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Phosphatidylinositol 3-Kinase/Akt Signaling Is Neither Required for Hypoxic Stabilization of HIF-1α nor Sufficient for HIF-1-dependent Target Gene Transcription*

Open AccessPublished:February 21, 2002DOI:https://doi.org/10.1074/jbc.M111162200
      The serine/threonine kinase Akt/PKB and the oxygen-responsive transcription factor HIF-1 share the ability to induce such processes as angiogenesis, glucose uptake, and glycolysis. Akt activity and HIF-1 are both essential for development and implicated in tumor growth. Upon activation by products of phosphatidylinositol 3-kinase (PI3K), Akt phosphorylates downstream targets that stimulate growth and inhibit apoptosis. Previous reports suggest that Akt may achieve its effects on angiogenesis and glucose metabolism by stimulating HIF-1 activity. We report here that, whereas serum stimulation can induce a slight accumulation of HIF-1α protein in a PI3K/Akt pathway-dependent fashion, hypoxia induces much higher levels of HIF-1α protein and HIF-1 DNA binding activity independently of PI3K and mTOR activity. In addition, we find the effects of constitutively active Akt on HIF-1 activity are cell-type specific. High levels of Akt signaling can modestly increase HIF-1α protein, but this increase does not affect HIF-1 target gene expression. Therefore, the PI3K/Akt pathway is not necessary for hypoxic induction of HIF-1 subunits or activity, and constitutively active Akt is not itself sufficient to induce HIF-1 activity.
      Oxygen (O2) is critical for mammalian cells, which have evolved a variety of molecular mechanisms to sense and respond to both physiologic and pathophysiologic changes in O2 (
      • Zhu H.
      • Bunn H.F.
      ). From the standpoint of cellular survival, lack of O2(hypoxia) requires a metabolic switch from oxidative to glycolytic metabolism coupled with corresponding changes in the vasculature that compensate for reduced ATP-generating efficiency. To accomplish this, cells respond to low O2 by transactivating genes that increase glucose uptake (GLUT1), glycolysis (glycolytic enzymes), red blood cell production (erythropoietin), and vasodilation (inducible nitric-oxide synthase). However, these responses are short term survival strategies and do not address the fundamental problem of O2 deficiency. To restore O2 delivery to starved tissues, new blood vessel growth, known as angiogenesis or neovascularization, is required. Angiogenesis, the sprouting and growth of new vessels from existing ones, is activated primarily by vascular endothelial growth factor, a potent and highly endothelial cell-specific mitogen, whose expression is induced by hypoxia (
      • Ferrara N.
      ).
      The transcriptional response to hypoxia is regulated by the hypoxia-inducible factors (HIFs),
      The abbreviations used are: HIF
      hypoxia-inducible factor
      PI3K
      phosphatidylinositol 3-kinase
      mTOR
      mammalian target of rapamycin
      bHLH-PAS
      basic helix-loop-helix/Per-Arnt-Sim homology
      HRE
      hypoxia response element
      mHRE
      mutant HRE
      wHRE
      wild type HRE
      E3
      ubiquitin-protein isopeptide ligase
      VHL
      von Hippel-Lindau
      pVHL
      VHL tumor suppressor protein
      myr
      myristoylated
      FBS
      fetal bovine serum
      tet
      tetracycline
      EMSA
      electrophoretic mobility shift assay
      PGK
      phosphoglycerate kinase
      β-gal
      β-galactosidase
      LY
      LY294002
      EF1α
      elongation factor 1α
      MAPK
      mitogen-activated protein kinase
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      ERK
      extracellular signal-regulated kinase
      1The abbreviations used are: HIF
      hypoxia-inducible factor
      PI3K
      phosphatidylinositol 3-kinase
      mTOR
      mammalian target of rapamycin
      bHLH-PAS
      basic helix-loop-helix/Per-Arnt-Sim homology
      HRE
      hypoxia response element
      mHRE
      mutant HRE
      wHRE
      wild type HRE
      E3
      ubiquitin-protein isopeptide ligase
      VHL
      von Hippel-Lindau
      pVHL
      VHL tumor suppressor protein
      myr
      myristoylated
      FBS
      fetal bovine serum
      tet
      tetracycline
      EMSA
      electrophoretic mobility shift assay
      PGK
      phosphoglycerate kinase
      β-gal
      β-galactosidase
      LY
      LY294002
      EF1α
      elongation factor 1α
      MAPK
      mitogen-activated protein kinase
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      ERK
      extracellular signal-regulated kinase
      heterodimeric transcription factors consisting of a regulated α subunit (HIF-1α or HIF-2α/EPAS1) and a constitutive β subunit (ARNT/HIF-1β or ARNT2), all of which are bHLH-PAS proteins. The bHLH-PAS superfamily contains many proteins involved in sensing and responding to changes in environmental conditions (
      • Gu Y.Z.
      • Hogenesch J.B.
      • Bradfield C.A.
      ), and the HIF family exemplifies this role (
      • Semenza G.L.
      ). The HIF-α subunits are constitutively transcribed and translated, but protein levels are controlled by ubiquitination and proteasomal degradation. HIF-α subunits are ubiquitinated in direct proportion to cellular O2 concentrations, providing a molecular rheostat whereby levels of hypoxia-responsive genes are finely and expeditiously regulated by O2. Stabilized HIF-αs translocate to the nucleus where they dimerize with a β subunit, usually ARNT, and the heterodimer then binds to hypoxia response elements (HREs) (
      • Firth J.D.
      • Ebert B.L.
      • Pugh C.W.
      • Ratcliffe P.J.
      ) in the promoters and enhancers of target genes resulting in their transactivation. HIF-mediated gene expression is critical for both embryonic development (
      • Iyer N.V.
      • Kotch L.E.
      • Agani F.
      • Leung S.W.
      • Laughner E.
      • Wenger R.H.
      • Gassmann M.
      • Gearhart J.D.
      • Lawler A.M.
      • Yu A.Y.
      • Semenza G.L.
      ,
      • Maltepe E.
      • Schmidt J.V.
      • Baunoch D.
      • Bradfield C.A.
      • Simon M.C.
      ,
      • Ryan H.E.
      • Lo J.
      • Johnson R.S.
      ) and tumor growth (
      • Kung A.L.
      • Wang S.
      • Klco J.M.
      • Kaelin W.G.
      • Livingston D.M.
      ,
      • Ryan H.E.
      • Poloni M.
      • McNulty W.
      • Elson D.
      • Gassmann M.
      • Arbeit J.M.
      • Johnson R.S.
      ).
      Much is known mechanistically about the way HIF-α subunits are modified and destroyed. Newly translated HIF-α is hydroxylated at two proline residues in its oxygen-dependent degradation domain (ODD) (
      • Huang L.E.
      • Gu J.
      • Schau M.
      • Bunn H.F.
      ) by HIF-prolyl hydroxylases (
      • Bruick R.K.
      • McKnight S.L.
      ,
      • Epstein 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.
      ,
      • Ivan M.
      • Kondo K.
      • Yang H.
      • Kim W.
      • Valiando J.
      • Ohh M.
      • Salic A.
      • Asara J.M.
      • Lane W.S.
      • Kaelin Jr., W.G.
      ,
      • Jaakkola 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.
      ,
      • Masson N.
      • Willam C.
      • Maxwell P.H.
      • Pugh C.W.
      • Ratcliffe P.J.
      ,
      • Yu F.
      • White S.B.
      • Zhao Q.
      • Lee F.S.
      ). This modification allows HIF-α to be recognized by a ubiquitin E3 ligase complex containing pVHL, a tumor suppressor that recognizes hydroxylated HIF-α and allows it to be targeted to the 26 S proteasome (
      • Ivan M.
      • Kaelin W.G.
      ). In this fashion, low O2 leads to the accumulation of HIF-α subunits. The HIF-prolyl hydroxylases are among many proposed oxygen sensors, including the mitochondria (
      • Chandel N.S.
      • Maltepe E.
      • Goldwasser E.
      • Mathieu C.E.
      • Simon M.C.
      • Schumacker P.T.
      ,
      • Chandel N.S.
      • McClintock D.S.
      • Feliciano C.E.
      • Wood T.M.
      • Melendez J.A.
      • Rodriguez A.M.
      • Schumacker P.T.
      ), but where precisely cellular oxygen sensing takes place and by what remains a contentious issue.
      While the mechanism of HIF-α degradation is becoming clear, the signaling pathways that influence HIF-α stability and nuclear translocation and, subsequently, HIF-1 transcriptional activity remain somewhat confusing and controversial. Many published reports suggest that the regulation of HIF-1α stabilization and nuclear translocation and HIF-1 transcriptional activity are distinct events that are coordinately controlled by hypoxia (
      • Semenza G.L.
      ), although evidence also exists that hypoxic signal transduction is not necessary for HIF activity when HIF-1α is overexpressed (
      • Hofer T.
      • Desbaillets I.
      • Hopfl G.
      • Gassmann M.
      • Wenger R.H.
      ) or constitutively stable (
      • Masson N.
      • Willam C.
      • Maxwell P.H.
      • Pugh C.W.
      • Ratcliffe P.J.
      ).
      Particular scrutiny has been focused on the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Akt (also known as protein kinase B or PKB), a proto-oncogene, is a major downstream effector of growth factor signaling and has a wide array of progrowth, antiapoptotic effects when activated by growth factors through PI3K (for review, see Ref.
      • Kandel E.S.
      • Hay N.
      ). Most intriguingly Akt shares with HIF-1 the ability to induce vascular endothelial growth factor and angiogenesis (
      • Jiang B.H.
      • Zheng J.Z.
      • Aoki M.
      • Vogt P.K.
      ,
      • Zundel 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.
      ) and to increase cellular glucose uptake and glycolysis (
      • Barthel A.
      • Okino S.T.
      • Liao J.
      • Nakatani K.
      • Li J.
      • Whitlock Jr., J.P.
      • Roth R.A.
      ,
      • Plas D.R.
      • Talapatra S.
      • Edinger A.L.
      • Rathmell J.C.
      • Thompson C.B.
      ,
      • Gottlob K.
      • Majewski N.
      • Kennedy S.
      • Kandel E.
      • Robey R.B.
      • Hay N.
      ). Many nonhypoxic stimuli including the inflammatory mediators NO, tumor necrosis factor-α (
      • Sandau K.B.
      • Faus H.G.
      • Brune B.
      ,
      • Sandau K.B.
      • Zhou J.
      • Kietzmann T.
      • Brune B.
      ), and thrombin (
      • Gorlach A.
      • Diebold I.
      • Schini-Kerth V.B.
      • Berchner-Pfannschmidt U.
      • Roth U.
      • Brandes R.P.
      • Kietzmann T.
      • Busse R.
      ); growth factors including insulin (
      • Zelzer E.
      • Levy Y.
      • Kahana C.
      • Shilo B.Z.
      • Rubinstein M.
      • Cohen B.
      ), insulin-like growth factor (
      • Zundel 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.
      ,
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ), epidermal growth factor (
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ,
      • Zhong H.
      • Chiles K.
      • Feldser D.
      • Laughner E.
      • Hanrahan C.
      • Georgescu M.M.
      • Simons J.W.
      • Semenza G.L.
      ), hepatocyte growth factor/scatter factor (
      • Tacchini L.
      • Dansi P.
      • Matteucci E.
      • Desiderio M.A.
      ), platelet-derived growth factor, and transforming growth factor-β (
      • Gorlach A.
      • Diebold I.
      • Schini-Kerth V.B.
      • Berchner-Pfannschmidt U.
      • Roth U.
      • Brandes R.P.
      • Kietzmann T.
      • Busse R.
      ); and heregulin (
      • Laughner E.
      • Taghavi P.
      • Chiles K.
      • Mahon P.C.
      • Semenza G.L.
      ) have been reported to induce HIF activity in PI3K-dependent or -related ways. In certain cell types, positive stimulation of the PI3K/Akt pathway using constitutively active molecules can induce or enhance HIF activity. Conversely inhibition of the pathway using dominant negative peptides, small molecule inhibitors, or overexpression of PTEN, a phosphatase that reverses PI3K-catalyzed phosphorylation, can block HIF activity (
      • Zundel 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.
      ,
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ,
      • Zhong H.
      • Chiles K.
      • Feldser D.
      • Laughner E.
      • Hanrahan C.
      • Georgescu M.M.
      • Simons J.W.
      • Semenza G.L.
      ,
      • Baek S.H.
      • Lee U.Y.
      • Park E.M.
      • Han M.Y.
      • Lee Y.S.
      • Park Y.M.
      ,
      • Blancher C.
      • Moore J.W.
      • Robertson N.
      • Harris A.L.
      ,
      • Mazure N.M.
      • Chen E.Y.
      • Laderoute K.R.
      • Giaccia A.J.
      ,
      • Sodhi A.
      • Montaner S.
      • Miyazaki H.
      • Gutkind J.S.
      ), although this remains controversial (
      • Jones A.
      • Fujiyama C.
      • Blanche C.
      • Moore J.W.
      • Fuggle S.
      • Cranston D.
      • Bicknell R.
      • Harris A.L.
      ).
      Previous reports suggest that PI3K and Akt are central in the regulation of HIF activity. We set out to test this link using a variety of different cell types and assays and to test both the necessity and sufficiency of Akt or PI3K activity to lead to HIF-1α stability or HIF-1 activity. We report here that, although inhibitors of the PI3K/Akt pathway can block serum-induced accumulation of HIF-1α, they do not block hypoxic induction. In transient transfections, constitutively active (myristoylated) Akt (myrAkt) is able to increase HIF-dependent transcription in glioblastoma cells but not in hepatoma cells, suggesting a fundamental difference in the way these two cell lines respond to constitutive Akt activity. In addition, we show in a murine pro-B cell line (FL5.12) that even at elevated levels of Akt signaling, HIF-1α protein levels are only slightly affected, and target gene expression is not induced, indicating that Akt activity itself is not sufficient in these cells to stimulate HIF-1 target gene transcription.

      DISCUSSION

      It has been tempting to speculate, based on published reports, that the demonstrable interactions between HIF-1 and the PI3K/Akt/mTOR pathway represent a common mechanism whereby growth factor signaling or oncogenic transformation could lead to increased angiogenesis, glucose transport, and glycolysis. Constitutively active Akt and PI3K have remarkable effects on angiogenesis in chick chorioallantoic membrane assays (
      • Jiang B.H.
      • Zheng J.Z.
      • Aoki M.
      • Vogt P.K.
      ) and on metabolism in mammalian cells (
      • Barthel A.
      • Okino S.T.
      • Liao J.
      • Nakatani K.
      • Li J.
      • Whitlock Jr., J.P.
      • Roth R.A.
      ,
      • Plas D.R.
      • Talapatra S.
      • Edinger A.L.
      • Rathmell J.C.
      • Thompson C.B.
      ,
      • Gottlob K.
      • Majewski N.
      • Kennedy S.
      • Kandel E.
      • Robey R.B.
      • Hay N.
      ). HIF-1 transactivates many genes that would be useful to a rapidly proliferating cell mass, including genes and processes known to be impacted by Akt, and is regulated post-translationally in a way that could easily be modulated by a kinase such as Akt. In addition, both HIF-1 and Akt are responsive to the environment of the cell, enacting cellular responses to changes in the cellular milieu. The fact that so many of the responses enacted by growth factor signaling through PI3K and Akt overlap with those regulated by HIF-1 makes HIF an attractive candidate as a downstream effector of Akt.
      In fact, HIF-1α protein and/or HIF-1 DNA binding levels have been shown to be responsive to a variety of growth factors and oncogenes in certain cell types (for review, see Ref.
      • Semenza G.L.
      ), and these responses are often PI3K- and mTOR-dependent. Constitutively active PI3K and Akt, as well as loss of PTEN, appear to enhance HIF-1 activity (
      • Zundel 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.
      ,
      • Jiang B.H.
      • Jiang G.
      • Zheng J.Z.
      • Lu Z.
      • Hunter T.
      • Vogt P.K.
      ,
      • Zhong H.
      • Chiles K.
      • Feldser D.
      • Laughner E.
      • Hanrahan C.
      • Georgescu M.M.
      • Simons J.W.
      • Semenza G.L.
      ). In addition, overexpression of wild type PTEN in U373 glioblastoma cells, normally mutated for this gene, can completely ablate hypoxic induction of HIF-1 (
      • Zundel 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.
      ). It has been shown that growth factors and oncogenes can induce HIF-1α, but we demonstrate here that this induction is of a much lower magnitude than hypoxia and is context- and cell type-dependent in marked contrast to hypoxia. Previous reports have relied on prostate cancer and glioblastoma lines, which are known to have high mutation frequencies in the PTEN gene and elsewhere in the PI3K pathway (
      • Cantley L.C.
      • Neel B.G.
      ). The mutational backgrounds of these cells may predispose or sensitize them to additional perturbations in the PI3K pathway. As we have shown in hepatocytes and in glioblastoma cells, PI3K activity is not necessary for hypoxic accumulation of HIF-1α or activation of HIF-1, and in FL5.12 cells myrAkt does not affect HIF-1 target genes. A recent report from Semenza and colleagues (
      • Laughner E.
      • Taghavi P.
      • Chiles K.
      • Mahon P.C.
      • Semenza G.L.
      ) convincingly shows that, in breast cancer and 3T3 cells, heregulin and HER2 stimulation of HIF-1α occurs via an increase in the rate of protein synthesis not by blocking its degradation. This is an attractive model for all growth factor-induced increases in HIF-1 activity since it invokes a mechanism that does not involve VHL, HIF-prolyl hydroxylase, or hypoxia.
      Many signaling pathways other than PI3K/Akt have been studied with respect to hypoxic signal transduction and HIF activity, and it has been proposed that HIF-1α stability, translocation, dimerization with ARNT, and transcriptional activity are separable (and perhaps separately regulated) events. MAPK/MEK/ERK (
      • Hofer T.
      • Desbaillets I.
      • Hopfl G.
      • Gassmann M.
      • Wenger R.H.
      ,
      • Sodhi A.
      • Montaner S.
      • Miyazaki H.
      • Gutkind J.S.
      ,
      • Hur E.
      • Chang K.Y.
      • Lee E.
      • Lee S.K.
      • Park H.
      ,
      • Minet E.
      • Arnould T.
      • Michel G.
      • Roland I.
      • Mottet D.
      • Raes M.
      • Remacle J.
      • Michiels C.
      ,
      • Richard D.E.
      • Berra E.
      • Gothie E.
      • Roux D.
      • Pouyssegur J.
      ,
      • Sodhi A.
      • Montaner S.
      • Patel V.
      • Zohar M.
      • Bais C.
      • Mesri E.A.
      • Gutkind J.S.
      ,
      • Treins C.
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Van Obberghen E.
      ) c-Jun NH2-terminal kinase/p38 (
      • Tacchini L.
      • Dansi P.
      • Matteucci E.
      • Desiderio M.A.
      ,
      • Sodhi A.
      • Montaner S.
      • Patel V.
      • Zohar M.
      • Bais C.
      • Mesri E.A.
      • Gutkind J.S.
      ), Ras (
      • Blancher C.
      • Moore J.W.
      • Robertson N.
      • Harris A.L.
      ,
      • Chen C.
      • Pore N.
      • Behrooz A.
      • Ismail-Beigi F.
      • Maity A.
      ), and Rac1 (
      • Hirota K.
      • Semenza G.L.
      ) have all been implicated in aspects of HIF-1 regulation. In worms and flies, however, the loss of HIF-prolyl hydroxylase activity prevents HIF-αs from being ubiquitinated, which not only renders HIF-1α constitutively stable but causes HIF-1 target genes to be up-regulated as well (
      • Bruick R.K.
      • McKnight S.L.
      ,
      • Epstein 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.
      ). These results suggest that hypoxic signaling is not necessary for HIF-1 activity. In support of this, VHL-null tumor lines (
      • Ivan M.
      • Kondo K.
      • Yang H.
      • Kim W.
      • Valiando J.
      • Ohh M.
      • Salic A.
      • Asara J.M.
      • Lane W.S.
      • Kaelin Jr., W.G.
      ,
      • Jaakkola 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.
      ) and embryonic stem cells
      F. Mack, personal communication.
      possess constitutively stable and transcriptionally active HIF-1. In addition, rendering HIF-1α constitutively stable (
      • Masson N.
      • Willam C.
      • Maxwell P.H.
      • Pugh C.W.
      • Ratcliffe P.J.
      ) or overexpressing it under normoxic conditions are each sufficient to activate target genes, although the latter appears to be partially dependent on MAPK for full activity (
      • Hofer T.
      • Desbaillets I.
      • Hopfl G.
      • Gassmann M.
      • Wenger R.H.
      ). Thus it appears that HIF-1 is at least partially competent to activate transcription of target genes in the absence of any additional hypoxic signaling. The activities of other pathways implicated in or activated by hypoxic signaling may be required for additional activation but do not appear to be necessary for basal function.
      We show here that 1c1c7 hepatocyte and U373 glioblastoma cell lines, both of which have robust HIF responses, behave differently with respect to myrAkt. While myrAkt can induce modest HIF-dependent transcriptional responses in the U373 cells, it is unable to do so in the 1c1c7 line. In addition, FL5.12 cells, which show marked survival and metabolic phenotypes in response to myrAkt (
      • Plas D.R.
      • Talapatra S.
      • Edinger A.L.
      • Rathmell J.C.
      • Thompson C.B.
      ), demonstrate no consistent HIF-1α response and no HIF-1 target gene induction by increased myrAkt expression. This suggests both that myrAkt alone is not sufficient to induce HIF-1 and that the metabolic and antiapoptotic effects of myrAkt in these cells are not dependent on HIF-1.
      Hypoxia and Akt have profound effects on cellular glucose metabolism, leading to increased glucose uptake and lactate secretion over time. Consequent changes in the culture medium can themselves affect gene expression and must be controlled for. In our experimental systems, the differences between glucose and lactate in the medium of normoxic and hypoxic 1c1c7 cells is less than 5% at 24 h (data not shown). In the myrAkt-expressing FL5.12 cells, there is a slightly more significant change in glucose and lactate (on the order of 10%) compared with vector controls at 24 h.
      K. A. Frauwirth, J. L. Riley, M. H. Harris, R. V. Parry, J. C. Rathmell, D. R. Plas, R. L. Elstrom, C. H. June, and C. B. Thompson, manuscript submitted.
      However, we do not believe that these changes are sufficiently pronounced to alter the metabolism of the cells over the time periods studied as the medium is still replete with glucose after 24 h, and any accumulation of lactate is similar between Akt and vector control cells. In addition, we feel that in any experiments where these factors might affect the results, the appropriate controls (i.e.±doxycycline, ±hypoxia, and ±Akt) have been included to rule out such variables.
      Hypoxia induces HIF-α levels in all cell lines or primary cell cultures tested (except those lacking pVHL), demonstrating the importance and primacy of hypoxic regulation of HIF. MyrAkt, in contrast, seems only to affect HIF in certain cell types. Whether this is a function of PTEN mutation or other dysregulation in the PI3K pathway is an open question. Sufficiently high levels of Akt signaling can increase the levels of HIF-1α protein in the FL5.12 system, although this observation was not consistent among different clones. We propose that constitutive up-regulation of PI3K signaling, either through oncogenic activation of the pathway or through inactivation of the PTEN tumor suppressor, can lead to increased HIF-α levels in certain tumor contexts. However, the relevance of this to nontumor-related HIF function remains unclear. There may be important distinctions between physiological and pathophysiological regulation and effects of HIF-1, and further understanding of the role of the HIF family in both normal development and physiology and in tumor biology will depend on elucidation of these distinctions.

      Note Added in Proof

      While this manuscript was under review, del Peso and colleagues reported similar findings using different cell lines and experimental approaches (Alvarez-Tejado, M., Alfranca, A., Aragones, J., Vara, A., Landazuri, M. O., and del Peso, L. (January 28, 2002) J. Biol. Chem.10.1074/jbc.M200017200).

      Acknowledgments

      We thank Brian Keith for helpful discussions and critical reading of this manuscript.

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