t3-Dependent Transcriptional Suppression of HIF-1α
as I. Papadakis1,2, Efrosyni Paraskeva5, Philippos Peidis1, Hala Muaddi1,2, Suiyang Li3,
Raptis8, Kostas Pantopoulos1,2, George Simos6,7, and Antonis E. Koromilas1,2,4
ractHyp
progreglobal(eIF2αOur inicant rtionaltranscwas intor of
translatissful con
s' Affiliationity, Sir Morental MedCenter, an
University, Mboratory oical ResearLIS; Larissology andity Cancer In
upplementa(http://cance
is and H. M
ponding Aul Researchl, Montrea97; Fax: 514
.1158/0008-
American A
r Res; 70(
oxia within the tumor microenvironment promotes angiogenesis, metabolic reprogramming, and tumorssion. In addition to activating hypoxia-inducible factor-1α (HIF-1α), cells also respond to hypoxia byly inhibiting protein synthesis via serine 51 phosphorylation of translation eukaryotic initiation factor 2α). In this study, we investigated potential roles for stress-activated eIF2α kinases in regulation of HIF-1α.vestigations revealed that the double-stranded RNA–dependent protein kinase R (PKR) plays a signif-ole in suppressing HIF-1α expression, acting specifically at the level of transcription. HIF-1α transcrip-repression by PKR was sufficient to impair the hypoxia-induced accumulation of HIF-1α andriptional induction of HIF-1α–dependent target genes. Inhibition of HIF-1A transcription by PKRdependent of eIF2α phosphorylation but dependent on inhibition of the signal transducer and activa-transcription 3 (Stat3). Furthermore, HIF-1A repression required the T-cell protein tyrosine phospha-hich acts downstream of PKR, to suppress Stat3. Our findings reveal a novel tumor suppressor
important means by which cells respond to environ-l stress is the inhibition of mRNA translation (1). Aharacterized mechanism of inhibition of protein syn-is through the phosphorylation of the α subunit ofnslation eukaryotic initiation factor 2 (eIF2) at serine1; ref. 2). Phosphorylated eIF2α acts as a dominant in-r of the guanine exchange factor eIF2B, which preventscycling of eIF2 between succeeding rounds of proteinsis and eventually leads to a global obstruction of
on initiation (2). This allows cells to adaptditions by economizing on energy expended
trol n(ER)–RNAare acimprodsRNApressein allkinaseexplaitheirstrongcan aphospA c
normincludstagesin micto hy
s: 1Lady Davis Institute for Medical Research, McGilltimer B. Davis-Jewish General Hospital; 2Division oficine, 3Department of Biochemistry and Goodmand 4Department of Oncology, Faculty of Medicine,ontreal, Quebec, Canada; 5Laboratory of Physiology
f Biochemistry, School of Medicine, and 7Institute ofch and Technology (BIOMED), University of Thessaly,a, Greece; and 8Departments of Microbiology andPathology and Molecular Medicine, and Queen'sstitute, Queen's University, Kingston, Ontario, Canada
ry data for this article are available at Cancer Researchrres.aacrjournals.org/).
uaddi contributed equally to this work.
thor: Antonis E. Koromilas, Lady Davis Institute for, Room 508, Sir Mortimer B. Davis-Jewish Generall, Quebec, Canada H3T 1E2. Phone: 514-340-8222-340-7576; E-mail: [email protected].
5472.CAN-10-0215
ssociation for Cancer Research.
20) October 15, 2010
tein synthesis (2). The adaptation process of eIF2αorylation involves the selective translation of transcrip-ctors such as activating transcription factor 4 (ATF4)d ATF5 (4), which induce the expression of genes thatte adaptation. In cases of prolonged stress, the induc-f eIF2α phosphorylation leads to cell death through theion of apoptotic pathways (2).ammalian cells, eIF2α phosphorylation is mediated byily of protein kinases, each of which responds to dis-forms of environmental stress (2). The eIF2α kinaseincludes heme-regulated inhibitor (HRI), general con-on-derepressible 2 (GCN2), endoplasmic reticulumresident protein kinase (PERK), and double-stranded(dsRNA)–dependent protein kinase R (PKR), whichtivated by heme deficiency, the absence of amino acids,perly folded proteins accumulated in the ER, and, respectively (2). Whereas HRI protein is mainly ex-d in erythroid cells, GCN2, PERK, and PKR are foundtissues. Despite their diverse regulatory domains, thedomains of these enzymes are significantly conservedning their specificity toward eIF2α (2). In addition tofunction in phosphorylating eIF2α, there has beenevidence to suggest that mammalian eIF2α kinases
lso mediate biological effects independent of eIF2αhorylation (5–7).ommon stress condition encountered by cells duringal development, but also in many pathologic casesing cancer, is the lack of oxygen or hypoxia. At certainduring tumorigenesis, cancer cells find themselves
roenvironments of low oxygen. The ability to adaptpoxic conditions has important effects on tumor
pment, which determines disease progression andl prognosis (8). At the molecular level, coordinatedr responses allow tumor cells to alter gene expressionduce survival pathways in response to hypoxic stressloiting transcriptional and translational machinery (9).ia-inducible factor 1 (HIF-1) is a key transcription fac-mediating responses to oxygen-deficient conditions.plays a major role in tumorigenesis by activating manythat promote angiogenesis [e.g., vascular endothelialh factor (VEGF)], mediate metabolic reprogramminglucose transporter-1 (GLUT-1)], and facilitate metas-e.g., 3-phosphoinositide-dependent protein kinase-11); ref. 8]. As such, HIF-1α levels correlate with tumorssion and poor clinical prognosis (8). Because of itsl role in cancer development, HIF-1α is regarded asractive target for therapeutics (8).-1 consists of an α subunit and a β subunit, which het-erize, bind to DNA, and induce transcription of targetWhereas HIF-1β (also known as ARNT) is constitu-expressed, HIF-1α levels are tightly regulated (8).normal oxygen conditions, HIF-1α is modified by pro-roxylases (PHD) at specific proline residues, whichs binding of the tumor suppressor Von Hippel-Lindauprotein and subsequent ubiquitination and proteaso-egradation (8). In contrast, oxygen-dependent hydrox-does not occur in hypoxic conditions leading to theulation of HIF-1α and induction of HIF-1 activity (8).transcriptional activity is also controlled by oxygenn, as asparaginyl hydroxylation of HIF-1α by FIH-1r inhibiting HIF-1) impairs its association with theriptional coactivator CBP/p300 (10).as been long considered that regulation of HIF-1α isively a posttranscriptional process (8). However, verydata suggest that transcriptional control of HIF-1αsynthesis can also be important, at least under certainions or in certain cell types, and can involve distinctription factors such as NF-κB (11, 12), SP-1 (13), or sig-nsducer and activator of transcription 3 (Stat3; ref. 14).ation of HIF-1α gene transcription may not affectα protein levels during normoxia because the PHD-roteasome system is still limiting, but it can signifi-alter the HIF-1α protein expression levels on hypoxiaeatly affect the corresponding cellular response.oxia has been previously shown to induce the unfoldedn response (UPR), which leads to PERK activation (15,any aspects of the UPR are cytoprotective, and severals indicate that it plays a positive role in facilitating tu-rowth (17). Given that induction of eIF2α phosphory-and upregulation of HIF-1α represent importantnisms of cell adaptation to hypoxic stress, we were in-ed to examine whether the eIF2α phosphorylationay is involved in regulating HIF-1α expression. Herein,ow that the eIF2α kinase PKR plays a specific role inessing HIF-1α levels through mechanisms that areendent of eIF2α phosphorylation and translationall. More specifically, our data reveal that PKR can sup-
the transcription of the HIF-1A gene via a mechanisming Stat3.
GCAGGGTT
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rials and Methods
ulture and treatments−/−, PERK−/−, GCN2−/−, eIF2αA/A mouse embryoniclasts (MEF) and their isogenic wild-type MEFs wereed as previously described (6). T-cell protein tyrosinehatase−/− (TC-PTP−/−) MEFs and their isogenic coun-ts were cultured as described (18). HT1080 expressingPKR were maintained as previously described (19).cells were maintained in DMEM (Wisent) supplemen-th 10% fetal bovine serum (Wisent) and 100 units/mLicillin-streptomycin (Wisent). NiCl2 (Sigma), CoCl2r Scientific), and cycloheximide (Sigma) were dissolvedilled H2O. CPA-7 (20) and 2-methoxyestradiol (Sigma)dissolved in DMSO. Coumermycin (Sigma) was dis-in DMSO. For hypoxic treatments, cells were incubat-he hypoxic chamber (Coy Laboratory Products, Inc.) inesence of 1% O2, 5% CO2, and 94% N2 at 37°C.
in extraction, immunoblot analysis,mmunoprecipitationtein extraction, immunoblotting, and immunoprecipi-were performed as described (18). For immunoblot-nd/or immunoprecipitation, the following antibodiesused: mouse monoclonal antibody for mouse HIF-1αSystems), rabbit HIF-2α (Novus Biologicals), anti-TC-ouse monoclonal antibody (18), mouse monoclonaldy for actin (Clone C4, ICN Biomedicals, Inc), rabbitubulin (Chemicon), mouse monoclonal against PKRf. 18), rabbit polyclonal phosphospecific against S512α (Invitrogen), mouse monoclonal to eIF2α (Cell Sig-), rabbit anti–iron regulatory protein 2 (IRP2; ref. 21),e anti-Stat3 (Cell Signaling), and antiphoshorylatede 705 (Y705) Stat3 (Santa Cruz). All antibodies weret a final concentration of 0.1 to 1 μg/mL. After incuba-ith antimouse IgG or antirabbit IgG antibodies conju-to horseradish peroxidase, proteins were visualizedhe enhanced chemiluminescence reagent (Thermoific) detection system according to the manufacturer'sctions. Quantification of protein bands was performedsitometry using Scion Image from NIH.
solation and real-time PCRal RNA was isolated by Trizol reagent (Invitrogen)ing to the manufacturer's protocol. Total RNA (1 μg)verse transcribed with the high-capacity cDNA reverseription kit (Applied Biosystems International). Quanti-real-time–PCR (RT-PCR) was performed in a Mini-on RT-PCR system (BIORAD) using the IQ SYBRSupermix (BIORAD) and primers for mouse HIF-1α,, and GLUT-1. The levels of mRNA were normalizeduse β-actin mRNA. cDNA of three independent experi-s was analyzed in duplicates. The primers usedantitative RT-PCR were mouse HIF-1α sense GCACTA-AAGTTCACCTGAGA, mouse HIF-1α antisenseATCCACATCAAAGCAA, mouse VEGF sense
CTTGAGTTAAACGAACG, mouse VEGF antisenseCCCGAAACCCTGAG, mouse SLC2A1 (GLUT-1) sense
Cancer Res; 70(20) October 15, 2010 7821
ATGGsenseCTAAsensetion w
PKR tFor
5 ′-GCTAGTexpreswere szeocinwere u
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with 0geneLuc; rcontrotainswere6D), l24 hou
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Papadakis et al.
Cance7822
ATCCCAGCAGCAAG, mouse SLC2A1 (GLUT-1) anti-CCAGTGTTATAGCCGAACTGC, mouse β-actin senseGGCCAACCGTGAAAAG, and mouse β-actin anti-ACCAGAGGCATACAGGGACA. Relative quantifica-as done with the REST-MCS software.
argeting by short hairpin RNAtargeting of human PKR by short hairpin RNA (shRNA)AGGGAGTAGTACTTAAA-3 ′ and 5 ′-GGCAGT-CCTTTATTA-3′ were subcloned into pCXS U6/Zeosion vector. H1299 cells harboring the target vectorelected for resistance to 400 μg/mL zeocin. As control,-resistant cells harboring empty pCXS/zeo DNAsed.
ter gene assayss were transfected by Lipofectamine Plus (Invitrogen).5 μg of pGL3 vector containing the firefly luciferaseunder the control of the HIF-1A promoter (HIF-1Aef. 22) or pGL3 vector alone (control). As an internall, 0.1 μg of pRL-TK vector (Promega Corp.), which con-the Renilla luciferase reporter gene, was used. Cellseither lysed 48 hours posttransfection (Figs. 4B and
eft untreated or treated with 20 μmol/L CPA-7 forrs, or cotransfected with 0.4 μg of Stat3D (previously
higher(Fig. 1
r Res; 70(20) October 15, 2010
bed in ref. 23) or the control pcDNA and lysed 48 hoursansfection (Fig. 5D), as indicated in the figure legends.and Renilla luciferases were determined in protein
ts using the dual-luciferase reporter system (Promegaaccording to the manufacturer's specifications, andluciferase was normalized to Renilla luciferase. Relativease activity refers to normalized firefly luciferase activ-the luciferase activity measured in cells transfectedontrol vector.
lts
educes the levels of HIF-1α protein expression inic cellst, we checked the protein expression levels of HIF-1αn in PKR+/+ and PKR−/− MEFs maintained under nor-(20% O2) or hypoxic conditions (1% O2) for 24 hours.ssion of HIF-1α was barely detectable under normoxictions but readily induced on hypoxic treatmentA). However, hypoxic HIF-1α expression was substan-higher in PKR−/− MEFs compared with PKR+/+ MEFsA). When hypoxia-mimetic compounds, such as CoCl2r NiCl2 (25), were used to treat cells, we also observed a
1. PKR reduces HIF-1αaccumulation and activityoxic treatment. PKR+/+ andMEFs were incubated inic (N, 21% O2) or hypoxicns (H, 1% O2) for 24 h (A)ed with either cobalt(Co2+, 200 μmol/L) orhloride (Ni2+, 500 μmol/L)(B). A and B, protein
s (70 μg) were subjected tooblot analysis for thed proteins. The ratio ofto actin for each lane isd. C, RNA was isolatedKR+/+ and PKR−/− MEFsed to normoxia or hypoxia) for 24 h. Quantitativewas performed usingdetecting transcripts ofVEGF and GLUT-1. Resultsresentative of an average ofdependent experiments.cal analysis was performed,alues compared within normoxia are indicated.
− than in PKR+/+ MEFsexpression was readily
Cancer Research
detectlevelsThe lain lineregulaever, uby hypPKR−/
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that tsignifimodesGLUT-ing amousHIF-2αand Gsion oless runderTo
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FigureHIF-1αconditio(A, B) aMEFs (C(N, 21%conditioeither cnickel c(B, D).subjectHIF-1αbandsare ind
Transcriptional Inhibition of HIF-1A Gene by PKR
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able in normoxic cells and was maintained in similarwhen cells were kept under hypoxia (Fig. 1A and B).ck of an induction of HIF-2α in the hypoxic MEFs iswith previous findings showing that HIF-2α is not up-ted in mouse embryonic cells under hypoxia (26). How-nlike HIF-2α, we found that the IRP2, which is inducedoxia (21), was efficiently induced in both PKR+/+ and− MEFs (Fig. 1A, c) supporting a specific role of PKR inssing HIF-1α in hypoxic cells.as next important to determine whether the higher ex-on of HIF-1α in PKR−/− MEFs also resulted in increasedactivity. To do so, we examined the expression of HIF-1genes under normoxic or hypoxic conditions by
itative RT PCR. We found that the mRNA levels ofGF and GLUT-1 genes were higher in PKR−/− than in+ MEFs under hypoxia (Fig. 1C), providing evidencehe difference in HIF-1α protein levels has functionalcance in these cells. We also noticed that VEGF wastly induced in hypoxic PKR+/+ MEFs compared with1 (Fig. 1C). This is in line with previous studies show-higher induction of GLUT-1 than VEGF by HIF-1α ine fibroblasts under hypoxia (27, 28). Given thatalso targets HIF-1–dependent genes such as VEGF
LUT-1 (29), it is also possible that constitutive expres-f HIF-2α in MEFs (Fig. 1A, b; ref. 26) renders these cellsesponsive to HIF-1α–dependent gene transcriptionhypoxic conditions.determine whether inhibition of HIF-1α expression inpoxic cells is specific for PKR, we examined the roleer eIF2α kinases in this process. To do so, we useddeficient in PERK or GCN2 together with their genet-
matched wild-type MEFs. We found that HIF-1α wasy induced in control wild-type MEFs or
HIF-1
icated.
acrjournals.org
(Fig. 2A) or GCN2 (Fig. 2C) under hypoxic condi-Consistent with these observations, treatment
the hypoxia mimetic compounds CoCl2 and NiCl2d in an equal induction of HIF-1α in wild-type MEFsEFs lacking either PERK (Fig. 2B) or GCN2 (Fig. 2D).together, these data supported a specific role of
n the negative regulation of HIF-1α under hypoxicions.t, we attempted to determine whether inhibition ofα expression by PKR in hypoxic cells was due tophosphorylation. To this end, we used MEFs con-g either a wild-type allele of eIF2α (eIF2αS/S) or a-in S51A mutant allele (eIF2αA/A), which produces an that cannot be phosphorylated by the eIF2α ki-We observed similar induction of HIF-1α between
S/S and eIF2αA/A MEFs under hypoxic conditionsA) or after treatment with CoCl2 or NiCl2 (Fig. 3B),ting that eIF2α phosphorylation is not playing a ma-le in the induction of HIF-1α under these treatments.termine whether the decreased inducibility of HIF-1αR+/+ MEFs was not a side effect of the genetic back-d or immortalization of the cells that dampened theirsiveness to hypoxic treatment, we compared HIF-1αion in different wild-type MEFs that were isogenic to/−, GCN2−/−, or eIF2αA/A MEFs. We found that treat-of the various wild-type MEFs with NiCl2 resulted inparable induction of HIF-1α, ruling out the possibil-a defective response of PKR+/+ MEFs to hypoxic en-ent (Supplementary Fig. S1). Also, PKR did notHIF-1α stability in hypoxic cells because inhibitiontein synthesis by cycloheximide treatment caused
α destabilization in both PKR+/+ and PKR−/− MEFs
MEFs lacking (Supplementary Fig. S2).
2. PERK and GCN2 do not affectexpression under hypoxicns. PERK+/+ and PERK−/− MEFss well as GCN2+/+ and GCN2−/−
, D) were incubated under normoxicO2) or hypoxic (H, 1% O2)ns for 24 h (A, C) or treated withobalt chloride (Co2+, 200 μmol/L) orhloride (Ni2+, 500 μmol/L) for 20 h(A–D) Protein extracts (70 μg) wereed to immunoblot analysis for(a) and actin (b). The intensity of thewas normalized, and ratios (a/b)
script30). AactivitwhichConsisphospMEFsincrea(Fig. 55A). NeIF2αand 4necesscientPKR, neither PERK nor GCN2 displayed a role in regulatingStat3 phosphorylation in MEFs (Supplementary Fig. S3).
Figurephosphin normwere trchloride(70 μg) were subjected to immunoblot analysis for HIF-1α (a) and actin (b).The no
FigureA, RNAeIF2αS/
hypoxiaprimers***, P <companormoxconstrucontrolcontaininterna
Papadakis et al.
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educes HIF-1α expression at theriptional levelfurther address the mechanism of inhibition of HIF-1αsion by PKR, we examined the HIF-1α mRNA levels in+ and PKR−/− MEFs by quantitative real-time PCR. Wethat HIF-1α mRNA was more highly expressed in− than in PKR+/+ MEFs under normoxic conditions,is difference in mRNA levels was maintained afteric treatment (Fig. 4A). We also found that the status2α phosphorylation did not have a significant effectF-1α mRNA levels as determined by the analysis ofS/S and eIF2αA/A MEFs (Fig. 4A). These data indicatedible transcriptional regulation of HIF-1α expressionR in normoxic as well as hypoxic cells.confirm that HIF-1A gene was under transcriptionall by PKR, we performed transient transactivationin PKR+/+ and PKR−/− MEFs using a luciferase
er gene under the control of the HIF-1A promoter.tent with the upregulation of HIF-1α mRNA levelsPKR−/− MEFs (Fig. 1A), the reporter gene assays
ed an ∼3-fold increase of HIF-1A promoter activityR−/− MEFs compared with PKR+/+ MEFs supporting
rmalized ratio (a/b) of band intensity is indicated.
scriptional effect of PKR on HIF-1α expressionB).
Relativethe actanalysi
r Res; 70(20) October 15, 2010
ontrols HIF-1A gene transcription through Stat3ent reports have suggested that HIF-1A gene tran-ion can be directly induced by activated Stat3 (14,dditionally, our group has previously shown that Stat3y is impaired by PKR due to activation of TC-PTP,targets the phosphorylation of Stat3 at Y705 (18).tent with this finding, we observed a higher (10-fold)horylation of Stat3 at Y705 in PKR−/− than in PKR+/+
under normoxia (Fig. 5A). We also observed anse of Stat3 phosphorylation in the eIF2α A/A MEFsB), which was not as high as in PKR−/− MEFs (Fig.evertheless, increased Stat3 phosphorylation in theA/A MEFs did not affect HIF-1α expression (Figs. 3A). These data indicate that eIF2α phosphorylation isary for a partial inhibition of Stat3, which is not suffi-to decrease the transactivation of HIF-1A gene. Unlike
3. HIF-1α induction by hypoxia is not affected by the eIF2αorylation status. The eIF2αS/S and eIF2αA/A MEFs were incubatedoxic (N, 21% O2) or hypoxic (H, 1% O2) conditions for 24 h (A) oreated with either cobalt chloride (Co2+, 200 μmol/L) or nickel(Ni2+, 500 μmol/L) for 20 h (B). A and B, whole-cell extracts
4. PKR inhibits HIF-1α expression at the transcriptional level.was isolated from PKR+/+ and PKR−/− MEFs as well as
Sand eIF2αA/A MEFs subjected to normoxia (N, 21% O2) or(H, 1% O2) for 24 h. Quantitative RT-PCR was performed usingtargeting mouse HIF-1α. Statistical analysis was performed.0.001; **, P < 0.005 compared with PKR+/+ in normoxia; *, P < 0.05red with eIF2αS/S normoxia; #, P < 0.05 compared with eIF2αA/A
ia. B, PKR+/+ and PKR−/− MEFs were transiently transfected withcts containing the firefly luciferase reporter gene under theof the HIF-1α promoter or control vector. A second vectoring a Renilla luciferase reporter gene was cotransfected as anl control. Luciferase activity was assessed 48 h posttransfection.luciferase activity of the HIF-1α promoter was normalized to
ivity of the control vector. Error bar, SEM (n = 6). Statisticals was performed using the paired t test. **, P < 0.001.
Cancer Research
Figureuntreattotal SteIF2α S
S51 ph20 or 5absenctotal Stgene uinternalnormalipaired tStat3 dafter traStatistic
Transcriptional Inhibition of HIF-1A Gene by PKR
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5. PKR controls HIF-1A gene transcription through Stat3. A, Stat3 was immunoprecipitated from whole-cell extracts (750 μg of protein) fromed PKR+/+ and PKR−/− MEFs. Immunoprecipitated protein was immunoblotted with antibodies detecting phosphorylated Stat3 at Y705 (a) orat3 (b). The ratio a/b of band intensity was measured by densitometry, normalized, and indicated at the bottom. B, extracts from untreated/Sand A/A MEFs (50 μg of protein) were resolved and immunoblotted with antibodies targeting Y705 phosphorylated Stat3 protein (a), total Stat3 (b),osphorylated eIF2α (c), and total eIF2α (d). The ratio of the bands (a/b) for each lane is indicated. C, PKR−/− MEFs were untreated or treated with0 μmol/L of CPA-7 in hypoxic conditions (H, 1% O2) for 24 h (lanes 1–3) or left untreated or treated with CPA-7 (20 μmol/L) in the presence ore of CoCl2 (200 μmol/L) for 24 h (lanes 4–7). Protein extracts were subjected to immunoblotting for HIF-1α (a), Y705 phosphorylated Stat3 protein (b),at3 (c), and actin (d). D, PKR+/+ and PKR−/− MEFs were transiently transfected with pGL3 constructs containing the firefly luciferase reporternder the control of the HIF-1α promoter or control vector. A second vector containing a Renilla luciferase reporter gene was cotransfected as ancontrol. Cells were treated with CPA-7 (20 μmol/L) for 24 h and lysed 27 h after transfection. Relative luciferase activity of the HIF-1α promoter waszed to the basal activity of control vector with respect to each treatment. Error bar, SEM (n = 4). Statistical analysis was performed using thetest. #, P < 0.01; **, P < 0.001. Similarly, the MEFs were transiently cotransfected with HIF-1A Luc or control pGL3 construct with pcDNA encoding aominant negative or control vector and with the internal control vector containing the Renilla luciferase reporter gene. Cells were lysed 48 hnsfection. Relative luciferase activity of the HIF-1A Luc was normalized to the basal activity of the control vectors. Error bars, SEM (n = 4).
al analysis was performed using the paired t test. *, P < 0.05.
Cancer Res; 70(20) October 15, 2010acrjournals.org 7825
sequeall, wY705(Fig. 6of thisTC-PTor CoCof HIFTC-PTferencupregHIF-1MEFsportera negaoxia t
PKR iexpreIn a
1α exhumaby shRgulatioof Statary Fled to(Supp
urescrxtraFs wetintotao ofC-Pubatditiractlysian
-PTPμmteinanC-Psietainer ttrolillatrolractiferamalor bformed using the paired t test. **, P < 0.001.
Papadakis et al.
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examine whether the increased levels of phosphorylat-t3 were responsible for increased HIF-1α protein ex-on, PKR−/− MEFs were exposed to hypoxic conditionsabsence or presence of the Stat3 chemical inhibitor(20). CPA-7 inhibited Stat3 Y705 phosphorylation,is was associated with a decrease in HIF-1α expressionC). We also observed a similar regulation when PKR−/−
were treated with CoCl2 in the absence or presence of. That is, we found that CPA-7 impaired the induction-1α by CoCl2 concomitantly with an inhibition of Stat3horylation at Y705 (Fig. 5C).analyze the effect of Stat3 phosphorylation on HIF-1αsion, we measured HIF-1A promoter activity by lucifer-porter assays in PKR+/+ and PKR−/− MEFs. We foundharmacologic inhibition of Stat3 by CPA-7 resulted inhibition of HIF-1A promoter activity in PKR−/− MEFsD). Similarly, expression of Stat3D, a Stat3 mutantive in transactivation activity that exerts a dominantve effect (23), decreased HIF-1A promoter activity in− MEFs (Fig. 5D). On the other hand, HIF-1A promotery in PKR+/+ MEFs was not affected by either CPA-7 orexpression (Fig. 5D), indicating that the higher induc-
f Stat3 activity in PKR−/− MEFs was responsible for theriptional upregulation of the HIF-1A gene.
P is involved in the transcriptional control thatexerts on the HIF-1A geneen that induction of Stat3 activity in PKR−/− MEFs is
inactivation of TC-PTP (18), we wished to examine
er TC-PTP deficiency leads to Stat3 activation and sub-we exhuma
r Res; 70(20) October 15, 2010
nt upregulation of HIF-1A gene transcription. First ofe detected higher levels of Stat3 phosphorylation atin TC-PTP−/− MEFs compared with TC-PTP+/+ MEFsA), confirming that phosphorylated Stat3 is a targettyrosine phosphatase (18, 31). When TC-PTP+/+ andP−/− MEFs were subjected to either hypoxia (Fig. 6B)l2 treatment (Fig. 6C), we observed a higher induction-1α protein expression in cells that were deficient inP, in agreement with all our previous results. This dif-e in HIF-1α protein expression levels was due to theulation of HIF-1α gene transcription, because theA promoter displayed higher activity in TC-PTP−/−
than TC-PTP+/+ MEFs as shown by the luciferase re-assays (Fig. 6D). Taken together, these data supporttive role of TC-PTP in HIF-1α expression under hyp-hrough the inhibition of Stat3.
nhibits Stat3 phosphorylation and HIF-1αssion in human cancer cellsddition to MEFs, we looked at the role of PKR in HIF-pression in human cells. To this end, we used then lung cancer H1299 cells in which PKR was targetedNA (Supplementary Fig. S4A). We found that downre-n of PKR resulted in a higher tyrosine phosphorylationt3 and induction of HIF-1α under hypoxia (Supplemen-ig. S4B and C). Consistent with MEFs, PKR inactivationincreased transactivation of HIF-1A gene promoterlementary Fig. S4D). To verify these observations,
6. TC-PTP is involved in theiptional control of HIF-1A gene by Stat3.cts from TC-PTP+/+ and TC-PTP −/−
ere immunoblotted with antibodiesg the Y705 phosphorylated Stat3 proteinl Stat3 (b), TC-PTP (c), and actin (d). Thethe bands (a/b) for each lane is indicated.TP+/+ and TC-PTP−/− MEFs wereed in normoxic (N, 21% O2) or hypoxicons (H, 1% O2) for 24 h. Whole-cells (70 μg) were subjected to immunoblots with anti-HIF-1α (a), anti-TC-PTP (b),ti-actin (c) antibodies. C, TC-PTP+/+ and−/− MEFs were treated for 24 h withol/L CoCl2. Whole-cell extracts (70 μg of) were subjected to immunoblot analysisti-HIF-1α (a) and anti-actin (b) antibodies.TP+/+ and TC-PTP−/− MEFs werently transfected with constructsing the firefly luciferase reporter genehe control of the HIF-1α promoter orvector and a second vector containing aluciferase reporter gene as an internal. Luciferase activity was assessed froms obtained 48 h posttransfection. Relativese activity of the HIF-1A promoter wasized to the activity of the control vector.ar, SEM (n = 8). Statistical analysis was
of PKR activation on HIF-1α ina. To do so, we used the human
in partriguinenergyis efficendurmanysary tocellulacomprtionalkinaseprolifeindepeOur
modufamilyas it hsupprmoresthat Palso pgene,mor pto Stathe trarole foIn l
PKR f
vide gphoryHIF-1TC-PTuate cgressikinaseprolifetransimplichibitoTC-PTdothecells (by suin tumFro
conseloguesvationCaenoHIF-1servedthe otvidingAlso,bratescompakinaseevolution of PKR has been attributed to virus infection(37, 38
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Transcriptional Inhibition of HIF-1A Gene by PKR
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rcoma HT1080 cells, which were engineered to expressitionally active form of PKR in a fusion protein with(19). When HT1080 cells stably expressing GyrB.PKReated with the antibiotic coumermycin, the fusionPKR protein becomes dimerized and activated byhosphorylation leading to phosphorylation of endoge-IF2α (ref. 19; Supplementary Fig. S5A). We previouslyd that induction of GyrB.PKR in HT1080 cells leads totion of TC-PTP, which in turn inactivates Stat3 by de-horylation (18). When these cells were maintained inxic or hypoxic conditions for various periods of time,served that activation of GyrB.PKR resulted in theegulation of HIF-1α under hypoxic treatment, whichded with a substantial inhibition of Stat3 phosphoryla-upplementary Fig. S5B). Inhibition of HIF-1α expres-as mediated at the transcriptional level given thatpromoter activity was significantly reduced in nor-cells after the activation of GyrB.PKR (Supplementary5C). Overall, these data support that PKR activityvely regulates HIF-1α expression in human cells.
ssion
study shows that PKR suppresses HIF-1α expressionnsequently the transcription of its target genes. Thismenon is specific for PKR and mediated by a reductiontranscription of the HIF-1A gene itself. As illustrated inodel (Fig. 7), the ability of PKR to impair HIF-1α syn-is exerted through the activity of TC-PTP and Stat3.oxia affects gene expression at the level of translation,t through the eIF2α phosphorylation pathway (32). In-gly, despite the effort of the hypoxic cell to conserveby inhibiting global protein synthesis, HIF-1α mRNAiently translated under hypoxic conditions to promoteance to the assaulting stress (33). As is the case withsignaling pathways, feedback mechanisms are neces-maintain control, equilibrium, and plasticity of majorr processes. Herein, we present evidence that PKRomises expression of HIF-1α independent of transla-control consistent with previous reports that the eIF2αs can regulate key cell signaling pathways involved inration, apoptosis, viral replication, and tumorigenesisndent of their function in protein synthesis (5, 6).study reveals that PKR regulates HIF-1α expression bylating the activity of Stat3. Stat3 is a member of the Statthat has been described predominantly as oncogenic,as been shown to play a positive role in transformation,ession of apoptosis, proliferation, invasion, and che-istance (34). Our work supports our previous findingsKR inhibits Stat3 phosphorylation and activity (18). Werovide a specific example of an affected Stat3 targetHIF-1A, which is importantly a major facilitator of tu-rogression. Phosphorylated eIF2α partially contributest3 inhibition, although this is not sufficient to suppressnscription of the HIF-1A gene highlighting a dominantr PKR for this process.
ine with our previous work (18), our data show thatunctions to prevent basal activation of Stat3 and pro-
the inhpossiblHIF-1α
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enetic evidence supporting the notion that Stat3 phos-lation and its ability to upregulate transcription of theA gene is antagonized by TC-PTP. The function ofP as a tyrosine phosphatase has been shown to atten-ytokine signaling and negatively regulate cell cycle pro-on by inhibiting Janus-activated kinases, Src familys, and Stat3 (31). In addition to its ability to attenuateration, the ability of TC-PTP to decrease Stat3-mediatedcription of the HIF-1A gene may have importantations within the tumor microenvironment as an in-r of the angiogenic switch. In line with this notion,P has been recently shown to antagonize vascular en-lial growth factor receptor 2 signaling in endothelial35), suggesting that it can also inhibit angiogenesisppressing HIF-1α expression and secretion of VEGFor cells.m an evolutionary point of view, HIF-1α is highlyrved given that the majority of metazoa use homo-of HIF transcription factors to adapt to oxygen depri-. Furthermore, studies in mammals, Drosophila, andrhabditis elegans have shown that regulation ofα by PHD-mediated proteasomal degradation is con-, as well as a number of HIF target genes (36). Onher hand, PKR is not expressed in all metazoans, pro-evidence that HIF appeared before PKR in evolution.PKR evolved in more complex organisms. In verte-, the kinase domains of PKR evolved with a faster ratered with the kinase domains of the other three eIF2αs, namely, GCN2, PERK, and HRI (37). The accelerated
). More specifically, the rapid evolution of PKR kinase
7. PKR acts as a transcriptional suppressor of HIF-1A. PKRHIF-1α expression at the transcriptional level in normoxic cells.mediated by the ability of PKR to impair Stat3 phosphorylation atrough the activation of the tyrosine phosphatase TC-PTP.n of Stat3 phosphorylation is necessary and sufficient tose transcription of HIF-1A. This is a mechanism that accounts foribition of HIF-1α accumulation by PKR in hypoxic cells with
e implications in chemotherapies that activate PKR and impairexpression and function.
dancefunctiPKR gsion inThis isnalingdrugsshownization(44, 45of aposervedresultewas dvationangiogand fuas etotors le
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ns in vertebrates has been coupled with positive selec-f specific sites, particularly in residues near the eIF2αg site. In primates, positive selection was observed inf the three domains of PKR (dsRNA-binding domain,region, and kinase domain), consistent with the exten-istory of viral factors that bind PKR in these separatens (38). However, it is not presently known whetheradaptive changes of PKR play a role in the regulation-1α expression.novel role of PKR in the hypoxic response is in accor-with previous reports describing tumor-suppressingons for the kinase (39–42). Because mutations of theene have not been found in human tumors, its expres-tumor cells may indeed be of therapeutic potential.because PKR activity is induced by antioncogenic sig-(42) as well as by treatments with chemotherapeutic(43). In regard to hypoxia, 2-methoxyestradiol has beento act as an inhibitor of tumor growth and vascular-through its ability to downregulate Stat3 and HIF-1α). Interestingly, 2-methoxyestradiol is a potent inducerptosis in tumor cells by activating PKR (46). We ob-that 2-methoxyestradiol treatment of hypoxic MEFsd in the downregulation of HIF-1α in a manner thatependent on PKR (Supplementary Fig. S6). This obser-supports the notion that PKR is a valid target of anti-enic chemotherapies that disrupt HIF-1α expressionnction. Furthermore, chemotherapeutic drugs such
poside, doxorubicin, and related topoisomerase inhibi- Rece
ture 2008;453:807–11.laiba RS, Bonello S, Zahringer C, et al. Hypoxia up-regulatesoxia-inducible factor-1α transcription by involving phosphatidy-
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response to hypoxia (47–49). Given that PKR becomested by doxorubicin (50) and other chemotherapeutic(43), its activation may significantly contribute to sup-
on of tumor growth by drugs targeting HIF-1α.
osure of Potential Conflicts of Interest
otential conflicts of interest were disclosed.
owledgments
thank Dr. M. Tremblay (McGill University, Montreal, Canada) for the+/+ and TC-PTP−/− MEFs, Dr. D. Ron (Skirball Institute of Biomoleculare, New York, NY) for GCN2+/+, GCN2−/−, PERK+/+, and PERK−/− MEFs,aufman (University of Michigan Medical School, Ann Arbor, MI) forS and eIF2αA/A MEFs, and Dr. C Michiels (FUNDP-University ofBelgium) for the pGL3 HIF-1A promoter construct.
Support
dian Cancer Society Research Institute grant 17285 (A.E. Koromilas).adakis is a recipient of the Victor K. Lui Fellowship from McGill Uni-the Weekend to End Breast Cancer Studentship from Montreal Centrerimental Therapeutics in Cancer (MCETC), and the Doctoral FrederickCharles Best Canadian Graduate Scholarship from Canadian Institutesth Research. P. Peidis is a recipient of a MCETC postdoctoral award,Muaddi is a recipient of the FRSQ Master's Training award.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.
ived 01/18/2010; revised 06/28/2010; accepted 06/30/2010; published
ad to an inhibition of HIF-1 α synthesis and accumula- OnlineFirst 10/05/2010.
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