Direct Interactions between HIF-1α and Mdm2 Modulate p53 Function

Abstract

The p53 tumor suppressor is maintained at low levels in normal cells by Mdm2-mediated degradation and strongly stabilized in response to various types of stress including hypoxia. Although hypoxia-inducible factor 1α (HIF-1α) has been implicated to be involved in p53 stabilization, the precise mechanism by which HIF-1α regulates p53-mediated function remains unknown. Here, we found that HIF-1α directly binds Mdm2 both in vitro andin vivo; in contrast, p53 fails to directly interact with HIF-1α in vitro. Interestingly, Mdm2 expression can significantly enhance the in vivo association between p53 and HIF-1α, indicating that Mdm2 may act as a bridge and mediate the indirect interaction between HIF-1α and p53 in cells. Furthermore, HIF-1α protects p53 degradation mediated by Mdm2, and leads to activation of p53-mediated transcription in cells. To elucidate the mechanism of HIF-1α-mediated effect, we also found that HIF-1α can significantly suppress Mdm2-mediated p53 ubiquitination in vitro and blocks Mdm2-mediated nuclear export of p53. These results have significant implications regarding the molecular mechanism by which p53 is activated by HIF-1α in response to hypoxia. The p53 tumor suppressor is maintained at low levels in normal cells by Mdm2-mediated degradation and strongly stabilized in response to various types of stress including hypoxia. Although hypoxia-inducible factor 1α (HIF-1α) has been implicated to be involved in p53 stabilization, the precise mechanism by which HIF-1α regulates p53-mediated function remains unknown. Here, we found that HIF-1α directly binds Mdm2 both in vitro andin vivo; in contrast, p53 fails to directly interact with HIF-1α in vitro. Interestingly, Mdm2 expression can significantly enhance the in vivo association between p53 and HIF-1α, indicating that Mdm2 may act as a bridge and mediate the indirect interaction between HIF-1α and p53 in cells. Furthermore, HIF-1α protects p53 degradation mediated by Mdm2, and leads to activation of p53-mediated transcription in cells. To elucidate the mechanism of HIF-1α-mediated effect, we also found that HIF-1α can significantly suppress Mdm2-mediated p53 ubiquitination in vitro and blocks Mdm2-mediated nuclear export of p53. These results have significant implications regarding the molecular mechanism by which p53 is activated by HIF-1α in response to hypoxia. Cellular hypoxia is an important phenomenon in developmental biology, normal physiology, and many pathological conditions, including cancer. Hypoxia triggers a multifaceted adaptive response that is primarily mediated by the heterodimeric transcription factor hypoxia-inducible factor (HIF). 1The abbreviations used are: HIFhypoxia-inducible factorE3ubiquitin-protein isopeptide ligaseMEFmouse embryonic fibroblast cellDTTdithiothreitolPMSFphenylmethylsulfonyl fluoridePBSphosphate-buffered salineDAPI4′,6-diamidino-2-phenylindoleCMVcytomegalovirus HIF-1 is a heterodimer composed of two subunits, the rate-limiting factor HIF-1α and the constitutive expressed HIF-1β (1Wang G.L. Semenza G.L. J. Biol. Chem. 1995; 270: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar, 2Semenza G.L. Curr. Opin. Cell Biol. 2001; 13: 167-171Crossref PubMed Scopus (898) Google Scholar). HIF-1β has been characterized as an aryl hydrocarbon receptor nuclear translocator, and this family of proteins has previously been shown to heterodimerize with the aryl hydrocarbon receptor (3Hoffman E.C. Reyes H. Chu F.F. Sander F. Conley L.H. Brooks B.A. Hankinson O. Science. 1991; 252: 954-958Crossref PubMed Scopus (843) Google Scholar). On the other hand, HIF-1α specifically mediates hypoxic responses. In normoxia, HIF-1α is maintained at low and often undetectable levels. HIF-1α is targeted for degradation by the ubiquitination-proteasome pathway through directly binding to the von Hippel-Lindau tumor suppressor gene (pVHL), which forms the recognition component of an E3 ubiquitin-protein ligase leading to ubiquitination of HIF-1α (4Maxwell 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 PJ. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4152) Google Scholar, 5Kamura 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 (551) Google Scholar, 6Blagosklonny M.V. Oncogene. 2001; 20: 395-398Crossref PubMed Scopus (29) Google Scholar, 7Ohh 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 (1263) Google Scholar). Recent reports demonstrate that HIF-1α undergoes an iron- and oxygen-dependent modification before it can interact with pVHL. This modification is catalyzed by a specific family of enzymes termed HIF-1α-proline hydroxylases (8Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim Av. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4456) Google Scholar, 9Epstein 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 (2736) Google Scholar, 10Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2114) Google Scholar). During hypoxia, this specific hydroxylase is inactive since it requires dioxygen for its activity. As a result, HIF-1α accumulates due to the failure of pVHL to recognize its non-hydroxylated form. HIF-1α then translocates to the nucleus and dimerizes with the constitutively present HIF-1β (11Semenza G.L. Annu. Rev. Cell Dev. Biol. 1999; 15: 551-578Crossref PubMed Scopus (1676) Google Scholar). hypoxia-inducible factor ubiquitin-protein isopeptide ligase mouse embryonic fibroblast cell dithiothreitol phenylmethylsulfonyl fluoride phosphate-buffered saline 4′,6-diamidino-2-phenylindole cytomegalovirus The importance of the HIF-1α response pathway in human tumorigenesis is underscored by the finding that HIF-1α is overexpressed in multiple human cancers, because tumor cells, unlike normal cells from the same tissue, are often chronically hypoxic (12Harries A.L. Nat. Rev. Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4287) Google Scholar). The tumor suppressor protein p53 integrates numerous signals that control cell life and death (13Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5822) Google Scholar). Wild-type p53 is expressed at low levels in most cells because of its short half-life under normal conditions. In contrast, the p53 protein is stabilized, and its level increases in response to various stresses such as DNA damage, hypoxia, and inappropriate oncogene signaling (14Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1177) Google Scholar, 15Koumenis C. Alarcon R. Hammond E. Sutphin P. Hoffman W. Murphy M. Derr J. Taya Y. Lowe S.W. Giaccia K.M. Mol. Cell. Biol. 2001; 21: 1297-1310Crossref PubMed Scopus (304) Google Scholar). In its active form, p53 can bind DNA in a sequence-specific manner and activate transcription of target genes. p53 levels are regulated in large part by Mdm2, the product of a p53-inducible gene. Mdm2 can interact with the N terminus of p53, which also contains the major acidic transcriptional activation domain. The interaction between Mdm2 and p53 can inhibit p53 transcriptional activity by interfering with the ability of p53 to contact transcriptional coactivators such as p300/CBP (16Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1233) Google Scholar). Importantly, Mdm2 binding also promotes the ubiquitination of p53 and its export from the nucleus to the cytoplasm, where p53 is then degraded by cytoplasmic proteasomes (17Ryan K.M. Phillips A.C. Vousden K.H. Curr. Opin. Cell Biol. 2001; 13: 332-337Crossref PubMed Scopus (580) Google Scholar). Hypoxic induction of p53 requires concomitant induction of HIF-1α, whereby HIF-1α can then bind to and stabilize p53 (18An W.G. Kanekal M. Simon M.C. Maltepe E. Blagosklonny M.V. Neckers L.M. Nature. 1998; 392: 405-408Crossref PubMed Scopus (654) Google Scholar, 19Blagosklonny M.V. An W.G. Romanova L.Y. Trepel J. Fojo T. Neckers L. J. Biol. Chem. 1998; 273: 11995-11998Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 20Semenza G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar, 21Ravi R. Mookerjee B. Bhujwalla Z.M. Sutter C.H. Artemov D. Zeng Q. Dillehay L.E. Madan A. Semenza G.L. Redi A. Genes Dev. 2000; 14: 33-44Google Scholar). However, the molecular mechanism by which HIF-1α stabilizes p53 remains unknown. Moreover, it is not clear whether HIF-1α interacts with p53 directly despite previous indications that p53 associated with HIF-1α in cells. Nevertheless, an array of immobilized peptide assay showed that the core domain of p53 has an affinity for the oxygen-dependent degradation domain of HIF-1α (22Hansson L.O. Friedler A. Freund S. Rudiger S. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10305-10309Crossref PubMed Scopus (112) Google Scholar). As described in this report, we have found a strong interaction between HIF-1α and Mdm2, while we failed to detect any direct interaction of p53 with HIF-1α. Our data demonstrate that HIF-1α regulates p53 activity including stability and nuclear export through interactions with Mdm2. These results provide a potential mechanism for p53 stabilization by HIF-1α in response to hypoxia. p53-null H1299 lung carcinoma cells and mouse embryonic fibroblast cells (MEFs) were cultured in Dulbecco's modified Eagle's medium (Mediatech, Richmond, VA) supplemented with penicillin/streptomycin and 10% fetal bovine serum (Mediatech, VA). 1 × 106 or 2 × 105 cells were plated in 10-cm or six-well plates, respectively, and 24 h later transfections were done by calcium phosphate precipitation procedures. After a 24-h incubation with 20% O2, the cells were harvested. Full-length HIF-1α DNA was generated by PCR using hemagglutinin-tagged HIF-1α from David Livingston (Dana Farber Cancer Institute) and was subcloned into pcDNA3.1/v5-His-Topo (Invitrogen) or p3×FLAG-CMV-14 (Sigma). Green fluorescence protein (GFP)-tagged wild-type p53 was obtained from Yanping Zhang (M. D. Anderson Cancer Center). Plasmid DNA for transfections was isolated using Qiagen plasmid maxi kit (Qiagen). GST fusions of p53 and Mdm2 were expressed in Escherichia coli BL-21 (DE3) (Promega) and induced with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. Bacterial pellets were lysed in BC500 (25 mm Tris, pH 7.8, 500 mmNaCl, 1 mm EDTA, 1 mm DTT, 10% glycerol, 0.2% Nonidet P-40, fresh 1 mm PMSF) with sonication. Levels of expressed GST fusion proteins were estimated by incubation with glutathione-Sepharose beads, washing, and quantification by SDS-PAGE followed by staining with Coomassie Brilliant Blue R-250. Known amounts of bovine serum albumin were used as standards. 35S-Labeled in vitro translated HIF-1α was prepared by using the TNT system (Promega). Equal amounts (1 μg) of GST, GST-p53, and GST-Mdm2 immobilized on glutathione-Sepharose beads were incubated with in vitro translated HIF-1α in BC200 for 3 h at 4 °C. After washing, the bound proteins were eluted with SDS sample buffer and were separated by SDS-PAGE, followed by autoradiography. Cells were lysed in FLAG lysis buffer (50 mm Tris, 137 mm NaCl, 10 mm NaF, 1 mm EDTA, 1% Triton X-100, 0.2% Sarkosyl, 1 mm DTT, 10% glycerol, pH 7.8) with fresh protease inhibitors (1 mm PMSF, protease inhibitor mixture (Sigma)). Aliquots (30 μg) of cell extracts were resolved in SDS, 8% polyacrymide gels and then transferred to nitrocellulose membranes in 20 mm Tris-HCl, pH 8.0, 150 mm glycine, 20%(v/v) methanol. Membranes were blocked with 5% (v/v) nonfat dry milk, TBST (20 mm Tris-HCl, pH 7.6, 137 mmNaCl, 0.1% Tween 20), incubated with α-p53 (DO-1) antibody (Santa Cruz), or anti-GFP (Clontech), and detected with ECL reagents (Amersham Biosciences). Coimmunoprecipitation assay was performed essentially as described previously (23Gu W. Shi X.L. Roeder R.G. Nature. 1997; 387: 819-823Crossref PubMed Scopus (522) Google Scholar). In brief, 50 μl of proteasome inhibitorN-acetyl-leucyl-leucyl-norleucinal was added to the cotransfected culture 6 h before harvest. 24 h after transfection, the cells were lysed in BC100 (25 mm Tris, pH 7.8, 100 mm NaCl, 1 mm EDTA, 1 mmDTT, 10% glycerol, 0.2% Nonidet P-40, fresh 1 mm PMSF) and incubated with anti-FLAG M2 beads (Sigma) overnight at 4 °C. The beads were washed five times with 1 ml of lysis buffer, after which the associated proteins were eluted with BC100, 0.2% Nonidet P-40 plus 0.2 mg/ml FLAG peptide (Sigma). The eluted proteins were resolved on 8% SDS-PAGE and Western blot with the anti-p53 or anti-Mdm2 (SMP14, Santa Cruz) monoclonal antibody. Luciferase activity was determined using a dual luciferase assay system (Promega) following the manufacturer's protocol. Cells in six-well plates were removed by scraping into 100 μl of reporter lysis buffer. Cell lysate was collected by centrifugation for 15 min at 12,000 × g. Luciferase activity was measured using a Lumat LB 9507 luminometer (EG&G Wallac, Gaithersburg, MD). The in vitroubiquitination assay was performed as previously described (24Li M. Luo J. Brooks C.L. Gu W. J. Biol. Chem. 2002; 277: 50607-50611Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar) with some modifications. For a standard reactions, the purified FLAG-p53 proteins from H1299 cells were mixed with other purified components, including E1, E2 (GST-UbcH5C), E3 (GST-Mdm2), and His-ubiquitin in reaction buffer (40 mm Tris, 5 mmMgCl2, 2 mm ATP, 2 mm DTT, pH 7.6). The reaction was stopped after 60 min at 37 °C by additions of SDS sample buffer and subsequently resolved SDS-PAGE gels for Western blot analysis with α-p53 (DO-1). H1299 cells were plated on six-well-containing glass coverslips, and GFP-p53 was transfected as described. 50 μm proteasome inhibitorN-acetyl-leucyl-leucyl-norleucinal was added for 6 h before fixation. Twenty-four hours after transfection, cells on the coverslips were washed three times with phosphate-buffered saline (PBS) and then fixed in 4% paraformaldehyde for 10 min at room temperature. After fixation, cells were washed in PBS three times and then permeabilized in ice-cold PBS containing 0.2% Triton X-100 for 10 min. Cells were blocked in PBS containing 1% bovine serum albumin and 1 μg of DAPI (Sigma)/ml at room temperature for 30 min. Cells were washed three times with PBS, and the stained cells were mounted with mounting medium (Polysciences, Inc., Warrington, PA) and sealed with nail polish. Immunofluorescence was recorded using an immunofluorescence microscope. Although earlier studies indicated that p53 can bind HIF-1α in cells, we repeatedly failed to detect any direct interaction between these two protein in a GST pull-down assay (data not shown), suggesting that HIF-1α may interact with p53 through other factors. To examine the notion that p53 may interact with HIF-1α through Mdm2, we whether HIF-1α directly binds Mdm2 in vitro by GST pull-down GST-Mdm2 and GST were expressed in and purified to As shown in in vitro translated HIF-1α strongly bound to GST-Mdm2 not to immobilized GST However, using the same significant interaction was detected between and HIF-1α. Furthermore, we the interaction between HIF-1α and vivo by HIF-1α was with Mdm2 in H1299 cells. were performed with M2 beads, and the proteins were by Western blot with an anti-Mdm2 antibody. As shown in the were detected in cells cotransfected with and Mdm2 not by Mdm2 indicating that is a specific in vivo interaction between Mdm2 and HIF-1α. To examine the that the interaction is mediated by Mdm2, we whether Mdm2 expression is for the interaction in cells. p53 and were transfected with or Mdm2 in H1299 cells. were from cell extracts using M2 As shown in p53 was in the in the of Mdm2 expression the that p53 directly bind HIF-1α in In contrast, Mdm2 was expressed in the cells, p53 was with HIF-1α by the same These results that Mdm2 as a bridge the binding between HIF-1α and p53. Mdm2 is a of p53 and can both inhibit p53 transcriptional activity and target it for degradation (24Li M. Luo J. Brooks C.L. Gu W. J. Biol. Chem. 2002; 277: 50607-50611Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). the whether HIF-1α p53 from degradation mediated by Mdm2. The p53-null H1299 cells were transfected with and 24 h after the cells were lysed in a FLAG lysis buffer and by Western As shown in HIF-1α p53 from Mdm2-mediated of Mdm2 significantly induced p53 degradation 2 this degradation was in a manner of HIF-1α 3 and 4 Interestingly, protein can p53 degradation through the ligase However, of HIF-1α p53 degradation mediated by these results an of HIF-1α directly on Mdm2-mediated degradation of p53. Furthermore, we used p53 transcriptional activity assay to the To this we cotransfected with p53, Mdm2, and HIF-1α, with a reporter containing the As indicated in of Mdm2 with p53 strongly luciferase activity due to p53 However, HIF-1α significantly the of Mdm2 in a The data demonstrate that HIF-1α can strongly stabilize p53 by Mdm2-mediated which leads to activation of p53-mediated To whether HIF-1α can suppress p53 ubiquitination mediated by Mdm2, we an in vitro ubiquitination As shown in p53 was detected at the reaction with Mdm2 HIF-1α 2 However, in the of HIF-1α, the levels of p53 3 This that HIF-1α can significantly suppress Mdm2-mediated p53 that Mdm2 can also nuclear export of p53 R.K. Nat. Cell Biol. 2000; 2: PubMed Scopus Google T. Nat. Cell Biol. 2000; 2: PubMed Scopus Google Scholar). To the that HIF-1α can this we transfected H1299 cells with Mdm2 or HIF-1α and by immunofluorescence The of GFP-p53 was nuclear expressed it to the to with Mdm2. the by which p53 to the expressed or expressed with Mdm2 Cells cotransfected with p53 and Mdm2 a cytoplasmic expression p53 was transfected indicating that nuclear Mdm2 can the cytoplasmic of p53 R.K. Nat. Cell Biol. 2000; 2: PubMed Scopus Google Scholar, T. Nat. Cell Biol. 2000; 2: PubMed Scopus Google Scholar). Interestingly, of cytoplasmic expression in cells cotransfected with HIF-1α in to p53 and Mdm2 a and These results that HIF-1α can strongly Mdm2-mediated p53 nuclear cells in is a in In cells, of or hypoxia, leads to stabilization of a sequence-specific DNA binding transcriptional factor of are to such as and G.L. Genes Dev. 2000; 14: 1983-1991PubMed Google Scholar). On the other hand, hypoxia of the tumor suppressor p53. studies indicated that HIF-1α interacts with p53 in vivo (18An W.G. Kanekal M. Simon M.C. Maltepe E. Blagosklonny M.V. Neckers L.M. Nature. 1998; 392: 405-408Crossref PubMed Scopus (654) Google the of this interaction has not been In this we demonstrate for the that HIF-1α directly binds to Mdm2 both in vitro and in In the of Mdm2, the binding between HIF-1α and p53 is Furthermore, Mdm2 expression significantly the indirect interaction between p53 and HIF-1α in cells, indicating that Mdm2 may act as a bridge the In this we failed to detect any significant interaction between p53 and HIF-1α in cells, and stabilization of p53 induced by HIF-1α expression was also (data not Our at with the of binding between the domain of HIF-1α and the core domain of p53 by an array of immobilized peptide assay (22Hansson L.O. Friedler A. Freund S. Rudiger S. Fersht A.R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10305-10309Crossref PubMed Scopus (112) Google Scholar). is that the was on p53 which may not be to the However, the of Mdm2 the of p53 the of binding of p53 to HIF-1α. In results demonstrate that HIF-1α protects p53 from degradation mediated by Mdm2 and can p53 transcriptional by Mdm2. that p53 degradation is induced by Mdm2 in normal cells, we also found that Mdm2-mediated of p53 is significantly by HIF-1α. Mdm2-mediated p53 ubiquitination promotes its nuclear export R.K. Nat. Cell Biol. 2000; 2: PubMed Scopus Google Scholar, T. Nat. Cell Biol. 2000; 2: PubMed Scopus Google we demonstrate that HIF-1α expression can nuclear export of p53. these results have significant implications regarding the molecular mechanism by which HIF-1α p53 function in Our results are also with results indicating HIF-1α interacts with the wild-type p53 protein not the p53 in cells (18An W.G. Kanekal M. Simon M.C. Maltepe E. Blagosklonny M.V. Neckers L.M. Nature. 1998; 392: 405-408Crossref PubMed Scopus (654) Google Scholar). wild-type p53 protein is of Mdm2 expression in cells, the interaction between p53 and HIF-1α is most mediated by Mdm2. In contrast, because the p53 is inactive in transcriptional activation of Mdm2, HIF-1α failed to interact with p53 since is low Mdm2 in cells p53. Recent studies also that Mdm2 is involved in HIF-1α stability under hypoxic R. Mookerjee B. Bhujwalla Z.M. Sutter C.H. Artemov D. Zeng Q. Dillehay L.E. Madan A. Semenza G.L. Redi A. Genes Dev. 2000; 14: 33-44Google the notion that HIF-1α directly interacts with Mdm2 not p53. Mdm2 is a E3 ligase and both p53 degradation and nuclear export of p53 through it is that Mdm2 also directly mediates degradation and nuclear export of HIF-1α. Our with other studies C. Alarcon R. Hammond E. Sutphin P. Hoffman W. Murphy M. Derr J. Taya Y. Lowe S.W. Giaccia K.M. Mol. Cell. Biol. 2001; 21: 1297-1310Crossref PubMed Scopus (304) Google Scholar, W.G. Kanekal M. Simon M.C. Maltepe E. Blagosklonny M.V. Neckers L.M. Nature. 1998; 392: 405-408Crossref PubMed Scopus (654) Google Scholar, 21Ravi R. Mookerjee B. Bhujwalla Z.M. Sutter C.H. Artemov D. Zeng Q. Dillehay L.E. Madan A. Semenza G.L. Redi A. Genes Dev. 2000; 14: 33-44Google strongly the important of HIF-1α in the of the pathway in response to hypoxia. C. Brooks for the and other of W. Gu for data and David Livingston and Yanping Zhang for