Activated pp60c-Src Leads to Elevated Hypoxia-inducible Factor (HIF)-1α Expression under Normoxia

Hypoxia-inducible factor (HIF)-1 is a master transcription factor, which up-regulates glycolysis, erythropoiesis, and angiogenesis under hypoxia. HIF-1α accumulates in normoxic tumor cells, leading to glycolysis under aerobic conditions. This phenomenon, known as the “Warburg effect,” is caused by a yet unknown mechanism. Here we show that transformed cells that express constitutively active pp60c-Src (Src) express HIF-1α protein under normoxia, which results in the expression of multiple HIF-1α target genes. We show that this occurrence is due to an enhanced rate of HIF-1α protein synthesis and not due to reduced HIF-1α degradation. Furthermore, we show that the Src-induced increase in protein synthesis is due to the global increase in the rate of cap-dependent translation and does not involve inhibition of HIF-1α degradation. Hypoxia-inducible factor (HIF)-1 is a master transcription factor, which up-regulates glycolysis, erythropoiesis, and angiogenesis under hypoxia. HIF-1α accumulates in normoxic tumor cells, leading to glycolysis under aerobic conditions. This phenomenon, known as the “Warburg effect,” is caused by a yet unknown mechanism. Here we show that transformed cells that express constitutively active pp60c-Src (Src) express HIF-1α protein under normoxia, which results in the expression of multiple HIF-1α target genes. We show that this occurrence is due to an enhanced rate of HIF-1α protein synthesis and not due to reduced HIF-1α degradation. Furthermore, we show that the Src-induced increase in protein synthesis is due to the global increase in the rate of cap-dependent translation and does not involve inhibition of HIF-1α degradation. hypoxia-inducible factor vascular endothelial growth factor HIF-1-responsive element kinase-dead phosphatidylinositol 3-kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase mitogen-activated protein kinase hemagglutinin wild type extracellular signal-regulated kinase internal ribosome entry site secreted alkaline phosphatase untranslated region phosphate-buffered saline monoclonal antibody Hypoxia-inducible factor (HIF)1-1 is a master transcription factor, which regulates oxygen homeostasis by inducing glycolysis, erythropoiesis, and angiogenesis (1Semenza G.L. Cell. 2001; 107: 1-3Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). It has been suggested that pp60c-Src (Src) mediates the hypoxic induction of VEGF expression (2Mukhopadhyay D. Tsiokas L. Zhou X.M. Foster D. Brugge J.S. Sukhatme V.P. Nature. 1995; 375: 577-581Crossref PubMed Scopus (539) Google Scholar) and that the expression of v-Src enhances the expression of HIF-1α (3Jiang B.H. Agani F. Passaniti A. Semenza G.L. Cancer Res. 1997; 57: 5328-5335PubMed Google Scholar) which, in turn, up-regulates HIF-1 target genes. In view of the presence of activated Src in many malignant tumors, such as colon (4Mao W. Irby R. Coppola D., Fu, L. Wloch M. Turner J., Yu, H. Garcia R. Jove R. Yeatman T.J. Oncogene. 1997; 15: 3083-3090Crossref PubMed Scopus (180) Google Scholar), breast (5Verbeek B.S. Vroom T.M. Adriaansen-Slot S.S. Ottenhoff-Kalff A.E. Geertzema J.G. Hennipman A. Rijksen G. J. Pathol. 1996; 180: 383-388Crossref PubMed Scopus (241) Google Scholar), and lung (6Krystal G.W. DeBerry C.S. Linnekin D. Litz J. Cancer Res. 1998; 58: 4660-4666PubMed Google Scholar) cancers and in light of the observation that many of these tumors also express high levels of HIF-1α protein under normoxia, we examined the proposition that Src up-regulates HIF-1α and explored the molecular mechanism(s) by which Src may affect HIF-1α protein levels. Earlier reports have already pointed to a correlation between HIF-1α expression and Src activity. Studies have revealed hypoxia-induced activation of Src kinases (7Seko Y. Tobe K. Takahashi N. Kaburagi Y. Kadowaki T. Yazaki Y. Biochem. Biophys. Res. Commun. 1996; 226: 530-535Crossref PubMed Scopus (50) Google Scholar) and increased levels of HIF-1α in v-Src transformed cells (3Jiang B.H. Agani F. Passaniti A. Semenza G.L. Cancer Res. 1997; 57: 5328-5335PubMed Google Scholar). It has also been shown that inhibition of c-Src expression by antisense expression reduces VEGF expression (8Ellis L.M. Staley C.A. Liu W. Fleming R.Y. Parikh N.U. Bucana C.D. Gallick G.E. J. Biol. Chem. 1998; 273: 1052-1057Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), although it has been argued that c-Src is not involved in inducing hypoxic-regulated genes (9Gleadle J.M. Ratcliffe P.J. Blood. 1997; 89: 503-509Crossref PubMed Google Scholar). The well documented mechanism for HIF-1α regulation is by its stabilization under hypoxia and its rapid degradation via the ubiquitin-proteasome pathway under normoxia (10Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1417) Google Scholar, 11Maxwell 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 (4179) Google Scholar, 12Cockman 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 (924) Google Scholar). Here we report that HIF-1α protein accumulates due to elevated translation and not due to elevated mRNA levels or extended protein half-life. We show that the Src-induced increase in protein synthesis is not a HIF-1α-specific phenomenon, but is due to a general increase in cap-dependent translation. CSH12 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. HT29 and Saos-2 cells were grown in McCoy-5A medium supplemented with 10% fetal calf serum. All media were supplemented with penicillin and streptomycin. Saos-2 clones containing empty plasmid (pUSE(−), wild type c-Src (pUSE-WT Src), kinase-dead Src (K297R) (pUSE-KD Src) and active Src (Y529F) (pUSE-Active Src) were obtained by transfection of the above constructs (Upstate Biochemistry) into Saos-2 cells using FuGENE 6 (according to the manufacturer's instructions) and selection with 500 μg/ml G418. HeLa cells expressing HA-eIF4E were generated by transfection of HeLa cells with pCDNA-HA-eIF4E (a gift of Dr. Nahum Sonenberg, McGill University) and were grown in the presence of 500 μg/ml G418. PP1, PP2 and PP3 were synthesized by Dr. Aviv Gazit as described (34Schindler T. Sicheri F. Pico A. Gazit A. Levitzki A. Kuriyan J. Mol. Cell. 1999; 3: 639-648Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Transient transfections were performed with FuGENE 6 (according to the manufacturer's instructions) or polyethyleneimine (35Xing X. Wang S.C. Xia W. Zou Y. Shao R. Kwong K.Y., Yu, Z. Zhang S. Miller S. Huang L. Hung M.C. Nat. Med. 2000; 6: 189-195Crossref PubMed Scopus (161) Google Scholar) Transcription from the HRE was measured using plasmid p-tkp-luc, which contains five HRE sequences (17Zelzer E. Levy Y. Kahana C. Shilo B.Z. Rubinstein M. Cohen B. EMBO J. 1998; 17: 5085-5094Crossref PubMed Scopus (498) Google Scholar). Cells were seeded and 24 h later were transfected. Total DNA for transfection was 2.5 μg per well, comprising 1 μg of luciferase reporter constructs, 0.5 μg of β-galactosidase internal control vector and 1 μg of the Src construct, as indicated under “Results.” In the experiments utilizing the Src inhibitor PP1, cells were transfected only with p-tkp-luc and CMV-lac-Z plasmids; 24 h after transfection the medium was replaced with medium containing inhibitor for an additional 24 h, using PP1 concentrations as indicated in the results. In all the experiments cells were lysed 48 h after transfection with reporter lysis buffer (Promega) according to the manufacturer's instructions, and luciferase activity was measured and normalized to β-galactosidase activity. The reporter encoding secreted alkaline phosphatase and containing HIF-1α 5′-UTR was constructed by inserting 289 bp of HIF-1α 5′-UTR into plasmid pBKC/S in HindIII andXhoI sites. HIF-1α 5′-UTR was amplified using primers HIFI-5-R: 5′-GGTGAATCGGTCCCCGCGATG-3′ and HIFI-5-F: 5′-GTGCTGCCTCGTCTGAGGGGACA-3′. Cells were washed three times with PBS, then lysed with sample buffer (10% glycerol, 0.2 m Tris-HCl, pH 6.8, 5% β-mercaptoethanol, 3% SDS) and boiled for 5 min. The whole cell lysate was then subjected to SDS-PAGE and transferred to nitrocellulose. The membranes were blocked in TBST (10 mmTris-HCl, pH 7.5, 50 mm NaCl, and 0.1% Triton X-100) containing 5% low fat (1%) milk for 30 min, followed by incubation for 1.5 h with primary antibody (as indicated in the figure legends). Membranes were then washed extensively with TBST, and immunoreactive proteins were detected by incubation with either horseradish peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch 1:10000) for detection of monoclonal antibodies or horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (Jackson ImmunoResearch 1:10000) for detection of polyclonal antibodies. Proteins were visualized using enhanced chemiluminescence. Cells were grown on 10-cm dishes and treated with PP1 for 2 h or 24 h as indicated in the results. RNA was prepared using Trizol reagent (Sigma), and 10 μg of total RNA were denatured and loaded on a 1% agarose gel containing 8% formaldehyde. Following capillary blotting onto a nylon membrane, the RNA was UV-cross-linked (1200 j/m2), and the membrane stained with 0.1% methylene blue to verify equal loading and transfer. The blot was then hybridized overnight at 42 °C with 32P-labeled DNA probe, prepared with the Rediprime kit (Amersham Biosciences). After two washes at 60 °C with 2× SSC, 1% SDS the blot was exposed to a MS-sensitive film (Kodak) or a phosphorimaging plate (Fuji). 106 (HT29) or 5 × 105 (Saos-2) cells were seeded on 60-mm dishes, and 24 h later 100 μm cycloheximide was added for the length of time indicated in the results. Cells were then lysed with sample buffer and subjected to SDS-PAGE and Western blotting. The blots were probed with anti-HIF-1α (1:250 Transduction Laboratories), anti-actin (1:2000 Santa Cruz), and anti-c-Src (mAb 327) antibodies as indicated in the figure legends. 4 × 105 (HT29) and 2 × 105 (Saos-2) cells were seeded on 6-well plates and 24 h later were treated with PP1 (20 μm), PP2 (20 μm), PP3 (20 μm), LY294002 (20 μm, Calbiochem), PD98059 (50 μm,Calbiochem) wortmannin (200 nm, Sigma), and rapamycin (50 nm, Calbiochem) for 30 min. The medium was then replaced with medium containing ALLN (200 μm, Calbiochem) as well as the inhibitors mentioned above for an additional 2 h. Cells were then lysed with sample buffer. For labeling of HA-HIF-1α, CSH12 cells were grown on 10-cm dishes (1 × 106 cells/dish) and were transfected with 0.5 μg of HA-HIF-1α expression plasmid, together with 5 μg of pUSE(−), (Upstate Biochemistry) using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's instructions. 24 h later cells were starved for methionine and cysteine for 1 h in the presence of the inhibitors indicated. Cells were then labeled with [35S]methionine and [35S]cysteine Promix (Amersham Biosciences), 100 μCi/ml for 45 min in the presence of 200 μm ALLN and the inhibitors. Cells were then washed with PBS and lysed with RIPA buffer (20 mm Hepes, pH 7.4, 125 mm NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, 5 mm NaF, 100 μm NaVO3, 1 mm EDTA, 2 mm EGTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin and 5 μg/ml leupeptin). After Bradford protein quantification, HA-HIF-1α was immunoprecipitated from lysates containing 0.8 mg of total protein, using anti-HA antibody mAb 12CA5 produced from hybridoma cells or Rat mAb 3F10 (Roche Molecular Biochemicals). 40 μl of protein G-Sepharose beads (AmershamBiosciences) per sample were incubated with the anti-HA antibodies for 1.5 h and after washes were incubated with lysates for 2 h. After additional washes the beads were mixed with 2× sample buffer and boiled for 5 min. After Western blotting and blocking, membranes were exposed to a phosphorimaging device (Fuji) or to film. For detection of HA-HIF-1α protein levels, membranes were probed with anti-HIF-1α antibody (Transduction Laboratories). For labeling of total protein, Saos-2 cells (3 × 105 cells/well) and HT29 cells (5 × 105 cells/well) were seeded on six-well plates (Nunc). 24 h later, cells were washed with PBS, and the medium was replaced with methionine- and cysteine-deficient medium containing the indicated inhibitors for 1 h. Medium was then replaced with medium containing 50 μCi/ml [35S]Met/Cys Promix (AmershamBiosciences) and the indicated inhibitors. Cells were labeled for 10 min at 37 °C. Cells were then washed with PBS and lysed with sample buffer. Identical amounts of protein from the lysates of Saos-2 cells were run on 10% SDS-PAGE and exposed to film. To measure synthesis rates, lysates were loaded on Whatman No. 3MM paper squares. Paper squares were stained with Coomassie, and washed five times for 6 min with destain solution (20% methanol 7% acetic acid). Then the paper squares were dried, and radioactivity was counted with scintillation solution in a β-counter. In parallel, in another set of same samples, staining extracted from papers with 3% SDS and protein amounts were determined using a bovine serum albumin calibration curve, reading the absorbance at 590 nm. In the cell line CSH12 (NIH3T3 overexpressing the chimeric receptor, EGFR-HER-2 (13Lee J. Dull T.J. Lax I. Schlessinger J. Ullrich A. EMBO J. 1989; 8: 167-173Crossref PubMed Scopus (108) Google Scholar)) Src is constitutively active N. Levitzki A. J. Biochem. PubMed Scopus Google Scholar). We that genes such as and VEGF were under normoxia in CSH12 in cells 1 We also high levels of expression of these genes in cell such as the colon cell line HT29 1 S. Biol. 1996; Google Scholar), and Saos-2 cells not that high Src activity. of Src kinase activity by the Src kinase PP1 J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), in a in the mRNA levels of these genes 1 The mRNA levels of the transcription factor that regulates the expression of these PP1 1 To the regulation of by we a luciferase by a five HIF-1-responsive from the (17Zelzer E. Levy Y. Kahana C. Shilo B.Z. Rubinstein M. Cohen B. EMBO J. 1998; 17: 5085-5094Crossref PubMed Scopus (498) Google Scholar). We that the Src kinase PP1, the expression of the luciferase in HT29 and CSH12 cells and activated Src (Y529F) or wild type c-Src were with the expression of the luciferase was under normoxic kinase-dead Src (K297R) its expression and the of the Src kinase PP1, may not we examined the of c-Src on HIF-1α We examined the levels of HIF-1α expression in Saos-2 cells expressing activated wild type or shown in 2 Saos-2 cells expressing activated or overexpressing wild type Src express high HIF-1α protein levels as with cells expressing empty plasmid or in protein levels were not due to mRNA levels The for HIF-1α regulation stabilization under hypoxia and degradation via the ubiquitin-proteasome pathway under normoxia (10Salceda S. Caro J. J. Biol. Chem. 1997; 272: 22642-22647Abstract Full Text Full Text PDF PubMed Scopus (1417) Google Scholar). We the of HIF-1α protein in Saos-2 cells expressing the of Src and in the presence or of Src inhibitor PP1 in HT29 shown in and the of HIF-1α is in all clones and in the presence or of PP1 and is 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 (4179) Google Scholar). To Src kinase inhibition HIF-1α degradation or synthesis and to the of the and the of we blocked the degradation of HIF-1α in HT29 cells using the inhibitors or ALLN of the pathway in the of the HIF-1α This was blocked in the presence of HIF-1α was also by the inhibitor the inhibitor LY294002 and and to a by the inhibitor of in Saos-2 cells and a Src kinase inhibitor that is to PP1, the of HIF-1α in a to PP1, that is in but does not Src kinases F. M. W. S. A. 2000; PubMed Scopus Google Scholar), not HIF-1α results that Src is to HIF-1α protein synthesis in a of protein In of these we that the of HA-HIF-1α in the presence of inhibitors in CSH12 cells was also blocked by but not by active or empty plasmid Src not affect the mRNA levels or the of 1 and although inhibition of Src by PP1 or Src blocked the of we that Src the synthesis of We measured the of [35S]methionine into transfected HA-HIF-1α in CSH12 We that PP1 and LY294002 PD98059 on HIF-1α synthesis in CSH12 cells 4 the pathway and the pathway involved in cap-dependent translation B. N. 2001; 15: PubMed Scopus Google Scholar, B. R. N. 1999; PubMed Scopus Google Scholar, B. J. Biochem. Biol. 1999; PubMed Scopus Google Scholar) and to activated we examined the of active and PP1 on cap-dependent translation activity. We a of a luciferase reporter followed by an internal ribosome entry site with secreted alkaline phosphatase as a reporter We measured on luciferase activity to on activity We that active Src increased cap-dependent kinase-dead Src and PP1 cap-dependent translation 4 The two reporter genes were also as constructs, as to cap-dependent 4 Src and Src the and in a the of to the same that the of Src constructs on translation were of the of the reporter it was that the region of HIF-1α its synthesis E. P. K. Semenza G.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar), we the 5′-UTR of HIF-1α to of the reporter and measured the of Src on the reporter as in 4 C. We on the reporter activity by the Src To the Src on protein synthesis is HIF-1α-specific or it global protein we measured the of [35S]methionine into Saos-2 and HT29 cells in the or presence of Src kinase inhibitors and inhibitors of its PP1 and PP2 protein synthesis in Saos-2 and HT29 cells, which is in but does not Src F. M. W. S. A. 2000; PubMed Scopus Google Scholar), not and of by LY294002 and wortmannin or inhibition of its target by rapamycin also protein and the inhibitor the of the protein is the in the of cap-dependent translation B. N. Biochem. 1999; PubMed Scopus Google Scholar), we in HeLa cells and examined its on the levels of HIF-1α in expression of elevated the levels of of the of tumors is to glycolysis in the presence of the and to by of Semenza G.L. Biochem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. P. K. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar), which is under the control of We show that was reduced in the presence of and inhibitors and and 100 μm cycloheximide the rate of of was in CSH12 in cells, which the in mRNA levels 1 of has been to increase activation of the HIF-1α but molecular mechanism has been has been to increase the of HIF-1α protein and to expression of HIF-1α target genes in normoxia and hypoxia via Blood. 1997; PubMed Google Scholar, C. N. A. F. A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). expression of v-Src HIF-1α protein levels and target It has been that the of v-Src is the increase in HIF-1α mRNA (3Jiang B.H. Agani F. Passaniti A. Semenza G.L. Cancer Res. 1997; 57: 5328-5335PubMed Google Scholar). mitogen-activated protein kinase has been in HIF-1α and activation of has been shown to activation by HIF-1α D. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, E. E. D. J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In these as in and the activation of HIF-1α not to involve increased protein We that active Src the synthesis of the HIF-1α protein and as a of its target genes with on HIF-1α mRNA levels 1 and In this we show that the elevated levels of HIF-1α under normoxia, in cells which active results from enhanced cap-dependent protein translation and by the pathway on the cell also by the pathway It is that HIF-1α expression is by a mechanism in the activity of protein kinases Src enhanced (17Zelzer E. Levy Y. Kahana C. Shilo B.Z. Rubinstein M. Cohen B. EMBO J. 1998; 17: 5085-5094Crossref PubMed Scopus (498) Google Scholar, E. P. K. Semenza G.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, D. Agani F. B. G. Semenza G.L. Cancer Res. 1999; Google Scholar, E. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Google Scholar). the in the protein of HIF-1α that at oxygen levels is the of an elevated rate of global protein synthesis reduced of HIF-1α degradation S. A. PubMed Scopus Google Scholar) that in cells with the v-Src aerobic glycolysis “Warburg at the cells to to the a mechanism for this inhibition of protein synthesis in cells in the of the in a to which that inhibition of Src activity or inhibition of protein synthesis to reduced results that Src activation the of cap-dependent the of The control of HIF-1α was also in a by E. P. K. Semenza G.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar) a molecular that activation HIF-1α levels by HIF-1α translation the that the was HIF-1α-specific and was the of the 5′-UTR of HIF-1α mRNA E. P. K. Semenza G.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). The between and the of may due to conditions. The added by have In that Src activity was not We Src in cells and measured only the of We also a 5′-UTR only the 5′-UTR from to from the of which to in the of the experiments show that the HIF-1α 5′-UTR is not involved in the of translation by Src 4 and that the of HIF-1α translation results from Src-induced of the cap-dependent 4 and In of this we show that the expression of the of the cap-dependent translation the protein elevated the levels of HIF-1α in HeLa cells as in inhibition of Src kinase and its and in the inhibition of total protein synthesis in Saos-2 and HT29 Furthermore, Src activity levels and synthesis of proteins by the same mechanism as for Y. E. and A. in results also a mechanism for the HIF-1α by (17Zelzer E. Levy Y. Kahana C. Shilo B.Z. Rubinstein M. Cohen B. EMBO J. 1998; 17: 5085-5094Crossref PubMed Scopus (498) Google Scholar, E. P. K. Semenza G.L. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, D. Agani F. B. G. Semenza G.L. Cancer Res. 1999; Google Scholar, E. J. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Google Scholar). In we that the enhanced translation of HIF-1α to a to aerobic glycolysis 6 H. M. P. K. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). The molecular mechanism we for the expression of HIF-1α in many tumors under normoxia E. M. D. P. Semenza G.L. Cancer Res. 1999; Google Scholar) and to the of the which is a of cancers 6 We Dr. and Dr. for reading of the

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