Portal de Periódicos da CAPES

The Forkhead Transcription Factor FOXO4 Induces the Down-regulation of Hypoxia-inducible Factor 1α by a von Hippel-Lindau Protein-independent Mechanism

2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês

10.1074/jbc.m302042200

1083-351X

Tracy Tzu-Ling Tang, Laurence A. Lasky,

Cancer, Hypoxia, and Metabolism

Tumors utilize hyperactivation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway to cope with deleterious environmental conditions. Activation of the PI3K/AKT pathway has been shown to increase protein expression of the α subunit of the hypoxia-inducible factor (HIF) 1, a key regulator of oxygen homeostasis. Elevated levels of HIF-1α induce expression of genes with critical roles in angiogenesis, erythropoiesis, and glucose metabolism, processes that are essential for tumor expansion. Here we examine the involvement of FOXO4 (also known as AFX), a member of the forkhead transcription factor superfamily that is negatively regulated by the PI3K/AKT pathway, in the regulation of HIF-1α protein expression. Nuclear expression of FOXO4 results in the suppression of various responses to hypoxia, including decreased vascular endothelial growth factor, glucose transporter 1, and erythropoietin expression. Interestingly, FOXO4 down-regulates the HIF-1α protein levels, consistent with the lack of hypoxia responsiveness. Previous results have revealed a role for prolyl hydroxylation and resultant von Hippel-Lindau protein (pVHL) interactions in the ubiquitinproteasome-mediated degradation of HIF-1α. However, neither inhibition of prolyl hydroxylases nor mutation of HIF-1α-hydroxylated prolines involved with pVHL-mediated binding inhibits the observed FOXO4-mediated down-regulation of HIF-1α. These results suggest a novel alternate mechanism for hypoxic regulation that is dependent upon the level of activation of FOXO4-mediated transcription. Tumors utilize hyperactivation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway to cope with deleterious environmental conditions. Activation of the PI3K/AKT pathway has been shown to increase protein expression of the α subunit of the hypoxia-inducible factor (HIF) 1, a key regulator of oxygen homeostasis. Elevated levels of HIF-1α induce expression of genes with critical roles in angiogenesis, erythropoiesis, and glucose metabolism, processes that are essential for tumor expansion. Here we examine the involvement of FOXO4 (also known as AFX), a member of the forkhead transcription factor superfamily that is negatively regulated by the PI3K/AKT pathway, in the regulation of HIF-1α protein expression. Nuclear expression of FOXO4 results in the suppression of various responses to hypoxia, including decreased vascular endothelial growth factor, glucose transporter 1, and erythropoietin expression. Interestingly, FOXO4 down-regulates the HIF-1α protein levels, consistent with the lack of hypoxia responsiveness. Previous results have revealed a role for prolyl hydroxylation and resultant von Hippel-Lindau protein (pVHL) interactions in the ubiquitinproteasome-mediated degradation of HIF-1α. However, neither inhibition of prolyl hydroxylases nor mutation of HIF-1α-hydroxylated prolines involved with pVHL-mediated binding inhibits the observed FOXO4-mediated down-regulation of HIF-1α. These results suggest a novel alternate mechanism for hypoxic regulation that is dependent upon the level of activation of FOXO4-mediated transcription. Induction of the PI3K 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; VEGF, vascular endothelial growth factor; EPO, erythropoietin; HIF-1, hypoxia-inducible factor 1; GLUT-1, glucose transporter 1; pVHL, von Hippel-Lindau protein; DFO, deferoxamine mesylate; DOX, doxycycline hydrochloride; GFP, green fluorescent protein; AHR, aryl hydrocarbon receptor; PTEN, phosphatase with tensin homology.1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; VEGF, vascular endothelial growth factor; EPO, erythropoietin; HIF-1, hypoxia-inducible factor 1; GLUT-1, glucose transporter 1; pVHL, von Hippel-Lindau protein; DFO, deferoxamine mesylate; DOX, doxycycline hydrochloride; GFP, green fluorescent protein; AHR, aryl hydrocarbon receptor; PTEN, phosphatase with tensin homology. signaling pathway results in a diversity of survival-enhancing functions in both normal as well as oncogenically transformed cells (1Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1223) Google Scholar). A large body of work in a variety of systems has revealed that activation of this pathway results in the induction of a cascade of kinases that include PDK-1 and one or more of the AKT/protein kinase B kinases (2Toker A. Newton A.C. Cell. 2000; 103: 185-188Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). A major end result of AKT/protein kinase B kinase activation is phosphorylation of several sites in a family of forkhead-type transcription factors that includes FOXO4 (3Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1536) Google Scholar, 4Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5412) Google Scholar, 5Kops G.J. de Ruiter N.D. De Vries-Smits A.M. Powell D.R. Bos J.L. Burgering B.M. Nature. 1999; 398: 630-634Crossref PubMed Scopus (950) Google Scholar, 6Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 7Takaishi H. Konishi H. Matsuzaki H. Ono Y. Shirai Y. Saito N. Kitamura T. Ogawa W. Kasuga M. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11836-11841Crossref PubMed Scopus (217) Google Scholar, 8Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Crossref PubMed Scopus (495) Google Scholar, 9Tang T.T. Dowbenko D. Jackson A. Toney L. Lewin D.A. Dent A.L. Lasky L.A. J. Biol. Chem. 2002; 277: 14255-14265Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 10Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (941) Google Scholar). Phosphorylation of these transcription factors results in nuclear export and cytoplasmic sequestration by 14-3-3 proteins as well as other factors. Lack of nuclear localization of FOXO4-type forkhead family members is highly beneficial to the proliferative and survival states of the cell, because expression programs induced by this category of transcription factors include the expression of proteins that inhibit cell cycle progression and induce apoptosis (8Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Crossref PubMed Scopus (495) Google Scholar, 9Tang T.T. Dowbenko D. Jackson A. Toney L. Lewin D.A. Dent A.L. Lasky L.A. J. Biol. Chem. 2002; 277: 14255-14265Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 12Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1221) Google Scholar, 13Dijkers P.F. Medema R.H. Pals C. Banerji L. Thomas N.S. Lam E.W. Burgering B.M. Raaijmakers J.A. Lammers J.W. Koenderman L. Coffer P.J. Mol. Cell. Biol. 2000; 20: 9138-9148Crossref PubMed Scopus (542) Google Scholar, 14Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar, 15Modur V. Nagarajan R. Evers B.M. Milbrandt J. J. Biol. Chem. 2002; 277: 47928-47937Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 16Schmidt M. de Mattos S.F. van der Horst A. Klompmaker R. Kops G.J. Lam E.W. Burgering B.M. Medema R.H. Mol. Cell. Biol. 2002; 22: 7842-7852Crossref PubMed Scopus (467) Google Scholar, 17Ramaswamy S. Nakamura N. Sansal I. Bergeron L. Sellers W.R. Cancer Cell. 2002; 2: 81-91Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Thus, inhibition of FOXO4-type transcription factor nuclear localization and the subsequent lack of expression of a variety of proteins involved in cellular quiescence and death constitute a major end point for the activated PI3K pathway. This fact is highlighted by examination of a range of late-stage tumors, including glioblastoma multiforme, where this kinase cascade is hyperactivated by the mutational loss of PTEN, a lipid phosphatase that is the major negative regulator of this pathway (18Li D.M. Sun H. Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar, 19Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2512) Google Scholar, 20Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2590) Google Scholar, 21Wu X. Senechal K. Neshat M.S. Whang Y.E. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15587-15591Crossref PubMed Scopus (595) Google Scholar, 22Haas-Kogan D. Shalev N. Wong M. Mills G. Yount G. Stokoe D. Curr. Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar). The activation of this pathway in these tumors results in a high degree of resistance to many cytotoxic agents, including radiation and chemotherapy. It is, therefore, likely that examination of the diversity of functions regulated by this pathway may result in enhanced treatments for the many tumors that benefit from PI3K activation. In addition to intracellular survival mechanisms such as the PI3K pathway, rapidly proliferating cells in embryonic and oncogenically transformed tissues require an increased blood supply to maintain a normal nutritive and oxygenated state. This is accomplished by the induction of angiogenic and hematopoietic factors, including VEGF and EPO. When the cell is exposed to reduced oxygen levels or hypoxia as a result of tissue expansion or tumor growth, a transcription factor called hypoxia-inducible factor 1 (HIF-1) becomes stabilized and activates the expression of VEGF, EPO, GLUT-1, and others that are involved in angiogenesis, erythropoiesis, and glucose metabolism (23Semenza G.L. J. Appl. Physiol. 2000; 88: 1474-1480Crossref PubMed Scopus (1486) Google Scholar). HIF-1 consists of two subunits, HIF-1α and HIF-1β, and the steady-state quantity of the α subunit is tightly controlled by degradative processes in response to changing oxygen concentrations. The molecular mechanisms that link oxygen and HIF-1α levels have recently been elucidated (24Semenza G.L. Curr. Opin. Cell Biol. 2001; 13: 167-171Crossref PubMed Scopus (890) Google Scholar, 25Semenza G.L. Biochem. Pharmacol. 2002; 64: 993-998Crossref PubMed Scopus (744) Google Scholar). The importance of the von Hippel-Lindau (VHL) tumor suppressor protein, a component of a ubiquitin ligase complex, in the ubiquitin-mediated degradation of HIF-1α has been demonstrated, and a diversity of tumors, particularly renal carcinomas, shows loss of function of VHL and greatly enhanced angiogenesis (26Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 27Huang L.E. Gu J. Schau M. Bunn H.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7987-7992Crossref PubMed Scopus (1845) Google Scholar, 28Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4117) Google Scholar, 29Cockman M.E. Masson N. Mole D.R. Jaakkola P. Chang G.W. Clifford S.C. Maher E.R. Pugh C.W. Ratcliffe P.J. Maxwell P.H. J. Biol. Chem. 2000; 275: 25733-25741Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 30Kamura T. Sato S. Iwai K. Czyzyk-Krzeska M. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10430-10435Crossref PubMed Scopus (546) Google Scholar, 31Ohh M. Park C.W. Ivan M. Hoffman M.A. Kim T.Y. Huang L.E. Pavletich N. Chau V. Kaelin W.G. Nat. Cell Biol. 2000; 2: 423-427Crossref PubMed Scopus (1258) Google Scholar, 32Tanimoto K. Makino Y. Pereira T. Poellinger L. EMBO J. 2000; 19: 4298-4309Crossref PubMed Google Scholar, 33Berra E. Roux D. Richard D.E. Pouyssegur J. EMBO Rep. 2001; 2: 615-620Crossref PubMed Scopus (133) Google Scholar, 34Maranchie J.K. Vasselli J.R. Riss J. Bonifacino J.S. Linehan W.M. Klausner R.D. Cancer Cell. 2002; 1: 247-255Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 35Kondo K. Klco J. Nakamura E. Lechpammer M. Kaelin W.G. Cancer Cell. 2002; 1: 237-246Abstract Full Text Full Text PDF PubMed Scopus (652) Google Scholar). Furthermore, recent studies have demonstrated that a family of prolyl-4-hydroxylases are involved with sensing increased oxygen concentrations and converting this information into higher levels of HIF-1α prolyl hydroxylation (36Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3873) Google Scholar, 37Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4432) Google Scholar, 38Masson N. Willam C. Maxwell P.H. Pugh C.W. Ratcliffe P.J. EMBO J. 2001; 20: 5197-5206Crossref PubMed Scopus (856) Google Scholar, 39Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2106) Google Scholar, 40Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2720) Google Scholar). The VHL protein (pVHL) interacts with these hydroxylated proline residues and induces the ubiquitination and subsequent proteasome-dependent degradation of HIF-1α. These elegant regulatory mechanisms thus ensure that appropriate levels of cellular angiogenic responses are maintained under varying oxygen concentrations. Because both the PI3K and the hypoxic response pathways are regulated by the sensing of extracellular nutrient or oxygen levels, respectively, it seems likely that these two pathways may be connected. Previous data revealed a clear linkage between the level of activation of the PI3K pathway and hypoxia response as well as the steady-state quantities of HIF-1α (41Laughner E. Taghavi P. Chiles K. Mahon P.C. Semenza G.L. Mol. Cell. Biol. 2001; 21: 3995-4004Crossref PubMed Scopus (1124) Google Scholar, 42Zundel W. Schindler C. Haas-Kogan D. Koong A. Kaper F. Chen E. Gottschalk A.R. Ryan H.E. Johnson R.S. Jefferson A.B. Stokoe D. Giaccia A.J. Genes Dev. 2000; 14: 391-396Crossref PubMed Google Scholar, 43Jiang B.H. Zheng J.Z. Aoki M. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1749-1753Crossref PubMed Scopus (484) Google Scholar, 44Jiang B.H. Jiang G. Zheng J.Z. Lu Z. Hunter T. Vogt P.K. Cell Growth & Differ. 2001; 12: 363-369PubMed Google Scholar, 45Mazure N.M. Chen E.Y. Laderoute K.R. Giaccia A.J. Blood. 1997; 90: 3322-3331Crossref PubMed Google Scholar, 46Blancher C. Moore J.W. Robertson N. Harris A.L. Cancer Res. 2001; 61: 7349-7355PubMed Google Scholar, 47Zhong H. Chiles K. Feldser D. Laughner E. Hanrahan C. Georgescu M.M. Simons J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar, 48Chen E.Y. Mazure N.M. Cooper J.A. Giaccia A.J. Cancer Res. 2001; 61: 2429-2433PubMed Google Scholar). Inhibition of the PI3K pathway was accompanied by a clear decrease in HIF-1α protein levels and inhibition of VEGF induction. Here we demonstrate that the PI3K-regulated transcription factor FOXO4 can suppress cellular response to hypoxia at least in part by down-regulation of the steady-state levels of the HIF-1α protein. Analysis of the mechanism of this down-regulation revealed that, although mediated by the proteasome pathway, it appears to be independent of pVHL-induced ubiquitination. Our data provide not only a molecular explanation for the regulation of the angiogenic response by the PI3K pathway but also the first evidence for the involvement of forkhead transcription factor FOXO4 in the regulation of hypoxia response, suggesting alternate means to control the levels of HIF-1α and resultant angiogenesis. DNA Constructs—The full-length wild-type HIF-1α gene was PCR-amplified from human brain Marathon-Ready cDNA (Clontech) and subcloned into pCDNA3.1/V5-His TOPO vector (Invitrogen) to create an HIF-1α.V5 expression plasmid (pCDNA3.1.HIF-1α.V5). The pEGFP.N3.TM-FOXO4 (also known as pEGFP.N3.TM-AFX) DNA construct used in the transient transfection experiment and luciferase assays has been previously described (9Tang T.T. Dowbenko D. Jackson A. Toney L. Lewin D.A. Dent A.L. Lasky L.A. J. Biol. Chem. 2002; 277: 14255-14265Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). The VEGF and EPO promoter constructs (pGL3p.VEGF and pGL3p.EPO) were made by amplifying the promoter fragments from human genomic DNA (Clontech) and inserting them upstream of the SV40 promoter in the luciferase pGL3.promoter vector (Promega). The primers for amplifying the 948-bp (–1042 to –95) VEGF promoter fragment were 5′-CGA CGC GTC AGC AGG AAC AAG GGC CTC TGT CTG CCC-3′ and 5′-CCG CTC GAG GAC AGG CGA GCC TCA GCC CCT CCA C-3′, and those used for amplifying the 327-bp portion of the EPO promoter were 5′-GCT AGC GAT GCC CCC CAG GGG AGG TGT CC-3′ and 5′-CTC GAG GGT GGC CCA GGG ACT CTG CGG C-3′. To generate the HIF-1α.P402A/P564G.V5 expression plasmid, proline residues 402 and 564 were mutated to alanine and glycine, respectively, by a two-step PCR procedure. Cell Cultures and Stable Cell Lines—The HeLa Tet-on-inducible FOXO4 stable cell line, designated 15-14, was previously reported (9Tang T.T. Dowbenko D. Jackson A. Toney L. Lewin D.A. Dent A.L. Lasky L.A. J. Biol. Chem. 2002; 277: 14255-14265Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and was maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, l-glutamine, penicillin-streptomycin, Geneticin (200 μg/ml, Invitrogen), and hygromycin B (200 μg/ml, Clontech). The HeLa Tet-on cells were cultured in the same medium without hygromycin B. The renal carcinoma cell lines, RCC4-vector and RCC4-VHL-HA, previously described (28Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4117) Google Scholar), were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, glutamine (2 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), and G418 (1 mg/ml) for selection. TaqMan™ Quantitative PCR Analysis—Total RNA was isolated using Qiagen RNeasy mini kit (Qiagen), treated with DNase I (Amplification Grade, Invitrogen), and used at 100 ng per reaction. All the samples were performed in triplicates in each TaqMan™ experiment, and the mRNA levels were normalized to β-actin mRNA levels. Luciferase Assays—293E cells were plated in 24-well plates at subconfluent density and transiently transfected with 160 ng/well pEGFP.N3 or pEGFP.N3.TM-FOXO4, 36 ng/well pGL3p.VEGF or pGL3p.EPO promoter construct, and 4 ng/well pRLTK vector (Promega), using Effectene transfection reagent (Qiagen). When 16 ng/well pEGFP.N3.TM-FOXO4 was transfected, empty vector was added to equalize the final DNA contents. 9 h after transfection cells were incubated in the presence or absence of 100 μm deferoxamine mesylate (DFO; Sigma) for 16 h and then assayed for luciferase activities, which were measured by the dual-luciferase reporter assay system (Promega) as described by the manufacture's protocol. The inducible firefly luciferase activity was normalized to the constitutive Renilla luciferase activity. Western Blotting Analysis—For all HIF-1α down-regulation time course experiments, HeLa Tet-on or Tet-on-inducible FOXO4 stable cells (15-14) were transfected first with pCDNA3.1.HIF-1α.V5 or pCDNA3.1.HIF-1α.P402A/P564G.V5 (2 μg/10-cm dish), using Effectene transfection reagent (Qiagen), and then 8–9 h after transfection incubated in 2% oxygen or with deferoxamine mesylate (DFO, 100 μm, Sigma) to allow for the accumulation of the HIF-1α protein overnight. On the following day, the cells were treated with or without doxycycline hydrochloride (DOX, 2 μg/ml, Clontech) in 2% oxygen or in the presence of fresh 100 μm DFO to initiate the time course. When needed, zVAD-fmk (25 μm, Calbiochem), clastolactacystin β-lactone (20 μm, BIOMOL), MG-132 (20 μm, BIOMOL), or Me2SO was included. At the indicated times, both floating and adherent cells were harvested, and whole-cell extracts were resolved on 4–20% Tris-glycine polyacrylamide-SDS gels (Invitrogen) and transferred to nitrocellulose membranes (Invitrogen). Proteins were detected using antibodies directed against HIF-1α (1:250 dilution, Transduction Laboratories or H1α67, 1:250 dilution, Novus), V5 epitope tag (1:1000 dilution, Invitrogen), FLAG epitope tag (M5, 1:250 dilution, Sigma), β-tubulin (1:1000 dilution, BD Pharmingen), phosphorylated AKT (Phospho-AKT (Thr-308), 1:1000 dilution, and Phospho-AKT (Ser-473), 1:1000 dilution, both from Cell Signaling Technology), AKT (1:1000 dilution, Cell Signaling Technology), and pVHL (1:250 dilution, BD Pharmingen). The PI3K Signaling Pathway Regulates HIF-1α Levels in HeLa Cells—Studies in glioblastoma, prostate, and breast cancer cell lines have shown that activation of the PI3K/AKT signaling pathway results in increased expression of the HIF-1α protein and consequent up-regulation of the VEGF gene transcription (41Laughner E. Taghavi P. Chiles K. Mahon P.C. Semenza G.L. Mol. Cell. Biol. 2001; 21: 3995-4004Crossref PubMed Scopus (1124) Google Scholar, 42Zundel W. Schindler C. Haas-Kogan D. Koong A. Kaper F. Chen E. Gottschalk A.R. Ryan H.E. Johnson R.S. Jefferson A.B. Stokoe D. Giaccia A.J. Genes Dev. 2000; 14: 391-396Crossref PubMed Google Scholar, 43Jiang B.H. Zheng J.Z. Aoki M. Vogt P.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1749-1753Crossref PubMed Scopus (484) Google Scholar, 44Jiang B.H. Jiang G. Zheng J.Z. Lu Z. Hunter T. Vogt P.K. Cell Growth & Differ. 2001; 12: 363-369PubMed Google Scholar, 45Mazure N.M. Chen E.Y. Laderoute K.R. Giaccia A.J. Blood. 1997; 90: 3322-3331Crossref PubMed Google Scholar, 46Blancher C. Moore J.W. Robertson N. Harris A.L. Cancer Res. 2001; 61: 7349-7355PubMed Google Scholar, 47Zhong H. Chiles K. Feldser D. Laughner E. Hanrahan C. Georgescu M.M. Simons J.W. Semenza G.L. Cancer Res. 2000; 60: 1541-1545PubMed Google Scholar). Inhibition of the PI3K/AKT activity by the PI3K inhibitors LY294002 and wortmannin, as well as by overexpression of the phospholipid phosphatase PTEN, demonstrated a requirement for activation of the PI3K/AKT pathway in the increase of the HIF-1α protein levels. To determine whether the PI3K/AKT activity is also necessary in HeLa cells for the induction of HIF-1α-regulated genes under hypoxic condition, we treated the cells with LY294002 for 24 h in the presence or absence of a hypoxic mimic, deferoxamine mesylate (DFO), and analyzed the relative levels of VEGF mRNA by TaqMan™-quantitative PCR (Fig. 1A). DFO highly activated the expression of VEGF transcripts (∼6-fold, purple bars), and this increase was significantly inhibited by the addition of the PI3K inhibitor LY294002 (blue bars). The reduction in VEGF transcript levels by LY294002 was clearly associated with a decrease in the level of HIF-1α protein expression (Fig. 1B). HeLa cells transfected with a HIF-1α expression plasmid showed a decreased HIF-1α protein level in the presence of LY294002. This decline was observed even in the presence of DFO, which normally stabilizes the HIF-1α protein. Thus, the inhibition of the PI3K/AKT activity by LY294002 blocked DFO-induced up-regulation of the HIF-1α protein level. In the absence of DFO, the expression of HIF-1α was almost undetectable, and to see the change in the normoxic HIF-1α protein levels, the Western blot was subjected to longer exposure (indicated by "long"). Thus, PI3K/AKT activity is required in HeLa cells to induce the expression of the HIF-1α protein and allow for increased VEGF transcript levels in response to hypoxia. Similar results were observed when BJAB cells were treated with LY294002 for 24 h (Fig. 1A, data not shown). Nuclear FOXO4 Regulates HIF-1α Protein Levels—Because the forkhead transcription factor FOXO4 is one of the downstream targets of the PI3K/AKT signaling pathway, we examined if FOXO4 was involved in the regulation of the HIF-1α protein expression. To test this possibility, we used our previously reported HeLa Tet-on stable cell line, designated 15-14, which could be induced by doxycycline hydrochloride (DOX) to produce a constitutively active form of FOXO4 (9Tang T.T. Dowbenko D. Jackson A. Toney L. Lewin D.A. Dent A.L. Lasky L.A. J. Biol. Chem. 2002; 277: 14255-14265Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). All three of the AKT phosphorylation sites in this FOXO4 protein were mutated to alanines, allowing it to escape the phosphorylationinduced cytoplasmic sequestration by AKT and localize exclusively in the nucleus. This FOXO4 also contained a FLAG epitope tag at its amino terminus and the green fluorescent protein (GFP) at its carboxyl terminus. LY294002 treatment indicated that this inducible FOXO4 stable cell line 15-14 also required PI3K/AKT activity for hypoxia-induced increase in VEGF transcript levels (Fig. 1A). Nearly 8-fold up-regulation was seen in 15-14 cells when incubated with DFO, and addition of LY294002 suppressed not only up-regulation by DFO but also the basal level of VEGF transcript. Strikingly, induction of FOXO4 in 15-14 by DOX resulted in a dramatic down-regulation of the HIF-1α protein levels (Fig. 2A). This down-regulation occurred even in the presence of DFO, indicating that the stabilizing effect of DFO on the HIF-1α protein could be overridden by FOXO4. In the absence of DOX, FOXO4 expression was not induced, and the levels of the transfected HIF-1α-V5 fusion protein remained relatively constant as long as DFO was present. The down-regulation of the HIF-1α protein was not caused by DOX treatment, because the parental HeLa Tet-on cells, which did not express the mutant FOXO4 protein, did not exhibit down-regulation of the HIF-1α protein when treated with DOX (Fig. 2B). Transient transfection of the triple mutant FOXO4-GFP fusion protein (TM-FOXO4) into HeLa cells revealed a similar effect of FOXO4 on the expression of the HIF-1α protein (Fig. 2C). Cells transfected with a plasmid that expressed the mutant FOXO4-GFP protein showed much lower production of the co-transfected HIF-1α protein than cells transfected with a control GFP expression plasmid in both the presence and absence of the DFO. Expression of nuclear FOXO4 also induced the down-regulation of the HIF-1α protein under true hypoxic conditions (Fig. 2, D and E). Furthermore, in accordance with previous reports (reviewed in Ref. 24Semenza G.L. Curr. Opin. Cell Biol. 2001; 13: 167-171Crossref PubMed Scopus (890) Google Scholar), we observed that hypoxia significantly induced the expression of HIF-1α in HeLa Tet-on cells; this up-regulation was unaffected by DOX treatment (Fig. 2E). However, when expression of nuclear FOXO4 was induced by DOX in 15-14 cells the hypoxiainduced up-regulation of HIF-1α protein level was completely inhibited (compare the change in HIF-1α levels between lanes 7 and 5 to that between lanes 8 and 5). Importantly, although PI3K/AKT activity increases the expression of the HIF-1α protein, the FOXO4 transcription factor, being negatively regulated by AKT, induces the down-regulation of the HIF-1α protein. Because no change in the HIF-1α transcript levels was observed in the mutant FOXO4-inducible 15-14 cells when treated with DOX, it is clear that nuclear FOXO4 does not modulate the transcription of the HIF-1α gene (data not shown). The most likely conclusion from these studies is that FOXO4 initiates a transcriptional program that regulates either the stability or the rate of synthesis of the HIF-1α protein. Nuclear FOXO4 Inhibits the Induction of Hypoxia Response Genes—Because FOXO4 negatively regulates HIF-1α protein expression, we examined its effect on cellular response to hypoxia. Like the PI3K inhibitor LY294002, FOXO4 strongly suppressed the DFO-induced up-regulation of HIF-1α-modulated genes (Fig. 3A). In the absence of DOX, exposure to DFO resulted in a significant increase of the endogenous VEGF and GLUT-1 transcript levels in both 15-14 and parental HeLa Tet-on cell lines (purple bars). In the presence of DOX, approximately equal amounts of VEGF and GLUT-1 transcripts were still induced by DFO in HeLa Tet-on cells (blue bars in right graph). However, when the expression of nuclear FOXO4 was activated by DOX in 15-14 cells, DFO elicited only ∼13 and ∼5% VEGF and GLUT-1 transcript levels, respectively (blue bars in left graph). We next determined whether the FOXO4-induced decrease in transcript levels of the HIF-1α-regulated genes occurred at the level of transcription. Because FOXO4 inhibited the accumulation of the HIF-1α protein even in the presence of DFO, we would expect it to prevent the DFO-induced transcriptional activation of the VEGF and EPO promoters. As Fig. 3 (B and C) demonstrates, co-transfection of 160 ng of a control GFP expression plasmid allowed for ∼2-fold activation of VEGF and EPO promoters in response to DFO treatment. Approximately 50% of this activation was inhibited with only 16 ng of the TM-FOXO4 expression plasmid. Co-transfection of 160 ng of TM-FOXO4 expression plasm