofthe potent prodifferentiation transcriptional coactivator myocardinwith serum response factor at CArG elements in smooth muscle promo-ters (1, 35). Several studies raise the possibility that GATA-6 may func-tion in concert with myocardin to promote contractile gene expression inVSMCs. GATA-6 may have differential effects on contractile gene pro-moters, depending on the spacing between CArG and GATA elements inthe promoters, resulting in GATA-6 inhibition of or synergy with myo-cardin: GATA-6 represses the promoter activity of the CArG-dependentsmooth muscle gene telokin but induces the expression of SM-MHC,MLCK (myosin light chain kinase), and SM22a (36). Similarly, theclosely related GATA-4 can stimulate or suppress myocardin activity in agene-specific manner (37). We found that knockdown of GATA-6 alonereduced contractile protein abundance by up to 60% below baselineamounts. The magnitude of this effect is similar to the degree of inhibi-tion observed with myocardin knockdown (38, 39). Because the majorVSMC genes assessed in our study, including SM-MHC, calponin, andSM-a-actin, are regulated by CArG elements (1), our data suggest that inthe context of mTORC1 inhibition, GATA-6 may cooperate with myo-cardin to regulate these promoters. This will be an important area forfurther study.

mTORC1-regulated transcriptionOur work reveals that the mTORC1 pathway exerts tissue-specific tran-scriptional control, a finding with potential therapeutic relevance. Thereversible differentiation of smooth muscle is transcriptionally regulatedthrough incompletely understood mechanisms. We now implicate regu-lation of GATA-6 bymTORC1 and potentially Akt2 as a keymechanismunderlying the efficacy of rapamycin in promoting VSMC differentia-tion. mTORC1 coordinates growth and metabolism through transcrip-tional mechanisms, including regulation of HIF1a (hypoxia-induciblefactor 1a) and SREBP1c (sterol regulatory element–binding protein 1c)(40). Whereas knockout mouse studies have revealed specific mTORC1-regulated processes in different tissue types including the liver and skel-etal muscle (5), little is known about cell type–specific transcriptionalcontrol downstream of mTORC1. mTORC1 is required for adipocytedifferentiation through regulation of PGC1a [peroxisome proliferator–activated receptor g (PPARg) coactivator 1a] andPPARg (5, 41,42).Wehaveshown thatmTORC1 inhibition promotesVSMCdifferentiation (9, 10, 15).Together, these findings suggest that distinct cell type–specific transcrip-tion factor targets may be regulated by the mTORC1 pathway in differenttissues.



Therapeutic relevanceThe phenotypic modulation of vascular smooth muscle is critical not onlyfor normal growth and healing in blood vessels but also in pathologiesincluding atherosclerosis, restenosis, hypertension, and transplant arterio-sclerosis. Although rapamycin is highly efficacious as a stent therapeutic,it cannot be used systemically for long-term treatment or prevention ofvascular disease because the high doses required are associated with ad-verse effects including impaired wound healing, leukopenia, and thrombo-cytopenia (43). We showed that systemic rapamycin treatment increasedthe abundance of SM-MHC and GATA-6 in healthy, mature wild-typeaorta, and that overexpression of GATA-6 with the S290D mutant rescuedsevere intimal hyperplasia. Elucidating the smoothmuscle–specific signalingand downstream targets of mTORC1 inhibition such as Akt2 and GATA-6may give rise to new therapeutic strategies for cardiovascular diseases.

MATERIALS AND METHODS

Cell culturehCASMCs (Cascade Biologics or Lonza) (passages 3 to 6) were propa-gated in M199 medium with 10% fetal bovine serum (FBS) as described(9, 10). SMCswere culturedwith 2.5%FBS for 16 to 24 hours before drugtreatment. [hCASMCs proliferate slowly and do not spontaneously differ-entiate under these conditions (9, 10).] Rapamycin (20 nM) or ethanol ve-hicle was added at the indicated time points, up to 48 hours. HumanVSMCs from peripheral vessels were prepared by explant method (9). Ve-hicle was added for the maximum duration of treatment.

Cell lysis and Western blottingCells werewashed with phosphate-buffered saline (PBS) and harvested inradioimmunoprecipitation (RIPA) assay buffer with protease and phos-phatase inhibitors (Roche). Equal amounts of protein per lane were sep-arated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE), transferredonto a nitrocellulose membrane, and immunoblotted with primary antibodiesagainst SM2-MHC, SM-a-actin, calponin-1 (Sigma), GATA-6 (Abcam),GAPDH (glyceraldehyde-3-phosphate dehydrogenase), IRS-1, Akt1, Akt2,pan-Akt, Myc tag, phospho-Akt (Ser473), phospho-S6 (Ser240/244), CREB,phospho-CREB (Ser133), phospho-(Ser/Thr) Akt substrate (RXRXXS*/T* motif), phospho-(Ser/Thr) PKA substrate (RRXS*/T* motif) (CellSignaling), b-tubulin, p21cip, p27kip, DNA polymerase II (Santa Cruz Bio-technology), or hemagglutinin (HA) epitope and horseradish peroxidase–conjugated secondary antibodies (Pierce). Blots were developed usingenhanced chemiluminescence reagents (Pierce), and signals were quan-titated using ImageJ.

Quantitative RT-PCRRNAwas isolated from cells using the Qiagen RNeasy kit, and 1 mg of RNAwas reverse-transcribedusing an iScript cDNAsynthesis kit (Bio-Rad). qPCRwas performed using primers for GATA-6 (sense: 5′-GTGCCTTCAT-CACGGCGGCT, antisense: 5′-CACACGGGTTCACCCTCGGC) andMYH11 (Qiagen) using the DDCT method. GATA-6 and MYH11 productswere normalized to b-actin (ACTB) (sense: 5′-GACAGGATGCAGAAG-GAGA, antisense: 5′-CCACATCTGCTGGAAGGTGG) product, a house-keeping gene control.

Transient transfection of siRNA and plasmidTransient transfection of siRNA in hCASMCswas performed using Lipo-fectamine RNAiMAX (Life Technologies). Cells were plated in six-wellplates or 10-cm dishes the day before transfection to reach a confluency of70 to 80%. Transfection reagent and siRNA were mixed in Opti-MEM

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(Gibco) according to the manufacturer’s protocol and added to the cellscultured in Opti-MEM. M199 medium with 20% FBS was added to thecells after 6 hours. After 24 hours, the medium was changed toM199me-dium with 2.5% FBS, and the cells were cultured overnight before drugtreatment [vehicle or 20 nM rapamycin (Calbiochem)]. GATA-6 and non-silencing siRNAs (siControl) were purchased fromDharmacon, and siRNAsfor Akt1 and Akt2 were purchased fromQiagen. For overexpression, 8 mg ofplasmid DNAwas transfected into 1 million to 1.5 million hCASMCs usingNucleofector (Lonza) (10). Transient transfection of plasmid DNA toHEK293 cells was done using X-tremeGENE 9 (Roche).

Lentiviral expression of GATA-6 mutantspLVX-IRES-puro plasmids containing complementary DNAs (cDNAs)encoding Myc-tagged GATA-6 mutants were cotransfected with pack-aging vectors (pMDLg/pRRE, pRSV-Rev, pMD2.G) into HEK293 cells.Lentivirus secreted into themediawas isolated by ultracentrifugation, resus-pended in PBS, and used to infect hCASMCs in the presence of polybrene(8 mg/ml; Santa Cruz Biotechnology). Seventy-two hours after infection,transduced cells were selected in puromycin (0.75 mg/ml) (InvivoGen) for48 hours.

Luciferase reporter assayVSMCswere transfected with a firefly luciferase reporter construct drivenby a GATA element from the rat MYH11 promoter (14) and thymidinekinase–Renilla–luciferase construct (44) (transfection control). pcDNAvector or GATA-6 expression plasmids were cotransfected where indi-cated. Twenty-four hours after transfection, cells were treated with rapa-mycin. In the mutant MYH11 (SM-MHC) promoter, the GATA elementGTAATCATCACAwas mutated to GTAAcagcCACA using KODXtremeHot Start DNA Polymerase (Novagen). Luciferase assays were performedusing the Dual-Luciferase System (Promega).

ImmunofluorescencehCASMCs were cultured on glass coverslips, treated, washed with PBS,fixed in ice-cold 4% paraformaldehyde (PFA), blocked with 10% FBS inPBS with 0.1% Triton X-100 (PBS-T), and incubated with anti–GATA-6(Santa Cruz Biotechnology, clone H92) or anti–Myc epitope antibody inPBS-Tat 4°C overnight. Cells were then incubated with Alexa Fluor 488–or Alexa Fluor 594–conjugated secondary antibodies (Molecular ProbesInc.) in PBS-T for 1 hour, and 4′,6-diamidino-2-phenylindole (DAPI) stain-ing was performed before mounting and analysis using a spinning disc con-focal microscope (Perkin-Elmer).

Nuclear-cytosolic fractionationhCASMC cytosolic and nuclear fractions were separated using theSubcellular Protein Fractionation Kit (Pierce).

ImmunoprecipitationCells were harvested in 1× cell lysis buffer (Cell Signaling) containingprotease and phosphatase inhibitors. An equal amount of protein lysateper sample was incubated overnight with antibody to GATA-6 (R&DSystems Inc.) and then with protein G–Sepharose beads (AmershamBiosciences/GE Healthcare) at 4°C. Bound protein was washed, elutedby heating in 1× SDS sample buffer, and subjected to Western blotting.

Chromatin immunoprecipitation–quantitativepolymerase chain reactionFour confluent 10-cm dishes of hCASMCs (~4 × 106 cells) were treatedwith 1% formaldehyde to cross-link protein to DNA and were neutralizedwith glycine. Cells were scraped in cold PBS and subjected to ChIP assay



using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling)with antibody to GATA-6 (R&D Systems Inc.) or Myc tag (Cell Sig-naling). Precipitated DNA was analyzed by qPCR with the followingprimer sets: ACTA2 promoter, sense: 5′-AGGCCTCCGGCCACCCA-GATTA and antisense: 5′-GCCTGCTCTCCTCCCACTTGCT; MYH11promoter, sense: 5′-GACACAGAGACCAGAGACAAAG and antisense:5′-TCACTCTGATCATTGCTGTCTC. The amount of coprecipitatedDNAwas normalized to the amount of PCR product amplified from inputsamples reserved before immunoprecipitation. The negative control primersets were designed to amplify regions in the ACTA2 or MYH11 promotersthat do not contain GATA elements: ACTA2, sense: 5′-CCAGGCAG-TGTTCTAGGTGC and antisense: 5′-TCCCCTCTGTGCCTTAGCTT;MYH11, sense: 5′-ATTCCTGTTCCACCACTGCT and antisense: 5′-TCCCTCCCTACCCCCATTTT. For ChIP assays showing differencesin DNA binding between GATA-6 Ser290 mutants, the qPCR data werenormalized to expression ofMyc–GATA-6, which was assayed in parallelby Western blotting in the same lysates.

In vitro kinase assay and peptide synthesisGATA-6 overexpressed in HEK293 cells was immunoprecipitated andbound to protein G–Sepharose beads. Equal amounts of immunoprecipi-tated beads were mixed with recombinant active Akt1 or Akt2 (Abcam)protein or PBS or bovine serum albumin (BSA) as controls and incubatedat 30°C for 30 min with 1× kinase buffer (Cell Signaling). 2× SDS samplebuffer was added, and samples were heated at 95°C for 5 min andsubjected toWestern blottingwith anti–phospho-RXRXXS*/T* antibody.In parallel experiments, recombinant GSK3b protein (Cell Signaling) wasused as a substrate to verify the enzymatic activity of the recombinant ki-nases. For radioactive experiments, [33P]ATP (6 mCi per reaction) was in-cluded in the kinase assay using GST-GATA-6 substrates as describedbelow or recombinant GSK3b. Reactions were subjected to SDS-PAGE,Coomassie staining, gel drying (Promega kit), and autoradiography.

To generate the GST-tagged GATA-6 peptide, oligos encoding aminoacids 277 to 296 of human GATA-6 protein (LSRPLIKPQKRVPSSRRLGL)and the S290A mutant (LSRPLIKPQKRVPASRRLGL) were synthesizedand subcloned into the pGEX-4T-1 vector. The recombinant plasmid wastransformed to the BL21 (DE3) bacteria strain and induced by isopropyl-b-D-thiogalactopyranoside to produce GSTalone or the GST fusion pro-tein with the 20–amino acid (aa) peptide at the C-terminal end (namedGST-GATA-6_20aa_wild-type or GST-GATA-6_20aa_S290A). The pro-teins were then purified with glutathione–Sepharose 4B beads and elutedwith buffer containing glutathione.

MS analysisPeptides containing amino acids 281 to 292 of human wild-type GATA-6protein (LIKPQKRVPSSR) or a mutant with a phospho-serine substitutedat the Ser290 position were synthesized by GenScript. The peptides weredesalted using tC18 solid-phase extraction Sep-Pak columns (Waters).The unphosphorylated peptide (~1 pmol) was subjected to an in vitro ki-nase assay using purified Akt2 or no kinase as described above. The pep-tides from the reaction were concentrated and desalted using Stage Tips(45). Dried peptides were resuspended in 2.5% acetonitrile, 2.5% formicacid and subjected to liquid chromatography–tandemMSusing a SIL-201Prominence autosampler (Shimadzu), a Finnigan Surveyor MS PumpPlus (Thermo Fisher), and a linear ion trap–Orbitrap hybrid mass spec-trometer (Thermo Fisher) essentially as described (46). In addition to twodata-dependent MS2 scans per cycle, we collected MS2 scans targeted onthe triply charged precursor phosphopeptide ion. Only after collecting datafrom the peptides extracted from the in vitro kinase assays was the syntheticphosphopeptide analyzed.

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MTT assayhCASMCs transfected with siRNA or puromycin-selected hCASMCsexpressing GATA-6 lentiviral constructs were seeded at 6000 cells perwell (seven or eight replicate wells per condition) in 96-well plates, starved,and then cultured in 10% FBS for 60 or 72 hours (as indicated), and ana-lyzed with the CellTiter 96 Non-Radioactive Cell Proliferation Assay(MTT) (Promega). Cells seeded at the start of the experiment but harvestedjust after attachment served as an input control. The end-point reading wasnormalized to that of input control as fold change.

Animal experimentsAll experiments were approved by the Institutional Animal Care and UseCommittees of Yale University. For in vivo Gata-6 protein level or phos-phorylation induction by rapamycin, wild-type C57BL/6 or Akt2−/− micewere injected intraperitoneally with vehicle (dimethylacetamide diluted1:24 in 10% polyethylene glycol and 17% polyoxyethylene sorbitanmonooleate) or rapamycin (1 mg/kg). Mice were sacrificed at the in-dicated time points for tissue harvest. Tissues were homogenized usingTissueLyser II (Qiagen, 30 Hz, 3 min twice) in 1× cell lysis buffer (CellSignaling) with protease and phosphatase inhibitors. The supernatantafter centrifugation was collected for the following immunoprecipitationand/orWestern blot analysis. For femoralwire injurymodel, 8- to 12-week-old C57BL/6 (wild-type) or Akt2−/− mice were anesthetized with keta-mine (100 mg/kg) and xylazine (10 mg/kg), and femoral artery injurywas performed as previously described (47). For GATA-6 overexpression,control virus or lentivirus carryingGATA-6wild type, S290D, or S290A(1 × 108 plaque-forming units) was delivered to the adventitial side ofthe vessel in a 50-ml mixture containing 15 ml of lentivirus and 35 ml ofpluronic-127 gel immediately after injury. The animals were sacrificed6 or 21 days after surgery by cardiac perfusion with warm 4% PFA, andthe injured and uninjured contralateral femoral arteries were collected.Fixed femoral arteries were embedded in optimum cutting temperaturecompound (Tissue-Tek). For 21-day injury samples, cryosections (5mm)were obtained for elastic van Gieson staining, and morphometric analy-sis was performed on 10 sections per artery. For immunohistochemistry,sections were blocked with 2% BSA in 0.2% Triton X-100 (PBS-T) andthen incubated with rabbit anti–Myc tag antibody and Cy3-conjugatedanti–SM-a-actin at 4°C overnight. Cells were then incubated with AlexaFluor 594–conjugated anti-rabbit secondary antibody in PBS-T for1 hour, and DAPI staining was performed before mounting and analysisusing a fluorescence microscope (Nikon Eclipse 80i).

Statistical analysisAll values are presented asmean ± SEM. Formost data, comparisonswereanalyzedwith theMann-WhitneyU test. For protein decay assays, a pairedt test was used for comparison between two groups. P values were one-tailed. Analyses were performed with Prism 6.0 software (GraphPad).

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/8/376/ra44/DC1Fig. S1. GATA-6 is required for rapamycin-induced VSMC differentiation.Fig. S2. Rapamycin and GATA-6 transactivate a GATA-dependent MYH11 promoterreporter.Fig. S3. Rapamycin treatment does not affect GATA-6 mRNA.Fig. S4. GATA-6 accumulates in the nucleus after rapamycin treatment.Fig. S5. Rapamycin promotes the nuclear accumulation of GATA-6.Fig. S6. Rapamycin treatment stabilizes GATA-6 protein in the presence of cyclohexi-mide.Fig. S7. Phosphorylation of GATA-6 in cells is increased by coexpression of Akt2, butnot Akt1.Fig. S8. Inhibition of PKA activity does not block the rapamycin-induced phosphorylationof GATA-6.



Fig. S9. MS analysis shows that Akt2 could phosphorylate Ser290 in GATA-6.Fig. S10. The phosphorylation of wild-type but not S290A mutant GATA-6 is increased byAkt2 coexpression in cells.Fig. S11. The linker domain confers GATA-6 substrate specificity to Akt2.Fig. S12. Myc–GATA-6 is expressed in nearly 100% of cells after lentivirus infection andpuromycin selection.Fig. S13. Schematic of the femoral artery wire injury model.Fig. S14. GATA-6 inhibits SMC proliferation after injury in vivo.Fig. S15. Lentivirally delivered Myc-tagged GATA-6 cDNAs in wire-injured femoral artery(early time point).Fig. S16. Schematic of rapamycin signaling to Akt2 and GATA-6 in VSMCs.

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Acknowledgments: We thank K. Walsh for the GATA-6 wild type and DZF plasmidsand K. Hasegawa for the MYH11 promoter reporter plasmids. We thank B. Turk forhelpful discussions and H.Y.-F. Chiu and N. Garg for technical contributions to thisproject. Funding: Supported by grants from the NIH National Heart, Lung, and BloodInstitute to K.A.M. (RHL091013), J.H. (HL074190), J.Y. (R56HL117064), and R.J.P.(HL76612) and from the American Heart Association to J.Y. (SDG0930157N) and K.A.M.(SDG0230356N). B.A.B. was supported by the Vermont Genetics Network through NIHgrant P20 RR16462 from the INBRE [Institutional Development Award (IDeA) Networks

w

of Biomedical Research Excellence] program of the National Institute of General MedicalSciences. A.K.L. was supported by NIH Training Grant GM007324. Author contribu-tions: Y.X. and B.L.M. performed experiments, interpreted results, and wrote the manuscript;Y.J., M.D., K.M.F., R.J.W., A.M., S.G., D.F.T, A.K.L., B.A.B., and J.Y. performed and interpretedexperiments; M.J.B., W.C.S., E.M.R., R.J.P., and J.H. interpreted results and edited the man-uscript; H.Z. and L.H. performed statistical analysis; K.A.M. supervised the project, performedand interpreted experiments, and wrote the manuscript. Competing interests: Theauthors declare that they have no competing interests.

Submitted 14 May 2014Accepted 22 April 2015Final Publication 12 May 201510.1126/scisignal.2005482Citation: Y. Xie, Y. Jin, B. L. Merenick, M. Ding, K. M. Fetalvero, R. J. Wagner, A. Mai,S. Gleim, D. F. Tucker, M. J. Birnbaum, B. A. Ballif, A. K. Luciano, W. C. Sessa,E. M. Rzucidlo, R. J. Powell, L. Hou, H. Zhao, J. Hwa, J. Yu, K. A. Martin, Phosphorylationof GATA-6 is required for vascular smooth muscle cell differentiation after mTORC1 inhibition.Sci. Signal. 8, ra44 (2015).


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