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Hypoxia-inducible Factor 1α Is Regulated by the Mammalian Target of Rapamycin (mTOR) via an mTOR Signaling Motif *

Hypoxia-inducible Factor 1α Is Regulated by the Mammalian Target of Rapamycin (mTOR) via an mTOR... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 28, pp. 20534 –20543, July 13, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Hypoxia-inducible Factor 1 Is Regulated by the Mammalian Target of Rapamycin (mTOR) via an mTOR Signaling Motif Received for publication, December 22, 2006, and in revised form, May 10, 2007 Published, JBC Papers in Press, May 14, 2007, DOI 10.1074/jbc.M611782200 ‡ §1 Stephen C. Land and Andrew R. Tee From the Institute of Medical Genetics, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom and the Division of Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom Tumors that form as a result of heightened mammalian target two key angiogenic factors; vascular endothelial growth fac- of rapamycin (mTOR) signaling are highly vascularized. This tor (VEGF)-A (1) and angiopoietin-2 (Ang-2) (2). VEGF-A and process of angiogenesis is regulated through hypoxia-inducible Ang-2 encourage angiogenesis by stimulating the formation of factor (HIF)-mediated transcription of angiogenic factors. It is blood vessels that migrate into the tumor. recognized that inhibition of mTOR with rapamycin can dimin- As well as vascularization, HIF is known to control the ish the process of angiogenesis. Our work shows that activation expression of 70 genes involved in energy metabolism, apo- of mTOR by Ras homologue enriched in brain (Rheb) overex- ptosis/survival, and metastasis (for reviews see Refs. 3, 4). Low pression potently enhances the activity of HIF1 and vascular oxygen enhances the activity of HIF by stabilizing the -subunit endothelial growth factor (VEGF)-A secretion during hypoxia, of HIF. Oxygen-dependent prolyl hydroxylase domain (PHD) which is reversed with rapamycin. Mutants of Rheb, which do proteins become active in the presence of oxygen that sequen- not bind guanine nucleotide (D60K, D60V, N119I, and D122N) tially leads to hydroxylation of two proline residues within the and are unable to activate mTOR, inhibit the activity of HIF oxygen-dependent degradation domain (ODDD) of the HIF when overexpressed. We show that regulatory associated pro- -subunit. Proline hydroxylation results in ubiquitin-mediated tein of mTOR (Raptor) interacts with HIF1 and requires an degradation of the -subunit that requires the Von Hippel- mTOR signaling (TOS) motif located in the N terminus of Lindau (VHL) tumor suppressor protein, which is a component HIF1. Furthermore, a mutant of HIF1 lacking this TOS motif of an E3 ubiquitin ligase complex (5). VHL syndrome, caused dominantly impaired HIF activity during hypoxia and was through loss of function of this tumor suppressor, is a domi- unable to bind to the co-activator CBP/p300. Rapamycin treat- nantly inherited familial syndrome that predisposes the patient ments do not affect the stability of HIF1 and modulate HIF to renal cell carcinoma and cerebellar hemangioblastomas. In activity via a Von Hippel-Lindau (VHL)-independent mecha- conditions of low oxygen tension, the HIF protein is stable and nism. We demonstrate that the high levels of HIF activity in cells binds to HIF (also known as aryl hydrocarbon receptor devoid of TSC2 can be reversed by treatments with rapamycin or nuclear translocator-ARNT). The HIF/HIF dimer is then the readdition of TSC2. Our work explains why human cancers rapidly translocated to the nucleus. As a dimer, they bind to with aberrant mTOR signaling are prone to angiogenesis and RCGTG DNA sequences called hypoxia response elements. suggests that inhibition of mTOR with rapamycin might be a Transcription is sequentially activated when the C terminus suitable therapeutic strategy. transcriptional activation domain (C-TAD) of HIF binds to the co-activator CBP/p300 (for review see Ref. 6). The tuberous sclerosis complex (TSC), which is a hamarto- The supply of nutrients and oxygen is quickly depleted as mas syndrome, occurs through loss of function of the tumor solid tumors grow in size. Nutritional and oxygen homeostasis suppressor proteins TSC1 (hamartin) and TSC2 (tuberin). within solid tumors is reestablished by vascularization that Tumors that occur in TS patients are highly vascularized, consequently results in tumor expansion. Oxygen sensing is implying that HIF might be involved and is regulated down- known to control the mechanism of vasculogenesis through stream of TSC1/2. TSC is an autosomal genetic syndrome that hypoxia-inducible factor (HIF) -mediated transcription of occurs with an estimated prevalence of one in 6,000 newborns and is characterized by slow growing benign tumors that form in the heart, brain, kidneys, eyes, and skin (see Ref. 7 for review). * This research was supported by British Heart Foundation Intermediate Research Fellowship No. FS/04/022 and Association of International Can- These tumors can lead to both renal and neurological compli- cer Research Career Development Fellowship No 06-914/915 (to A. R. T.), and funding from Tenovus (to S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must human embryonic kidney; MEFs, mouse embryonic fibroblasts; mTOR, therefore be hereby marked “advertisement” in accordance with 18 U.S.C. mammalian target of rapamycin; TOS, mTOR signaling; NF1, neurofibro- Section 1734 solely to indicate this fact. min 1; PI3K, phosphoinositide 3-kinase; PHD, prolyl hydroxylases domain; To whom correspondence should be addressed. Tel.: 029-2074-4055; Fax: PTEN, phosphatase and tensin homolog; Raptor, regulatory associated 029-2074-6551; E-mail: [email protected]. protein of mTOR; Redd1/2, regulated in development and DNA damage The abbreviations used are: HIF, hypoxia-inducible factor; ARNT, aryl hydro- responses; Rheb, Ras homologue enriched in brain; Rictor, rapamycin-in- carbon receptor nuclear translocator; CHAPS, 3-(3-cholamidopropyl)dim- sensitive companion of mTOR; S6K1, ribosomal protein S6 kinase 1; STK11, ethylammonio-1-propanesulfonate; DFX, deferoxamine mesylate; DMOG, serine/threonine kinase 11; TSC, tuberous sclerosis complex; VEGF, vascu- dimethyloxalylglycine; DMEM, Dulbecco’s modified Eagle’s medium; DSP, lar endothelial growth factor; VHL, Von Hippel-Lindau; HA, hemagglutinin; dithiobis(succinimdyl propionate); 4E-BP1, eIF4E-binding protein 1; HEK, FITC, fluorescein isothiocyanate. This is an Open Access article under the CC BY license. 20534 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR cations. TSC1 and TSC2 function as a heterodimer that inhibits enhances the transcriptional activity of HIF1 that does not cell growth by impairing the Ser/Thr protein kinase called the involve increased HIF1 stabilization. Furthermore, mTOR mammalian target of rapamycin (mTOR) (see review in Ref. 8). activation of HIF1 requires the interaction of HIF1 with Rap- TSC1/TSC2 heterodimers function to inhibit mTOR, by acting tor. Given that Raptor functions as a scaffold protein that as a GTPase-activating protein (GAP) toward the small G-pro- recruits downstream mTOR substrates with a TOS motif to tein, Ras homologue enriched in brain (Rheb). Rheb has been mTOR (15), this work reveals that HIF1 is a downstream tar- shown to interact with mTOR, and promotes signal transduc- get of mTOR. tion when Rheb is in the active GTP-bound state (9). When in a EXPERIMENTAL PROCEDURES complex with TSC1, TSC2 inhibits Rheb-induced mTOR sig- 3 3 35 naling by reverting Rheb to its inactive GDP-bound state (10). Chemicals and Materials—[ H]GTP, [ H]GDP, and [ S]me- Therefore, when the normal function of TSC1/TSC2 het- thionine-radiolabeled reagents were purchased from Amer- erodimers become compromised, Rheb becomes constitutively sham Biosciences (GE Healthcare UK Ltd.). Dimethyloxalylg- GTP-bound and mTOR signaling is significantly enhanced and lycine was purchased from Frontier Scientific Europe Ltd contributes to the pathology associated with TSC. (Lancashire, UK). Deferoxamine mesylate, MG-132 (carboben- Within mammalian cells, mTOR functions as two distinct zoxy-L-leucyl-L-leucyl-L-leucinal), rapamycin, and LY294002 multi-protein kinase-signaling complexes. Rictor (rapamycin- were obtained from Merck Biosciences Ltd. (Nottingham, UK). insensitive companion of mTOR) and LST8 (also referred to as CHAPS was ordered from Pierce. DSP (dithiobis(succinimdyl G-protein -subunit-like protein (GL)) interact with mTOR propionate)) was purchased from Apollo Scientific Ltd. (Stock- to form an mTOR/Rictor/LST8 protein kinase complex (11). port, UK). Rat anti-HA antibodies were purchased from Roche mTOR also forms another multi-protein kinase complex with a Applied Science. Mouse anti-HA antibodies were kindly pro- different scaffold protein called Raptor (regulatory-associated vided by M. Chou (University of Pennsylvania, Philadelphia, protein of mTOR), where signaling from this (12, 13) promotes PA). Cell Signaling Technology anti-S6K1 and anti-S6K1 phos- mTOR-mediated phosphorylation of distinct downstream sig- pho-Thr antibodies were purchased from New England Bio- naling targets, such as the ribosomal S6 protein kinase 1 (S6K1) labs Ltd. (Hertfordshire, UK). Anti-HIF1 antibodies (clone: and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), H1alpha67) were obtained from Novus Biologicals (Stratech which is potently impaired by treatments with the immunosup- Scientific Ltd.). Anti-Coactivator p300 (C-20) antibodies were pressant drug rapamycin. mTOR regulates cell growth and pro- bought from Santa Cruz Biotechnology, Inc. (Heidelberg, liferation and is known to involve both S6K1 and 4E-BP1 (for Germany). Anti-FLAG antibodies (clone: M2), anti-Myc review see Ref. 8). It is believed that Raptor functions as a scaf- (clone: 9E10) and all other reagents (unless stated) were fold protein that recruits mTOR to its downstream substrates obtained from Sigma-Aldrich. through their mTOR signaling (TOS) motifs. The TOS motif is Plasmids and Molecular Biology—N-terminal FLAG-tagged a 5 amino acid peptide found within rapamycin-sensitive tar- pRK7/Rheb was generated as described in Ref. 10. Human HA- gets of mTOR (FDL/IDL and FEMDI within S6K1 (14) and tagged 4E-BP1 was a kind gift from N. Sonenberg (McGill Uni- 4E-BP1 (15), respectively). Mutation of this TOS motif (espe- versity, Montreal, Canada). HIF1 was cloned into a modified cially the highly conserved phenylalanine at position 1) pre- pRK7 vector so it expressed a triple N-terminal HA tag using vents mTOR-directed phosphorylation of both S6K1 and human HIF1 cDNA as a template (obtained from Novus Bio- 4E-BP1 (14, 15). logicals (Stratech Scientific Ltd.)). Myc-tagged Raptor/pRK5 Interestingly, a common feature of hamartomas syndromes was a kind gift from D. M. Sabatini (Whitehead Institute for caused by the loss of function of TSC1, TSC2 (16), PTEN (phos- Biomedical Research, Boston, MA). Human 4E-BP1 and phatase and tensin homolog) (17), LKB1 (also known as STK11 human Rheb were subcloned into pGEX-2T/GST to generate (serine/threonine kinase 11)) (18), or NF1 (neurofibromin 1) GST-tagged recombinant protein. Point mutations were gener- (19) tumor suppressors is that signaling through mTOR is sig- ated as described in the QuikChange site-directed mutagene- nificantly enhanced. Furthermore, VEGF levels are increased in sis kit (Stratagene). The N-terminal FLAG-tagged pRK7/TSC2 these syndromes (16, 20–22). It is possible that increased VEGF construct was generated as previously described (26). The secretion caused through loss of function of these tumor sup- pVHL-HA vector was kindly provided by P. Ratcliffe (Henry pressors is mediated via an mTOR-dependent mechanism. Wellcome Building of Molecular Physiology, Oxford Univer- Indeed, it has been shown that rapamycin has anti-angiogenic sity, UK) as described (27). The firefly luciferase reporter pGL2- properties by reducing the expression of VEGF (23, 24). Fur- TK-HRE plasmid was generated by subcloning three copies of thermore, VEGF expression within cells lacking TSC1 or TSC2 the hypoxia response element (HRE) (5-GTGACTACGT- is highly sensitive to treatments of rapamycin (16). The mech- GCTGCCTAG-3) from the inducible nitric-oxide synthase anism by how mTOR enhances VEGF expression has not been promoter into the promoter region of the pGL2-TK vector as fully elucidated. It is postulated that HIF becomes more stable previously described (28) and was kindly provided by G. Melillo by heightened mTOR activity, which causes enhanced VEGF (National Cancer Institute at Frederick, Maryland). expression (25). We wanted to explore the mTOR-specific acti- Tissue Culture and Analysis of Cell Lysates—Human embry- vation of HIF1 in greater detail. In this study, we potently onic kidney 293 (HEK293) cells were cultured (at 37 °C within activate mTOR by overexpressing Rheb. We have previously 5% CO ) and maintained in Dulbecco’s modified Eagle’s shown that Rheb can bind to and enhance mTOR signaling to medium (DMEM) supplemented with 10% fetal bovine serum. downstream targets (9, 10). We show that mTOR directly DMEM and fetal bovine serum were purchased from Invitro- JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20535 Regulation of HIF1 by mTOR gen Ltd. (Paisley, UK). TSC2 mouse embryonic fibroblasts O for 18 h on glass coverslips. Cells were then fixed in metha- (MEFS) were a kind gift from D. J. Kwiatkowski (Harvard Med- nol at20 °C, quenched in 0.1% Na borohydrate in Tris-buff- ical School, Boston, MA). The human renal carcinoma VHL ered saline (TBS; 50 mM Tris-Cl (pH 7.4), 150 mM NaCl) and cells (786-O) were bought from ATCC. Cell transfections were then blocked for1hinTBS containing 10% goat serum. Mono- carried out as described in the SuperFect manufacturer’s pro- clonal Anti-HA (clone HA-7) FITC conjugate (Sigma) was then tocol (purchased from Qiagen Ltd. (West Sussex, UK)). applied to the cells in TBS containing 0.1% bovine serum albu- HEK293 cells were transfected with 95% efficiency, while min overnight at 4 °C and at 1:5000 dilution. After washing, transfection of TSC2 MEFS and human renal carcinoma coverslips were counterstained with DAPI antifade (Q-Bio- VHL cells were 10% efficient. Hypoxic treatments were gene) and mounted. The intracellular distribution of FITC and carried out in a MACS VA500 Microaerophilic Work station DAPI fluorescence was observed using a Zeiss LSM 510 confo- (Don Whitley Scientific, Shipley, West Yorkshire) containing a cal microscope. humidified atmosphere equilibrated to 1% O ,5%CO , and 2 2 Raptor Binding to mTOR Targets—To generate Raptor pro- 94% N . To create cell lysates, cells were washed twice in phos- tein to be used in the overlay assays, Myc-tagged Raptor was phate-buffered saline, and then harvested with lysis buffer (10 overexpressed in HEK293 cells. These cells were lysed with 50 mM KH PO ,1mM EDTA, 10 mM MgCl ,50mM -glycero- 2 4 2 mM -glycerophosphate, pH 7.4; 1 mM EDTA; 1 mM EGTA; 0.5 phosphate, 5 mM EGTA, 0.5% Nonidet P-40, 0.1% Brij 35, 1 mM mM Na VO ;1mM benzamidine hydrochloride; 1 mM dithio- 3 4 sodium orthovanadate, 40 mg/ml phenylsulfonyl fluoride, 10 threitol; 0.1 mM phenylmethane sulfonyl fluoride; 1% (v/v) Tri- g/ml leupeptin, 5 g pepstatin, pH 7.2). Cell lysates were son- ton X-100, and 1 g/ml each of pepstatin, antipain, and leupep- icated to help break down the nucleus and then spun at 14, 000 tin. Proteins were transferred to Immobilon-P and blocked rpm for 8 min at 4 °C to remove the cell debris. For Western blot with 5% (w/v) milk in Tris-buffered saline were incubated over- analysis, these lysates were prepared as described for running night at 4 °C with 1:10 diluted Myc-tagged Raptor lysate. Myc- NuPage Novex gels (bought from Invitrogen Ltd.) Proteins tagged Raptor that bound to proteins immobilized on Immo- resolved on Novex gels were transferred to Millipore Immo- bilon-P was then detected by incubating the blot with anti-Myc bilon-P (purchased from Upstate Ltd. (Hampshire, UK)) and antibodies for2hin2% (w/v) bovine serum albumin Tris-buff- blotted with the appropriate antibody followed by horseradish ered saline followed by anti-mouse horseradish peroxidase- peroxidase-conjugated secondary antibodies. Enhanced conjugated secondary antibodies and ECL as described for chemiluminescence was carried out with Amersham Bio- TM sciences ECL Western blotting detection reagents (pur- Western blotting. Raptor association with mTOR substrates chased from GE Healthcare UK Ltd.). within cells was determined as previously described (15). Immunoprecipitation—Proteins were immunoprecipitated Monitoring HIF1 Stability in Vivo—HEK293 cells trans- using the relevant antibodies coupled to protein G-Sepharose fected with HA-HIF1 (grown on 6-cm plates) were incubated (Amersham Biosciences). HIF1 immunoprecipitations were in methionine-free medium and labeled with 10 Ci of carried out overnight at 4 °C, and the protein G-Sepharose was S]methionine for4hinthe hypoxic chamber set at 1% O . added for 1 h prior to being washed. Immunoprecipitates were The cells were washed twice in fresh media and then incubated washed twice each with both buffer A (50 mM Hepes (pH 7.4), for 0, 0.5, 1, and2hin DMEM before cell lysates were gener- 100 mM NaCl, 10 mM MgCl , 1 mg/ml bovine serum albumin, 1 ated. HA-HIF1 was immunoprecipitated for 1 h with anti-HA mM dithiothreitol, 1% Triton) and buffer B (50 mM Hepes (pH antibodies bound to protein G-Sepharose. Immunoprecipi- 7.4), 100 mM NaCl, 10 mM MgCl , 0.1% Triton) in the presence tated HIF1 was subjected to SDS-PAGE. The gel was stained, of protease inhibitors. fixed, incubated with Amplify (Amersham Biosciences), dried, Luciferase Reporter Assay—The Dual-Luciferase Reporter and then exposed to x-ray film. Assay System (purchased from Promega UK Ltd. (Southamp- Rheb Nucleotide Binding—To examine [ H]GTP and ton UK)) was carried out using cell lysis buffer as recommended [ H]GDP binding to Rheb in vitro, 2.5 g of recombinant Rheb by the manufacturer’s protocol with a Wallac Victor 2 1420 protein was incubated with either 1 Ci of [ H]GTP or Multi-label counter. We ran luciferase assays using an empty [ H]GDP in 25 l of loading buffer (50 mM HEPES, pH 7.5, 5 pGL3 vector (Promega) as a negative control. mM EDTA, and 5 mg/ml bovine serum albumin). After 30 min, VEGF Secretion—Secreted VEGF protein was detected using 0.5 l1 M MgCl and 25 l of cold 50 mM HEPES, pH 7.4, were the human VEGF DuoSet ELISA Development System (R&D 3 3 added. The [ H]GTP- or [ H]GDP-loaded Rheb was then Systems, Abingdon, UK) according to the manufacturer’s applied to 0.2 m cellulose nitrate membrane filter paper instructions. After treatment, the cell medium was recovered (Whatman) under suction in a vacuum manifold and washed following gentle agitation to maximize the recovery of secreted three times with Rheb wash buffer (50 mM HEPES, pH 7.5, 0.5 M proteins and immediately frozen in liquid N . The cells were NaCl, 0.1% Triton X-100, 5 mM MgCl , 0.005% SDS plus prote- then washed in phosphate-buffered saline and lysed for later 2 ase inhibitors). analysis of protein content using Bradford reagent. The Statistics—One-way analysis of variance with posthoc signif- quantity of VEGF secreted into the medium for each treat- icance assessed with Tukey’s honestly significant difference test ment was expressed as a fraction of the respective cellular using SigmaStat (version 2.0) was used on the data represented protein content. by bar charts. Values are given as means with S.D. A p value of Immunocytochemistry—Cells transfected with either empty vector, HA-HIF1, or HA-HIF1 (F99A) were cultured at 1% 0.05 was considered to be statistically significant. 20536 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR FIGURE 1. mTOR activation enhances HIF1 transcription during treat- ment with DMOG. A, HEK293 cells transfected with a HIF-inducible luciferase reporter and empty pRK7 or pRK7/FLAG-Rheb were serum-starved and where indicted were treated with 1 mM DMOG and/or 50 nM rapamycin for 18 h. Lysates prepared were analyzed for luciferase fluorescence. The HIF1 tran- scriptional activity from the DMOG transfected without Rheb was standard- ized to 100%. n  6. *, p and **, p  0.05 relative to activity obtained when Rheb was overexpressed in the presence of DMOG. B, protein levels of FLAG- Rheb, S6K1, and Thr phosphorylation of S6K1 were analyzed from the same lysates. Endogenous HIF1 was immunoprecipitated from these lysates and then subjected to Western blot analysis with HIF1 antibodies. RESULTS We wanted to examine whether the HIF1 transcriptional activity during treatment with dimethyloxalylglycine (DMOG) could be modulated by mTOR. A HIF1-induced FIGURE 2. mTOR activation enhances HIF1 transcription during hypoxia. A, serum-starved HEK293 cells transfected with a HIF-inducible luciferase reporter construct was co-transfected to allow us luciferase reporter and empty pRK7 or pRK7/FLAG-Rheb were transferred to to measure HIF1 transcriptional activities within these low oxygen (1%) or maintained at 21% O , where indicated, for 18 h in the cells. Cells were treated with 1 mM DMOG for 18 h to induce presence or absence of 50 nM rapamycin. Lysates prepared were analyzed for luciferase fluorescence. The HIF1 transcriptional activity from the hypoxic- the activity of HIF1 (Fig. 1A). Treatment with DMOG pre- treated cells transfected without Rheb was standardized to 100%. n  6. *, p vents proline hydroxylase-mediated proteasomal degrada- and **, p  0.05 relative to activity obtained when Rheb was overexpressed tion of HIF1 and thus increases the stability of HIF1 (29). during hypoxic treatment. B, protein levels of FLAG-Rheb, S6K1, and Thr phosphorylation of S6K1 was also analyzed. Total levels of endogenous HIF1 To enhance the activity of mTOR we overexpressed Rheb were analyzed after being immunoprecipitated with anti-HIF1 antibodies. within HEK293 cells. Interestingly, Rheb enhanced the levels C, levels of VEGF-A secretion within the cell media was measured as pg/mg cell protein . n  6. *, p and **, p  0.05 relative to levels of secretion of HIF1-mediated transcription during DMOG treatment obtained when Rheb was overexpressed during hypoxic treatment. (Fig. 1A). As a control, we show that Rheb potently stimu- lates mTOR-dependent phosphorylation of S6K1 at Thr (Fig. 1B). Rapamycin, which specifically inhibits mTOR, We wanted to examine whether the activity of HIF1 was markedly reduced the DMOG-induced transcriptional activ- enhanced by Rheb overexpression during hypoxic conditions. ity of HIF1 upon Rheb overexpression (Fig. 1A) and Similar to the DMOG-treated cells (Fig. 1A), Rheb enhanced blocked the phosphorylation of S6K1 at Thr (Fig. 1B). the activity of HIF1 during hypoxia, which was significantly Other studies have suggested that mTOR could stabilize reduced with treatment with rapamycin (Fig. 2A). Increased HIF1 (25). Therefore, we compared the total levels of activity of mTOR by Rheb overexpression, as observed by HIF1 within these cells. In DMOG-treated cells without increased Thr phosphorylation of S6K1, did not enhance the Rheb overexpression, we observed a reduction of HIF1 protein levels of endogenous HIF1 (Fig. 2B). We also observed upon treatment with rapamycin, which confirms previous a similar pattern of HIF1 expression within hypoxic cells (Fig. reports that inhibition of mTOR reduces the levels of endog- 2B), when compared with DMOG-treated cells (Fig. 1B). Given enous HIF1 within cells (Fig. 1B) (30). Interestingly, we that Rheb induced HIF1 transcription without increasing its observed that DMOG-treated cells expressing Rheb, which protein levels, we propose that mTOR targets the transcrip- had the highest activity of HIF1 (Fig. 1A), also had the low- tional activity of HIF1 rather than its stability. To confirm that est protein levels of HIF1 when compared with cells not Rheb overexpression was increasing the activity of HIF1 expressing Rheb (Fig. 1B, lower panel). Furthermore, rapa- within these cells, we analyzed the secreted protein levels of mycin treatment in cells expressing Rheb, which signifi- VEGF-A, which is a well-characterized downstream gene target cantly reduced the activity of HIF1, did not reduce the of HIF involved in angiogenesis (Fig. 2C). We observed that the HIF1 protein amount. levels of VEGF-A secretion paralleled the activity of HIF during JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20537 Regulation of HIF1 by mTOR FIGURE 4. HIF1 interacts with the Raptor and requires an mTOR signal- ing motif. A, equal levels of recombinant GST-4E-BP1 and GST-4E-BP1(F115A) were subjected to SDS-PAGE and a Raptor overlay assay (lower panel). Equal levels of protein are shown using anti-GST antibodies (upper panel). B, HEK293 cells were treated with 100 M DFX for 24 h, where indicated. Cells were treated with the MG-132 (50 M) for 30 min prior to prevent degradation of HIF1. Total lysates and purified HIF1 samples after immunoprecipitation with anti-HIF1 antibodies were resolved on SDS- PAGE and subjected to a Raptor overlay assay. The band that corresponds to HIF1 is marked. C, HEK293 cells transfected with HA-HIF1 and Myc- Raptor, where indicated, were lysed in the presence of reversible cross-linker DSP and non-ionic detergent (CHAPS). HA-HIF1 was immunoprecipitated FIGURE 3. Guanine nucleotide binding mutants of Rheb inhibit the tran- using anti-HA antibodies and associated Myc-Raptor was determined. 3 3 scription activity of HIF1. A, binding of both [ H]GTP and [ H]GDP to D, HEK293 cells were co-transfected with Myc-Raptor and either empty pRK7 recombinant GST-Rheb proteins (wild-type, D60V, D60K, N119I, and D121N) vector, HA-4E-BP1, HA-HIF1, or HA-HIF1(F99A), where indicated. Cells were or GST control were analyzed as described under “Experimental Procedures.” lysed in the presence of reversible cross-linker DSP and non-ionic detergent B, serum-starved HEK293 cells transfected with a HIF-inducible luciferase (CHAPS). Exogenous 4E-BP1 and HIF1 was immunoprecipitated using reporter and either empty pRK7 or pRK7 vector with FLAG-Rheb (wild-type, anti-HA antibodies as shown. Co-purifed Myc-Raptor was analyzed. D60V, D60K, N119I, or D121N mutants) were transferred to a hypoxic cham- ber set at 1% O for 18 h. Lysates prepared in the hypoxic chamber were described previously (32)), but do possess some ability to impair analyzed for luciferase fluorescence. The HIF1 transcriptional activity from the empty pRK7 was standardized to 100%. n  6. *, p  0.05 relative to mTOR (33). We wanted to investigate whether these Rheb activity of empty pRK7 vector control. C, protein levels of S6K1 and Thr mutants, which are deficient at binding guanine nucleotide, phosphorylation to measure mTOR activation was analyzed. FLAG-Rheb expression was analyzed with anti-FLAG antibodies to show equal expression could alter the levels of HIF1 transcriptional activity during of the Rheb mutants. hypoxia. Interestingly, these Rheb mutants dominantly inhib- ited the transcriptional activity of HIF1 (Fig. 3B), when hypoxia. Interestingly, Rheb overexpression enhanced the lev- expressed to the same level as wild-type (Fig. 3C). This shows els of VEGF-A secretion during conditions of normoxia that that these Rheb mutants can sufficiently impair the basal was sensitive to rapamycin. mTOR-mediated activation of HIF1 within HEK293 cells. Small G-protein mutants that do not bind guanine nucleo- Indeed, these mutants of Rheb were unable to induce Thr tide typically form inactive complexes with their upstream reg- phosphorylation of S6K1 (Fig. 3C). ulators, i.e. the putative guanine exchange factor (GEF), which Well-characterized downstream targets of mTOR, eIF4E- impedes small G-protein-mediated signal transduction. Stud- binding protein 1 (4E-BP1) and S6K1, are recruited to mTOR ies in yeast identified dominant-negative mutants of Rheb through their association with Raptor. To examine Raptor (D60V and D60K), which were unable to efficiently bind to interaction, we utilized a Raptor overlay assay to detect Raptor- guanine nucleotides and impaired TOR signaling (31). We also interacting proteins resolved on SDS-PAGE. This Raptor overlay generated two additional Rheb(N119I) and Rheb(D122N) assay specifically detects proteins containing a TOS motif. For mutants where these two mutated residues lie within the instance, the Raptor-overlay assay only detected wild-type NKXD nucleotide binding motif of Rheb. Guanine nucleotide 4E-BP1 and not a mutant of 4E-BP1 where the phenylalanine binding assays reveal that these D60V, D60K, N119I, and within the TOS motif at position 115 was mutated to an alanine D122N mutants of Rheb are unable to bind to guanine nucleo- (Fig. 4A). To determine whether Raptor interacted with HIF1, tides in vitro (Fig. 3A). These guanine binding-deficient we carried out a Raptor overlay assay on immunoprecipitated mutants of Rheb are unable to efficiently impair acute activa- HIF1. HEK293 cells were treated with 100 M deferoxamine tion of S6K1 by insulin treatment (data not shown, and mesylate (DFX) for 24 h and then incubated with the proteoso- 20538 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR mal inhibitor MG-132 prior to lysis to increase the protein lev- els of HIF1. DFX is an iron chelator and is known to stabilize HIF1 (34). We detected the HIF1 band with the Raptor over- lay assay (Fig. 4B). This data suggests that Raptor interacts with HIF1 (Fig. 4B). To confirm this interaction, we investigated whether HIF1 co-immunoprecipitated with Raptor. HEK293 expressing HA-HIF1 and Myc-Raptor, where indicated, were treated with DMOG and then lysed in the presence of reversible crosslinker DSP and non-ionic detergent (CHAPS). Immuno- precipitated HA-HIF1 was observed to associate with Myc- Raptor (Fig. 4C). We were unable to co-purify HA-HIF1 with Myc-Raptor using immunoprecipitation buffers containing Nonidet P-40 (data not shown). Interestingly, HIF1 contains a potential conserved TOS motif (FVMVL) within an area of unknown function of HIF1 that is immediately 3 of the Period-ARNT-Sim conserved domain-A (PAS-A). We mutated the phenylalanine at position 99 within the TOS motif of HIF1 and examined whether this FIGURE 5. The HIF1(F99A) TOS motif mutant dominantly inhibits HIF1 prevented Raptor interaction (Fig. 4D). We also examined the transcription during hypoxia. A, serum-starved HEK293 cells transfected interaction of Raptor with a well-known Raptor-interacting with a HIF-inducible luciferase reporter with either empty pRK7, HA-HIF1,or HA-HIF1(F99A) in the presence of absence of FLAG-Rheb were treated with protein, 4E-BP1. HIF1 interacted less well with raptor when 50 nM rapamycin, where indicated, and transferred to a hypoxic chamber set compared with 4E-BP1, but the interaction we observed was at 1% O for 18 h. Lysates prepared in the hypoxic chamber were analyzed for luciferase fluorescence. The HIF1 transcriptional activity of the cells trans- prevented when the TOS motif within HIF1 was mutated. fected with empty pRK7 vector and FLAG-Rheb was standardized to 100%. To examine whether this motif is essential for mTOR-medi- n  6. *, p  0.05 relative to activity of empty pRK7 vector control group. **, ated HIF1 transcriptional we compared the transcriptional p  0.01 relative to activity of wild-type HA-HIF1 vector control group. B, protein levels of HA-HIF1, FLAG-Rheb, S6K1, and Thr phosphorylation activity of HIF1(F99A) to wild-type within hypoxic HEK293 of S6K1 were analyzed. cells (Fig. 5A). Mutation of the TOS motif rendered HIF1 transcriptionally inactive during hypoxia. Furthermore, overexpres- sion of HIF1(F99A) significantly reduced the activity of endogenous HIF1 showing that mutation of the TOS motif yields a dominant nega- tive HIF1 mutant. We show that equal levels of wild-type HIF1 and HIF1(F99A) are being expressed and that Rheb over-expression is sufficient to enhance mTOR-medi- ated S6K1 phosphorylation at Thr (Fig. 5B). It was postulated that mTOR sig- naling enhances the stability of HIF1 (25). To examine whether the stability of HIF1 is regulated by mTOR, we pulse-chased cells overexpressing either HA-HIF1 or HIF1(F99A) and Rheb with [S ]methionine under hypoxia (Fig. 6). If mTOR enhanced the stability of HIF1, we would expect the HIF1(F99A) mutant to be less sta- FIGURE 6. mTOR does not modulate the stability of HIF1. HEK293 cells co-expressing FLAG-Rheb with ble. We would also expect that rap- either wild-type HA-HIF1 or HA-HIF1(F99A) were grown at 1% O in media containing [ S]methionine for 4 h, which was then replaced with unlabeled media in the presence or absence of rapamycin, where indicated. mycin would reduce the stability of These cells were lysed after 0, 0.5, 1, and2hinthe1%O hypoxic chamber. Exogenous HA-HIF1 was immu- HIF1. Instead, we found that the noprecipitated with anti-HA antibodies and the relative level of [ S]methionine incorporation into HA-HIF1 protein half-life of the HIF1(F99A) was determined as described under “Experimental Procedures.” Protein levels of HA-HIF1, FLAG-Rheb, S6K1, 389 35 and Thr phosphorylation of S6K1 were also analyzed. The level of [ S]methionine-labeled HIF1 wild-type mutant was similar to that of the and HIF1(F99A) at the zero time point was standardized at 100%. n 3. There was no significant difference of 35 wild-type and treatments with rapa- [ S]methionine incorporation when comparing wild-type HIF1 with the F99A mutant or when comparing cells treated with or without rapamycin at similar time point. mycin did not alter the stability of JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20539 Regulation of HIF1 by mTOR FIGURE 7. Rapamycin inhibits HIF transcription independently of VHL. VHL MEFS were transiently transfected with a HIF-inducible luciferase reporter and either empty pRK7 or VHL vector (where indicated). These cells were maintained at 21% O for 18 h in the presence or absence of 50 nM rapamycin. Lysates were analyzed for luciferase fluorescence and the HIF transcriptional activity from the VHL cells was standardized to 100%. n 5. *, p 0.05 relative to activity of the VHL control. To detect the presence of HA-VHL, HA-VHL was immunoprecipitated from these lysates, then sub- FIGURE 8. HIF1 and HIF1 (F99A) localize to the nucleus in hypoxia. Cells jected to Western blot analysis using anti-HA antibodies. were exposed to 1% O for 18 h following transfection and then processed for immunocytochemistry. Anti HA-FITC conjugated antibody was used to either the wild-type or the F99A mutant of HIF1. This exper- observe the distribution of HA-HIF1/HIF1(F99A) expression relative to the DAPI-stained chromatin. The merged image illustrates that the HA-FITC anti- iment supports our hypothesis that mTOR does not directly body was entirely nuclear in transfected cells. Images are representative of modulate the stability of HIF1. four independent experiments. It is known that VHL is required for the ubiquitin-mediated degradation of the -subunit of HIF (35). To verify that signal- ing through mTOR does not modulate the stability of HIF through a VHL-dependent mechanism, we analyzed HIF tran- scription within human renal carcinoma VHL cells. In nor- moxic conditions these VHL-null cells possess heightened transcriptional activity of HIF (Fig. 7) showing that HIF is sta- bilized and active in these cells during high oxygen tensions. The transcriptional activity of HIF in these cells was due to the loss of VHL as the levels of HIF activity was completely ablated when VHL was transfected back. We observed that rapamycin potently reduced the levels of HIF transcription in the absence of VHL. This experiment shows that rapamycin inhibits HIF transcription independently of VHL and again supports the FIGURE 9. Co-activator p300 interaction with HIF1 is impaired in the HIF1(F99A) mutant. Serum-starved HEK293 cells transfected with empty notion that TOR does not influence the stability of HIF1. pRK7, HA-HIF1, or HA-HIF1(F99A) where transferred to a hypoxic chamber Given that mTOR does not appear to affect the stability of set at 1% O for 18 h before being lysed. Exogenous HA-HIF1 was immuno- HIF1, the increased transcriptional activity of HIF1 by Rheb precipitated with anti-HA antibodies, and the amount of co-purified p300 was quantified by Western blot analysis followed by densitometry (Image J, must be either caused by enhanced nuclear translocation of version 1.37V). Numbers below the panel indicate the ratios of the signals for HIF1 or increased binding of HIF1 to other components of p300 relative to HIF. the transcription activation complex. First of all we examined whether the cellular distribution of the HIF1(F99A) mutant these cells during normoxic and hypoxic conditions (Fig. 10). was different to that of wild-type HIF1. Confocal immunoflu- We observed that rapamycin robustly inhibited the hypoxia- orescence studies showed that both wild-type and HIF1a(F99A) induced activation of HIF in cells without TSC2 by 70%. This mutant was mainly nuclear (Fig. 8), as observed by co-localiza- rapamycin-sensitive level of HIF transcriptional activity during tion with the nuclear stain, DAPI. We next examined if the hypoxia was caused by the loss of TSC2 because transient HIF1(F99A) mutant was deficient at forming transcriptional expression of TSC2 also potently blocked the activity of HIF by complexes. To examine this possibility, we measured the inter- 70%. This result demonstrates that the loss of function of TSC2 action of p300 with both the HIF1(F99A) mutant and wild- potently drives HIF-mediated transcription during conditions type HIF1 (Fig. 9). We observed a significant loss of p300 bind- of low oxygen and is sensitive to treatments with rapamycin. ing to the HIF1(F99A) mutant, which suggests that mTOR It is known that HIF-mediated gene expression is regulated enhances HIF1 transcription through assembly of the HIF1 through PI3K- and mTOR-dependent mechanisms (35–37). transcriptional machinery. To investigate PI3K and mTOR induced activation of HIF in We wanted to examine the levels of HIF transcription within more detail, we treated HEK293 cells with insulin for 18 h in the cells lacking TSC2. It is known that the loss of function of TSC2 presence and absence of LY294002 (to inhibit PI3K) and rapa- potently enhances cell signaling through mTOR (8). We trans- mycin (to inhibit mTOR) during hypoxia. We investigated the fected TSC2 MEFS with the HIF reporter construct to activity of HIF (Fig. 11A) and the amount of VEGF-A protein measure the relative levels of HIF transcriptional activity within that was secreted by these cells (Fig. 11B). Insulin increased the 20540 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR sufficient to activate PI3K/mTOR signaling after 18 h of treat- ment. Treatments with either LY294002 or rapamycin were sufficient to block insulin-induced phosphorylation of S6K1 showing that we were inhibiting PI3K and mTOR-mediated signaling in these cells. It is important to note that LY294002 also inhibits mTOR. Therefore, the difference of inhibition we observe with rapamycin and LY294002 represents the level of HIF transcriptional activity (Fig. 11A) and VEGF-A secretion (Fig. 11B) that is dependent on PI3K. Upon insulin stimulation, we observed a marked increased in the HIF transcriptional activity within cells during hypoxia that was completely blocked by treatments with LY294002 and significantly impaired by rapamycin (Fig. 11A). Similarly, LY294002 blocked FIGURE 10. Hypoxia-induced HIF transcription in TSC2-null cells are insulin-induced VEGF-A secretion during hypoxia while highly sensitive to rapamycin. Serum-starved TSC2 MEFS were tran- VEGF-A secretion was significantly repressed by rapamycin siently transfected with a HIF-inducible luciferase reporter and either empty pRK7 or pRK7/FLAG-TSC2 (where indicated). These cells were serum-starved (Fig. 11B). These experiment shows that mTOR is necessary for and were transferred to low oxygen (1%) or maintained at 21% O , where the maximal activation of HIF-mediated transcription upon indicated, for 18 h in the presence or absence of 50 nM rapamycin. Lysates prepared were analyzed for luciferase fluorescence and the HIF transcrip- insulin stimulation. tional activity from the TSC2 cells during hypoxia was standardized to 100%. n  6. *, p and **, p  0.05 relative to activity of the TSC2 control. DISCUSSION Protein levels of FLAG-TSC2 were determined by Western blot analysis after immunoprecipitation with anti-FLAG antibodies. The asterisk indicates a non- We show that mTOR positively enhances the level of HIF- specific band that migrates above FLAG-TSC2. mediated transcription. Rheb-specific activation of mTOR enhanced the transcriptional activity of HIF during condi- tions that favored HIF1 stabilization, i.e. during hypoxia (Fig. 2A) and treatments with DMOG (Fig. 1A). This enhanced HIF activity was blocked by treatments with rapa- mycin showing that Rheb-induced HIF activity was caused by the heightened activity of mTOR. A potential function of mTOR would be to regulate the expression levels of HIF1. Dogma has it that stability is the rate-limiting factor that determines the protein levels of HIF1. However, it is important to appreciate that the regulation of HIF1 expres- sion is multifaceted with additional inputs that function at the level of transcription and translation (30, 35–37). Indeed, it is known that the translation of HIF1 can be modulated by mTOR through cap-dependent mechanisms that is driven by eIF4E and repressed by the translation repressor, 4E-BP1 (30). The reduction of endogenous HIF1 that we observe after rapamycin treatment in the presence of DMOG or hypoxia conditions (Figs. 1B and 2B, respectively), could be accountable by the reduced rates of HIF1 protein synthesis. For added complexity, the translation of HIF1 can also be maintained through an internal ribosomal entry site (IRES). IRES-mediated translation does not require eIF4E and so FIGURE 11. Insulin-induced HIF activation and VEGF-A secretion is partially confers a rapamycin-insensitive mechanism to promote blocked by rapamycin. A, serum-starved HEK293 cells transfected with a HIF- inducible luciferase reporter were transferred to low oxygen (1%) or maintained HIF1 translation during times when mTOR signaling is at 21% O . Where indicated, cells where stimulated with 100 nM insulin in the switched off (38). Given that rapamycin did not reduce the presence or absence of either 50 nM rapamycin or 50M LY294002. After 18 h, the cells were harvested, and the lysates were analyzed for luciferase fluorescence. levels of HIF1 protein in cells over-expressing Rheb (Figs. 1B The HIF1 transcriptional activity from hypoxic unstimulated cells was standard- and 2B), the reduction of Rheb-induced HIF transcriptional ized to 100%. B, levels of VEGF-A secretion within the cell media was measured as activity by rapamycin was not caused by a loss of the HIF1 pg/mg cell protein . The level of VEGF-A secretion from hypoxic-unstimulated cells was standardized to 100%. For both graphs; n  6. *, p and **, p  0.05 protein. This rules out the possibility that mTOR modulates the relative to the levels of HIF activity or VEGF-A secretion obtained from stimulated stability of HIF1 and is supported by the observation that cells during hypoxia. C, S6K1 phosphorylation was analyzed by a mobility shift on SDS-PAGE. p70 and p80 isoforms of S6K1 are marked. rapamycin treatment did not alter the rates of protein break- down of HIF1 (Fig. 6). Furthermore, rapamycin potently phosphorylation of S6K1 (as observed by a mobility shift of the impaired the activity of HIF in VHL-null cells (Fig. 7). VHL is p70 and p85 isoforms of S6K1 to the higher phosphorylated required for the ubiquitin-mediated degradation of the -sub- bands) (Fig. 11C), showing that treatment with 100 nM insulin is unit of HIF. Therefore, our data suggests that mTOR promotes JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20541 Regulation of HIF1 by mTOR the transcriptional activity of HIF1 and does not involve VHL- with each of these hamartomas syndromes are known to have mediated degradation of HIF1. high levels of mTOR activation (see review in Ref. 8). Interest- The mechanism by how mTOR modulates HIF within cells ingly, we observed high levels of HIF activity in cells lacking has remained elusive to date. In this manuscript, we have iden- TSC2, which was reversed when we added back TSC2 or inhib- tified a potential FVMVL TOS motif within a previously unde- ited mTOR with rapamycin (Fig. 9). Our work suggests that the fined region of HIF1. This motif is similar to well known TOS high degree of vascularization observed in tumors arising from motifs found in both 4E-BP1 and S6K1, which are FEMDI and these syndromes could be the direct consequence of high levels FD(L/I)DL, respectively. Raptor is thought to bind to the TOS of mTOR activity. For instance, aberrant signaling through motif within mTOR substrates. This interaction recruits these mTOR would enhance the activity of HIF and encourage the substrates to mTOR for their optimal phosphorylation. For process of angiogenesis during hypoxia. instance, mutation of the TOS motif by alanine substitution of It is known that a negative feedback loop, which is activated the phenylalanine rendered both 4E-BP1 and S6K1 unrespon- by HIF, inhibits the mTOR pathway and HIF function. This sive to mTOR (14, 15). We show that raptor interacts with feedback loop is regulated by Redd1/2 (Regulated in Develop- HIF1 (Fig. 4, B and C) and this interaction is inhibited when ment and DNA damage responses), which are also referred to as the phenylalanine within the TOS motif is mutated to an ala- RTP801/801L and are transcriptionally up-regulated by HIF nine (Fig. 4D). We observed a lower level of raptor interaction (40). The inhibition of mTOR by either Redd1 or Redd2 with HIF1 when compared with 4E-BP1 (Fig. 4D) and this requires TSC2 (41) and suggests that Redd1/2 activates might be caused by the differences in the cellular distribution of TSC1/2. The mechanism by how Redd1/2 signals through both HIF1 and 4E-BP1, i.e. 4E-BP1 is cytoplasmic while HIF1 TSC2 is currently undefined. This negative feedback loop is mainly nuclear. We show that mutation of the TOS motif makes physiological sense, as it would be unfavorable for cellu- renders HIF1 transcriptionally inactive and this TOS mutant lar HIF responses to be maintained for long periods of time. In is unresponsive to enhanced mTOR signaling when Rheb is our experiments, we override this Redd1/2 negative feedback overexpressed (Fig. 5A). The HIF1 TOS mutant was predom- loop by overexpressing Rheb, which is sufficient to potently inantly nuclear (Fig. 8). These data suggest that raptor interac- enhance mTOR signaling during long term conditions of tion with HIF1 does not influence the nuclear translocation of hypoxia (Fig. 2B). Cells lacking functional TSC1/TSC2 would HIF, a process that requires the interaction of HIF with also lack this hypoxic induced negative feedback loop through HIF1. Interestingly, mutation of the TOS motif within HIF1, Redd1/2. The inability of Redd1/2 to activate the TSC1/TSC2 transforms HIF1 into a dominant negative mutant that inhib- heterodimer in TSC2-null cells might be the reason why we its endogenous HIF activity (Fig. 5A). It is possible that the observe high levels of HIF activity that is potently suppressed by HIF1(F99A) mutant sequesters the HIF subunit to form treatments with rapamycin (Fig. 9). This study suggests that inactive heterodimers, and thus competes with the interaction inhibition of mTOR might be a suitable strategy to treat har- of HIF with endogenous HIF1. Mutation of the TOS motif matomas syndromes to repress mTOR-mediated angiogenesis significantly impaired the interaction of HIF1 with the co- through HIF. activator CBP/p300 (Fig. 9) and supports our view that the HIF1(F99A) mutant is unable to form functional transcription Acknowledgments—We thank Prof. Grahame Hardie and the Divi- complexes. sion of Molecular Physiology for their support. HIF1 is a reported phosphoprotein. 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Hypoxia-inducible Factor 1α Is Regulated by the Mammalian Target of Rapamycin (mTOR) via an mTOR Signaling Motif *

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American Society for Biochemistry and Molecular Biology
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0021-9258
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DOI
10.1074/jbc.m611782200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 28, pp. 20534 –20543, July 13, 2007 © 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Hypoxia-inducible Factor 1 Is Regulated by the Mammalian Target of Rapamycin (mTOR) via an mTOR Signaling Motif Received for publication, December 22, 2006, and in revised form, May 10, 2007 Published, JBC Papers in Press, May 14, 2007, DOI 10.1074/jbc.M611782200 ‡ §1 Stephen C. Land and Andrew R. Tee From the Institute of Medical Genetics, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom and the Division of Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom Tumors that form as a result of heightened mammalian target two key angiogenic factors; vascular endothelial growth fac- of rapamycin (mTOR) signaling are highly vascularized. This tor (VEGF)-A (1) and angiopoietin-2 (Ang-2) (2). VEGF-A and process of angiogenesis is regulated through hypoxia-inducible Ang-2 encourage angiogenesis by stimulating the formation of factor (HIF)-mediated transcription of angiogenic factors. It is blood vessels that migrate into the tumor. recognized that inhibition of mTOR with rapamycin can dimin- As well as vascularization, HIF is known to control the ish the process of angiogenesis. Our work shows that activation expression of 70 genes involved in energy metabolism, apo- of mTOR by Ras homologue enriched in brain (Rheb) overex- ptosis/survival, and metastasis (for reviews see Refs. 3, 4). Low pression potently enhances the activity of HIF1 and vascular oxygen enhances the activity of HIF by stabilizing the -subunit endothelial growth factor (VEGF)-A secretion during hypoxia, of HIF. Oxygen-dependent prolyl hydroxylase domain (PHD) which is reversed with rapamycin. Mutants of Rheb, which do proteins become active in the presence of oxygen that sequen- not bind guanine nucleotide (D60K, D60V, N119I, and D122N) tially leads to hydroxylation of two proline residues within the and are unable to activate mTOR, inhibit the activity of HIF oxygen-dependent degradation domain (ODDD) of the HIF when overexpressed. We show that regulatory associated pro- -subunit. Proline hydroxylation results in ubiquitin-mediated tein of mTOR (Raptor) interacts with HIF1 and requires an degradation of the -subunit that requires the Von Hippel- mTOR signaling (TOS) motif located in the N terminus of Lindau (VHL) tumor suppressor protein, which is a component HIF1. Furthermore, a mutant of HIF1 lacking this TOS motif of an E3 ubiquitin ligase complex (5). VHL syndrome, caused dominantly impaired HIF activity during hypoxia and was through loss of function of this tumor suppressor, is a domi- unable to bind to the co-activator CBP/p300. Rapamycin treat- nantly inherited familial syndrome that predisposes the patient ments do not affect the stability of HIF1 and modulate HIF to renal cell carcinoma and cerebellar hemangioblastomas. In activity via a Von Hippel-Lindau (VHL)-independent mecha- conditions of low oxygen tension, the HIF protein is stable and nism. We demonstrate that the high levels of HIF activity in cells binds to HIF (also known as aryl hydrocarbon receptor devoid of TSC2 can be reversed by treatments with rapamycin or nuclear translocator-ARNT). The HIF/HIF dimer is then the readdition of TSC2. Our work explains why human cancers rapidly translocated to the nucleus. As a dimer, they bind to with aberrant mTOR signaling are prone to angiogenesis and RCGTG DNA sequences called hypoxia response elements. suggests that inhibition of mTOR with rapamycin might be a Transcription is sequentially activated when the C terminus suitable therapeutic strategy. transcriptional activation domain (C-TAD) of HIF binds to the co-activator CBP/p300 (for review see Ref. 6). The tuberous sclerosis complex (TSC), which is a hamarto- The supply of nutrients and oxygen is quickly depleted as mas syndrome, occurs through loss of function of the tumor solid tumors grow in size. Nutritional and oxygen homeostasis suppressor proteins TSC1 (hamartin) and TSC2 (tuberin). within solid tumors is reestablished by vascularization that Tumors that occur in TS patients are highly vascularized, consequently results in tumor expansion. Oxygen sensing is implying that HIF might be involved and is regulated down- known to control the mechanism of vasculogenesis through stream of TSC1/2. TSC is an autosomal genetic syndrome that hypoxia-inducible factor (HIF) -mediated transcription of occurs with an estimated prevalence of one in 6,000 newborns and is characterized by slow growing benign tumors that form in the heart, brain, kidneys, eyes, and skin (see Ref. 7 for review). * This research was supported by British Heart Foundation Intermediate Research Fellowship No. FS/04/022 and Association of International Can- These tumors can lead to both renal and neurological compli- cer Research Career Development Fellowship No 06-914/915 (to A. R. T.), and funding from Tenovus (to S. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must human embryonic kidney; MEFs, mouse embryonic fibroblasts; mTOR, therefore be hereby marked “advertisement” in accordance with 18 U.S.C. mammalian target of rapamycin; TOS, mTOR signaling; NF1, neurofibro- Section 1734 solely to indicate this fact. min 1; PI3K, phosphoinositide 3-kinase; PHD, prolyl hydroxylases domain; To whom correspondence should be addressed. Tel.: 029-2074-4055; Fax: PTEN, phosphatase and tensin homolog; Raptor, regulatory associated 029-2074-6551; E-mail: [email protected]. protein of mTOR; Redd1/2, regulated in development and DNA damage The abbreviations used are: HIF, hypoxia-inducible factor; ARNT, aryl hydro- responses; Rheb, Ras homologue enriched in brain; Rictor, rapamycin-in- carbon receptor nuclear translocator; CHAPS, 3-(3-cholamidopropyl)dim- sensitive companion of mTOR; S6K1, ribosomal protein S6 kinase 1; STK11, ethylammonio-1-propanesulfonate; DFX, deferoxamine mesylate; DMOG, serine/threonine kinase 11; TSC, tuberous sclerosis complex; VEGF, vascu- dimethyloxalylglycine; DMEM, Dulbecco’s modified Eagle’s medium; DSP, lar endothelial growth factor; VHL, Von Hippel-Lindau; HA, hemagglutinin; dithiobis(succinimdyl propionate); 4E-BP1, eIF4E-binding protein 1; HEK, FITC, fluorescein isothiocyanate. This is an Open Access article under the CC BY license. 20534 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR cations. TSC1 and TSC2 function as a heterodimer that inhibits enhances the transcriptional activity of HIF1 that does not cell growth by impairing the Ser/Thr protein kinase called the involve increased HIF1 stabilization. Furthermore, mTOR mammalian target of rapamycin (mTOR) (see review in Ref. 8). activation of HIF1 requires the interaction of HIF1 with Rap- TSC1/TSC2 heterodimers function to inhibit mTOR, by acting tor. Given that Raptor functions as a scaffold protein that as a GTPase-activating protein (GAP) toward the small G-pro- recruits downstream mTOR substrates with a TOS motif to tein, Ras homologue enriched in brain (Rheb). Rheb has been mTOR (15), this work reveals that HIF1 is a downstream tar- shown to interact with mTOR, and promotes signal transduc- get of mTOR. tion when Rheb is in the active GTP-bound state (9). When in a EXPERIMENTAL PROCEDURES complex with TSC1, TSC2 inhibits Rheb-induced mTOR sig- 3 3 35 naling by reverting Rheb to its inactive GDP-bound state (10). Chemicals and Materials—[ H]GTP, [ H]GDP, and [ S]me- Therefore, when the normal function of TSC1/TSC2 het- thionine-radiolabeled reagents were purchased from Amer- erodimers become compromised, Rheb becomes constitutively sham Biosciences (GE Healthcare UK Ltd.). Dimethyloxalylg- GTP-bound and mTOR signaling is significantly enhanced and lycine was purchased from Frontier Scientific Europe Ltd contributes to the pathology associated with TSC. (Lancashire, UK). Deferoxamine mesylate, MG-132 (carboben- Within mammalian cells, mTOR functions as two distinct zoxy-L-leucyl-L-leucyl-L-leucinal), rapamycin, and LY294002 multi-protein kinase-signaling complexes. Rictor (rapamycin- were obtained from Merck Biosciences Ltd. (Nottingham, UK). insensitive companion of mTOR) and LST8 (also referred to as CHAPS was ordered from Pierce. DSP (dithiobis(succinimdyl G-protein -subunit-like protein (GL)) interact with mTOR propionate)) was purchased from Apollo Scientific Ltd. (Stock- to form an mTOR/Rictor/LST8 protein kinase complex (11). port, UK). Rat anti-HA antibodies were purchased from Roche mTOR also forms another multi-protein kinase complex with a Applied Science. Mouse anti-HA antibodies were kindly pro- different scaffold protein called Raptor (regulatory-associated vided by M. Chou (University of Pennsylvania, Philadelphia, protein of mTOR), where signaling from this (12, 13) promotes PA). Cell Signaling Technology anti-S6K1 and anti-S6K1 phos- mTOR-mediated phosphorylation of distinct downstream sig- pho-Thr antibodies were purchased from New England Bio- naling targets, such as the ribosomal S6 protein kinase 1 (S6K1) labs Ltd. (Hertfordshire, UK). Anti-HIF1 antibodies (clone: and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), H1alpha67) were obtained from Novus Biologicals (Stratech which is potently impaired by treatments with the immunosup- Scientific Ltd.). Anti-Coactivator p300 (C-20) antibodies were pressant drug rapamycin. mTOR regulates cell growth and pro- bought from Santa Cruz Biotechnology, Inc. (Heidelberg, liferation and is known to involve both S6K1 and 4E-BP1 (for Germany). Anti-FLAG antibodies (clone: M2), anti-Myc review see Ref. 8). It is believed that Raptor functions as a scaf- (clone: 9E10) and all other reagents (unless stated) were fold protein that recruits mTOR to its downstream substrates obtained from Sigma-Aldrich. through their mTOR signaling (TOS) motifs. The TOS motif is Plasmids and Molecular Biology—N-terminal FLAG-tagged a 5 amino acid peptide found within rapamycin-sensitive tar- pRK7/Rheb was generated as described in Ref. 10. Human HA- gets of mTOR (FDL/IDL and FEMDI within S6K1 (14) and tagged 4E-BP1 was a kind gift from N. Sonenberg (McGill Uni- 4E-BP1 (15), respectively). Mutation of this TOS motif (espe- versity, Montreal, Canada). HIF1 was cloned into a modified cially the highly conserved phenylalanine at position 1) pre- pRK7 vector so it expressed a triple N-terminal HA tag using vents mTOR-directed phosphorylation of both S6K1 and human HIF1 cDNA as a template (obtained from Novus Bio- 4E-BP1 (14, 15). logicals (Stratech Scientific Ltd.)). Myc-tagged Raptor/pRK5 Interestingly, a common feature of hamartomas syndromes was a kind gift from D. M. Sabatini (Whitehead Institute for caused by the loss of function of TSC1, TSC2 (16), PTEN (phos- Biomedical Research, Boston, MA). Human 4E-BP1 and phatase and tensin homolog) (17), LKB1 (also known as STK11 human Rheb were subcloned into pGEX-2T/GST to generate (serine/threonine kinase 11)) (18), or NF1 (neurofibromin 1) GST-tagged recombinant protein. Point mutations were gener- (19) tumor suppressors is that signaling through mTOR is sig- ated as described in the QuikChange site-directed mutagene- nificantly enhanced. Furthermore, VEGF levels are increased in sis kit (Stratagene). The N-terminal FLAG-tagged pRK7/TSC2 these syndromes (16, 20–22). It is possible that increased VEGF construct was generated as previously described (26). The secretion caused through loss of function of these tumor sup- pVHL-HA vector was kindly provided by P. Ratcliffe (Henry pressors is mediated via an mTOR-dependent mechanism. Wellcome Building of Molecular Physiology, Oxford Univer- Indeed, it has been shown that rapamycin has anti-angiogenic sity, UK) as described (27). The firefly luciferase reporter pGL2- properties by reducing the expression of VEGF (23, 24). Fur- TK-HRE plasmid was generated by subcloning three copies of thermore, VEGF expression within cells lacking TSC1 or TSC2 the hypoxia response element (HRE) (5-GTGACTACGT- is highly sensitive to treatments of rapamycin (16). The mech- GCTGCCTAG-3) from the inducible nitric-oxide synthase anism by how mTOR enhances VEGF expression has not been promoter into the promoter region of the pGL2-TK vector as fully elucidated. It is postulated that HIF becomes more stable previously described (28) and was kindly provided by G. Melillo by heightened mTOR activity, which causes enhanced VEGF (National Cancer Institute at Frederick, Maryland). expression (25). We wanted to explore the mTOR-specific acti- Tissue Culture and Analysis of Cell Lysates—Human embry- vation of HIF1 in greater detail. In this study, we potently onic kidney 293 (HEK293) cells were cultured (at 37 °C within activate mTOR by overexpressing Rheb. We have previously 5% CO ) and maintained in Dulbecco’s modified Eagle’s shown that Rheb can bind to and enhance mTOR signaling to medium (DMEM) supplemented with 10% fetal bovine serum. downstream targets (9, 10). We show that mTOR directly DMEM and fetal bovine serum were purchased from Invitro- JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20535 Regulation of HIF1 by mTOR gen Ltd. (Paisley, UK). TSC2 mouse embryonic fibroblasts O for 18 h on glass coverslips. Cells were then fixed in metha- (MEFS) were a kind gift from D. J. Kwiatkowski (Harvard Med- nol at20 °C, quenched in 0.1% Na borohydrate in Tris-buff- ical School, Boston, MA). The human renal carcinoma VHL ered saline (TBS; 50 mM Tris-Cl (pH 7.4), 150 mM NaCl) and cells (786-O) were bought from ATCC. Cell transfections were then blocked for1hinTBS containing 10% goat serum. Mono- carried out as described in the SuperFect manufacturer’s pro- clonal Anti-HA (clone HA-7) FITC conjugate (Sigma) was then tocol (purchased from Qiagen Ltd. (West Sussex, UK)). applied to the cells in TBS containing 0.1% bovine serum albu- HEK293 cells were transfected with 95% efficiency, while min overnight at 4 °C and at 1:5000 dilution. After washing, transfection of TSC2 MEFS and human renal carcinoma coverslips were counterstained with DAPI antifade (Q-Bio- VHL cells were 10% efficient. Hypoxic treatments were gene) and mounted. The intracellular distribution of FITC and carried out in a MACS VA500 Microaerophilic Work station DAPI fluorescence was observed using a Zeiss LSM 510 confo- (Don Whitley Scientific, Shipley, West Yorkshire) containing a cal microscope. humidified atmosphere equilibrated to 1% O ,5%CO , and 2 2 Raptor Binding to mTOR Targets—To generate Raptor pro- 94% N . To create cell lysates, cells were washed twice in phos- tein to be used in the overlay assays, Myc-tagged Raptor was phate-buffered saline, and then harvested with lysis buffer (10 overexpressed in HEK293 cells. These cells were lysed with 50 mM KH PO ,1mM EDTA, 10 mM MgCl ,50mM -glycero- 2 4 2 mM -glycerophosphate, pH 7.4; 1 mM EDTA; 1 mM EGTA; 0.5 phosphate, 5 mM EGTA, 0.5% Nonidet P-40, 0.1% Brij 35, 1 mM mM Na VO ;1mM benzamidine hydrochloride; 1 mM dithio- 3 4 sodium orthovanadate, 40 mg/ml phenylsulfonyl fluoride, 10 threitol; 0.1 mM phenylmethane sulfonyl fluoride; 1% (v/v) Tri- g/ml leupeptin, 5 g pepstatin, pH 7.2). Cell lysates were son- ton X-100, and 1 g/ml each of pepstatin, antipain, and leupep- icated to help break down the nucleus and then spun at 14, 000 tin. Proteins were transferred to Immobilon-P and blocked rpm for 8 min at 4 °C to remove the cell debris. For Western blot with 5% (w/v) milk in Tris-buffered saline were incubated over- analysis, these lysates were prepared as described for running night at 4 °C with 1:10 diluted Myc-tagged Raptor lysate. Myc- NuPage Novex gels (bought from Invitrogen Ltd.) Proteins tagged Raptor that bound to proteins immobilized on Immo- resolved on Novex gels were transferred to Millipore Immo- bilon-P was then detected by incubating the blot with anti-Myc bilon-P (purchased from Upstate Ltd. (Hampshire, UK)) and antibodies for2hin2% (w/v) bovine serum albumin Tris-buff- blotted with the appropriate antibody followed by horseradish ered saline followed by anti-mouse horseradish peroxidase- peroxidase-conjugated secondary antibodies. Enhanced conjugated secondary antibodies and ECL as described for chemiluminescence was carried out with Amersham Bio- TM sciences ECL Western blotting detection reagents (pur- Western blotting. Raptor association with mTOR substrates chased from GE Healthcare UK Ltd.). within cells was determined as previously described (15). Immunoprecipitation—Proteins were immunoprecipitated Monitoring HIF1 Stability in Vivo—HEK293 cells trans- using the relevant antibodies coupled to protein G-Sepharose fected with HA-HIF1 (grown on 6-cm plates) were incubated (Amersham Biosciences). HIF1 immunoprecipitations were in methionine-free medium and labeled with 10 Ci of carried out overnight at 4 °C, and the protein G-Sepharose was S]methionine for4hinthe hypoxic chamber set at 1% O . added for 1 h prior to being washed. Immunoprecipitates were The cells were washed twice in fresh media and then incubated washed twice each with both buffer A (50 mM Hepes (pH 7.4), for 0, 0.5, 1, and2hin DMEM before cell lysates were gener- 100 mM NaCl, 10 mM MgCl , 1 mg/ml bovine serum albumin, 1 ated. HA-HIF1 was immunoprecipitated for 1 h with anti-HA mM dithiothreitol, 1% Triton) and buffer B (50 mM Hepes (pH antibodies bound to protein G-Sepharose. Immunoprecipi- 7.4), 100 mM NaCl, 10 mM MgCl , 0.1% Triton) in the presence tated HIF1 was subjected to SDS-PAGE. The gel was stained, of protease inhibitors. fixed, incubated with Amplify (Amersham Biosciences), dried, Luciferase Reporter Assay—The Dual-Luciferase Reporter and then exposed to x-ray film. Assay System (purchased from Promega UK Ltd. (Southamp- Rheb Nucleotide Binding—To examine [ H]GTP and ton UK)) was carried out using cell lysis buffer as recommended [ H]GDP binding to Rheb in vitro, 2.5 g of recombinant Rheb by the manufacturer’s protocol with a Wallac Victor 2 1420 protein was incubated with either 1 Ci of [ H]GTP or Multi-label counter. We ran luciferase assays using an empty [ H]GDP in 25 l of loading buffer (50 mM HEPES, pH 7.5, 5 pGL3 vector (Promega) as a negative control. mM EDTA, and 5 mg/ml bovine serum albumin). After 30 min, VEGF Secretion—Secreted VEGF protein was detected using 0.5 l1 M MgCl and 25 l of cold 50 mM HEPES, pH 7.4, were the human VEGF DuoSet ELISA Development System (R&D 3 3 added. The [ H]GTP- or [ H]GDP-loaded Rheb was then Systems, Abingdon, UK) according to the manufacturer’s applied to 0.2 m cellulose nitrate membrane filter paper instructions. After treatment, the cell medium was recovered (Whatman) under suction in a vacuum manifold and washed following gentle agitation to maximize the recovery of secreted three times with Rheb wash buffer (50 mM HEPES, pH 7.5, 0.5 M proteins and immediately frozen in liquid N . The cells were NaCl, 0.1% Triton X-100, 5 mM MgCl , 0.005% SDS plus prote- then washed in phosphate-buffered saline and lysed for later 2 ase inhibitors). analysis of protein content using Bradford reagent. The Statistics—One-way analysis of variance with posthoc signif- quantity of VEGF secreted into the medium for each treat- icance assessed with Tukey’s honestly significant difference test ment was expressed as a fraction of the respective cellular using SigmaStat (version 2.0) was used on the data represented protein content. by bar charts. Values are given as means with S.D. A p value of Immunocytochemistry—Cells transfected with either empty vector, HA-HIF1, or HA-HIF1 (F99A) were cultured at 1% 0.05 was considered to be statistically significant. 20536 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR FIGURE 1. mTOR activation enhances HIF1 transcription during treat- ment with DMOG. A, HEK293 cells transfected with a HIF-inducible luciferase reporter and empty pRK7 or pRK7/FLAG-Rheb were serum-starved and where indicted were treated with 1 mM DMOG and/or 50 nM rapamycin for 18 h. Lysates prepared were analyzed for luciferase fluorescence. The HIF1 tran- scriptional activity from the DMOG transfected without Rheb was standard- ized to 100%. n  6. *, p and **, p  0.05 relative to activity obtained when Rheb was overexpressed in the presence of DMOG. B, protein levels of FLAG- Rheb, S6K1, and Thr phosphorylation of S6K1 were analyzed from the same lysates. Endogenous HIF1 was immunoprecipitated from these lysates and then subjected to Western blot analysis with HIF1 antibodies. RESULTS We wanted to examine whether the HIF1 transcriptional activity during treatment with dimethyloxalylglycine (DMOG) could be modulated by mTOR. A HIF1-induced FIGURE 2. mTOR activation enhances HIF1 transcription during hypoxia. A, serum-starved HEK293 cells transfected with a HIF-inducible luciferase reporter construct was co-transfected to allow us luciferase reporter and empty pRK7 or pRK7/FLAG-Rheb were transferred to to measure HIF1 transcriptional activities within these low oxygen (1%) or maintained at 21% O , where indicated, for 18 h in the cells. Cells were treated with 1 mM DMOG for 18 h to induce presence or absence of 50 nM rapamycin. Lysates prepared were analyzed for luciferase fluorescence. The HIF1 transcriptional activity from the hypoxic- the activity of HIF1 (Fig. 1A). Treatment with DMOG pre- treated cells transfected without Rheb was standardized to 100%. n  6. *, p vents proline hydroxylase-mediated proteasomal degrada- and **, p  0.05 relative to activity obtained when Rheb was overexpressed tion of HIF1 and thus increases the stability of HIF1 (29). during hypoxic treatment. B, protein levels of FLAG-Rheb, S6K1, and Thr phosphorylation of S6K1 was also analyzed. Total levels of endogenous HIF1 To enhance the activity of mTOR we overexpressed Rheb were analyzed after being immunoprecipitated with anti-HIF1 antibodies. within HEK293 cells. Interestingly, Rheb enhanced the levels C, levels of VEGF-A secretion within the cell media was measured as pg/mg cell protein . n  6. *, p and **, p  0.05 relative to levels of secretion of HIF1-mediated transcription during DMOG treatment obtained when Rheb was overexpressed during hypoxic treatment. (Fig. 1A). As a control, we show that Rheb potently stimu- lates mTOR-dependent phosphorylation of S6K1 at Thr (Fig. 1B). Rapamycin, which specifically inhibits mTOR, We wanted to examine whether the activity of HIF1 was markedly reduced the DMOG-induced transcriptional activ- enhanced by Rheb overexpression during hypoxic conditions. ity of HIF1 upon Rheb overexpression (Fig. 1A) and Similar to the DMOG-treated cells (Fig. 1A), Rheb enhanced blocked the phosphorylation of S6K1 at Thr (Fig. 1B). the activity of HIF1 during hypoxia, which was significantly Other studies have suggested that mTOR could stabilize reduced with treatment with rapamycin (Fig. 2A). Increased HIF1 (25). Therefore, we compared the total levels of activity of mTOR by Rheb overexpression, as observed by HIF1 within these cells. In DMOG-treated cells without increased Thr phosphorylation of S6K1, did not enhance the Rheb overexpression, we observed a reduction of HIF1 protein levels of endogenous HIF1 (Fig. 2B). We also observed upon treatment with rapamycin, which confirms previous a similar pattern of HIF1 expression within hypoxic cells (Fig. reports that inhibition of mTOR reduces the levels of endog- 2B), when compared with DMOG-treated cells (Fig. 1B). Given enous HIF1 within cells (Fig. 1B) (30). Interestingly, we that Rheb induced HIF1 transcription without increasing its observed that DMOG-treated cells expressing Rheb, which protein levels, we propose that mTOR targets the transcrip- had the highest activity of HIF1 (Fig. 1A), also had the low- tional activity of HIF1 rather than its stability. To confirm that est protein levels of HIF1 when compared with cells not Rheb overexpression was increasing the activity of HIF1 expressing Rheb (Fig. 1B, lower panel). Furthermore, rapa- within these cells, we analyzed the secreted protein levels of mycin treatment in cells expressing Rheb, which signifi- VEGF-A, which is a well-characterized downstream gene target cantly reduced the activity of HIF1, did not reduce the of HIF involved in angiogenesis (Fig. 2C). We observed that the HIF1 protein amount. levels of VEGF-A secretion paralleled the activity of HIF during JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20537 Regulation of HIF1 by mTOR FIGURE 4. HIF1 interacts with the Raptor and requires an mTOR signal- ing motif. A, equal levels of recombinant GST-4E-BP1 and GST-4E-BP1(F115A) were subjected to SDS-PAGE and a Raptor overlay assay (lower panel). Equal levels of protein are shown using anti-GST antibodies (upper panel). B, HEK293 cells were treated with 100 M DFX for 24 h, where indicated. Cells were treated with the MG-132 (50 M) for 30 min prior to prevent degradation of HIF1. Total lysates and purified HIF1 samples after immunoprecipitation with anti-HIF1 antibodies were resolved on SDS- PAGE and subjected to a Raptor overlay assay. The band that corresponds to HIF1 is marked. C, HEK293 cells transfected with HA-HIF1 and Myc- Raptor, where indicated, were lysed in the presence of reversible cross-linker DSP and non-ionic detergent (CHAPS). HA-HIF1 was immunoprecipitated FIGURE 3. Guanine nucleotide binding mutants of Rheb inhibit the tran- using anti-HA antibodies and associated Myc-Raptor was determined. 3 3 scription activity of HIF1. A, binding of both [ H]GTP and [ H]GDP to D, HEK293 cells were co-transfected with Myc-Raptor and either empty pRK7 recombinant GST-Rheb proteins (wild-type, D60V, D60K, N119I, and D121N) vector, HA-4E-BP1, HA-HIF1, or HA-HIF1(F99A), where indicated. Cells were or GST control were analyzed as described under “Experimental Procedures.” lysed in the presence of reversible cross-linker DSP and non-ionic detergent B, serum-starved HEK293 cells transfected with a HIF-inducible luciferase (CHAPS). Exogenous 4E-BP1 and HIF1 was immunoprecipitated using reporter and either empty pRK7 or pRK7 vector with FLAG-Rheb (wild-type, anti-HA antibodies as shown. Co-purifed Myc-Raptor was analyzed. D60V, D60K, N119I, or D121N mutants) were transferred to a hypoxic cham- ber set at 1% O for 18 h. Lysates prepared in the hypoxic chamber were described previously (32)), but do possess some ability to impair analyzed for luciferase fluorescence. The HIF1 transcriptional activity from the empty pRK7 was standardized to 100%. n  6. *, p  0.05 relative to mTOR (33). We wanted to investigate whether these Rheb activity of empty pRK7 vector control. C, protein levels of S6K1 and Thr mutants, which are deficient at binding guanine nucleotide, phosphorylation to measure mTOR activation was analyzed. FLAG-Rheb expression was analyzed with anti-FLAG antibodies to show equal expression could alter the levels of HIF1 transcriptional activity during of the Rheb mutants. hypoxia. Interestingly, these Rheb mutants dominantly inhib- ited the transcriptional activity of HIF1 (Fig. 3B), when hypoxia. Interestingly, Rheb overexpression enhanced the lev- expressed to the same level as wild-type (Fig. 3C). This shows els of VEGF-A secretion during conditions of normoxia that that these Rheb mutants can sufficiently impair the basal was sensitive to rapamycin. mTOR-mediated activation of HIF1 within HEK293 cells. Small G-protein mutants that do not bind guanine nucleo- Indeed, these mutants of Rheb were unable to induce Thr tide typically form inactive complexes with their upstream reg- phosphorylation of S6K1 (Fig. 3C). ulators, i.e. the putative guanine exchange factor (GEF), which Well-characterized downstream targets of mTOR, eIF4E- impedes small G-protein-mediated signal transduction. Stud- binding protein 1 (4E-BP1) and S6K1, are recruited to mTOR ies in yeast identified dominant-negative mutants of Rheb through their association with Raptor. To examine Raptor (D60V and D60K), which were unable to efficiently bind to interaction, we utilized a Raptor overlay assay to detect Raptor- guanine nucleotides and impaired TOR signaling (31). We also interacting proteins resolved on SDS-PAGE. This Raptor overlay generated two additional Rheb(N119I) and Rheb(D122N) assay specifically detects proteins containing a TOS motif. For mutants where these two mutated residues lie within the instance, the Raptor-overlay assay only detected wild-type NKXD nucleotide binding motif of Rheb. Guanine nucleotide 4E-BP1 and not a mutant of 4E-BP1 where the phenylalanine binding assays reveal that these D60V, D60K, N119I, and within the TOS motif at position 115 was mutated to an alanine D122N mutants of Rheb are unable to bind to guanine nucleo- (Fig. 4A). To determine whether Raptor interacted with HIF1, tides in vitro (Fig. 3A). These guanine binding-deficient we carried out a Raptor overlay assay on immunoprecipitated mutants of Rheb are unable to efficiently impair acute activa- HIF1. HEK293 cells were treated with 100 M deferoxamine tion of S6K1 by insulin treatment (data not shown, and mesylate (DFX) for 24 h and then incubated with the proteoso- 20538 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR mal inhibitor MG-132 prior to lysis to increase the protein lev- els of HIF1. DFX is an iron chelator and is known to stabilize HIF1 (34). We detected the HIF1 band with the Raptor over- lay assay (Fig. 4B). This data suggests that Raptor interacts with HIF1 (Fig. 4B). To confirm this interaction, we investigated whether HIF1 co-immunoprecipitated with Raptor. HEK293 expressing HA-HIF1 and Myc-Raptor, where indicated, were treated with DMOG and then lysed in the presence of reversible crosslinker DSP and non-ionic detergent (CHAPS). Immuno- precipitated HA-HIF1 was observed to associate with Myc- Raptor (Fig. 4C). We were unable to co-purify HA-HIF1 with Myc-Raptor using immunoprecipitation buffers containing Nonidet P-40 (data not shown). Interestingly, HIF1 contains a potential conserved TOS motif (FVMVL) within an area of unknown function of HIF1 that is immediately 3 of the Period-ARNT-Sim conserved domain-A (PAS-A). We mutated the phenylalanine at position 99 within the TOS motif of HIF1 and examined whether this FIGURE 5. The HIF1(F99A) TOS motif mutant dominantly inhibits HIF1 prevented Raptor interaction (Fig. 4D). We also examined the transcription during hypoxia. A, serum-starved HEK293 cells transfected interaction of Raptor with a well-known Raptor-interacting with a HIF-inducible luciferase reporter with either empty pRK7, HA-HIF1,or HA-HIF1(F99A) in the presence of absence of FLAG-Rheb were treated with protein, 4E-BP1. HIF1 interacted less well with raptor when 50 nM rapamycin, where indicated, and transferred to a hypoxic chamber set compared with 4E-BP1, but the interaction we observed was at 1% O for 18 h. Lysates prepared in the hypoxic chamber were analyzed for luciferase fluorescence. The HIF1 transcriptional activity of the cells trans- prevented when the TOS motif within HIF1 was mutated. fected with empty pRK7 vector and FLAG-Rheb was standardized to 100%. To examine whether this motif is essential for mTOR-medi- n  6. *, p  0.05 relative to activity of empty pRK7 vector control group. **, ated HIF1 transcriptional we compared the transcriptional p  0.01 relative to activity of wild-type HA-HIF1 vector control group. B, protein levels of HA-HIF1, FLAG-Rheb, S6K1, and Thr phosphorylation activity of HIF1(F99A) to wild-type within hypoxic HEK293 of S6K1 were analyzed. cells (Fig. 5A). Mutation of the TOS motif rendered HIF1 transcriptionally inactive during hypoxia. Furthermore, overexpres- sion of HIF1(F99A) significantly reduced the activity of endogenous HIF1 showing that mutation of the TOS motif yields a dominant nega- tive HIF1 mutant. We show that equal levels of wild-type HIF1 and HIF1(F99A) are being expressed and that Rheb over-expression is sufficient to enhance mTOR-medi- ated S6K1 phosphorylation at Thr (Fig. 5B). It was postulated that mTOR sig- naling enhances the stability of HIF1 (25). To examine whether the stability of HIF1 is regulated by mTOR, we pulse-chased cells overexpressing either HA-HIF1 or HIF1(F99A) and Rheb with [S ]methionine under hypoxia (Fig. 6). If mTOR enhanced the stability of HIF1, we would expect the HIF1(F99A) mutant to be less sta- FIGURE 6. mTOR does not modulate the stability of HIF1. HEK293 cells co-expressing FLAG-Rheb with ble. We would also expect that rap- either wild-type HA-HIF1 or HA-HIF1(F99A) were grown at 1% O in media containing [ S]methionine for 4 h, which was then replaced with unlabeled media in the presence or absence of rapamycin, where indicated. mycin would reduce the stability of These cells were lysed after 0, 0.5, 1, and2hinthe1%O hypoxic chamber. Exogenous HA-HIF1 was immu- HIF1. Instead, we found that the noprecipitated with anti-HA antibodies and the relative level of [ S]methionine incorporation into HA-HIF1 protein half-life of the HIF1(F99A) was determined as described under “Experimental Procedures.” Protein levels of HA-HIF1, FLAG-Rheb, S6K1, 389 35 and Thr phosphorylation of S6K1 were also analyzed. The level of [ S]methionine-labeled HIF1 wild-type mutant was similar to that of the and HIF1(F99A) at the zero time point was standardized at 100%. n 3. There was no significant difference of 35 wild-type and treatments with rapa- [ S]methionine incorporation when comparing wild-type HIF1 with the F99A mutant or when comparing cells treated with or without rapamycin at similar time point. mycin did not alter the stability of JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20539 Regulation of HIF1 by mTOR FIGURE 7. Rapamycin inhibits HIF transcription independently of VHL. VHL MEFS were transiently transfected with a HIF-inducible luciferase reporter and either empty pRK7 or VHL vector (where indicated). These cells were maintained at 21% O for 18 h in the presence or absence of 50 nM rapamycin. Lysates were analyzed for luciferase fluorescence and the HIF transcriptional activity from the VHL cells was standardized to 100%. n 5. *, p 0.05 relative to activity of the VHL control. To detect the presence of HA-VHL, HA-VHL was immunoprecipitated from these lysates, then sub- FIGURE 8. HIF1 and HIF1 (F99A) localize to the nucleus in hypoxia. Cells jected to Western blot analysis using anti-HA antibodies. were exposed to 1% O for 18 h following transfection and then processed for immunocytochemistry. Anti HA-FITC conjugated antibody was used to either the wild-type or the F99A mutant of HIF1. This exper- observe the distribution of HA-HIF1/HIF1(F99A) expression relative to the DAPI-stained chromatin. The merged image illustrates that the HA-FITC anti- iment supports our hypothesis that mTOR does not directly body was entirely nuclear in transfected cells. Images are representative of modulate the stability of HIF1. four independent experiments. It is known that VHL is required for the ubiquitin-mediated degradation of the -subunit of HIF (35). To verify that signal- ing through mTOR does not modulate the stability of HIF through a VHL-dependent mechanism, we analyzed HIF tran- scription within human renal carcinoma VHL cells. In nor- moxic conditions these VHL-null cells possess heightened transcriptional activity of HIF (Fig. 7) showing that HIF is sta- bilized and active in these cells during high oxygen tensions. The transcriptional activity of HIF in these cells was due to the loss of VHL as the levels of HIF activity was completely ablated when VHL was transfected back. We observed that rapamycin potently reduced the levels of HIF transcription in the absence of VHL. This experiment shows that rapamycin inhibits HIF transcription independently of VHL and again supports the FIGURE 9. Co-activator p300 interaction with HIF1 is impaired in the HIF1(F99A) mutant. Serum-starved HEK293 cells transfected with empty notion that TOR does not influence the stability of HIF1. pRK7, HA-HIF1, or HA-HIF1(F99A) where transferred to a hypoxic chamber Given that mTOR does not appear to affect the stability of set at 1% O for 18 h before being lysed. Exogenous HA-HIF1 was immuno- HIF1, the increased transcriptional activity of HIF1 by Rheb precipitated with anti-HA antibodies, and the amount of co-purified p300 was quantified by Western blot analysis followed by densitometry (Image J, must be either caused by enhanced nuclear translocation of version 1.37V). Numbers below the panel indicate the ratios of the signals for HIF1 or increased binding of HIF1 to other components of p300 relative to HIF. the transcription activation complex. First of all we examined whether the cellular distribution of the HIF1(F99A) mutant these cells during normoxic and hypoxic conditions (Fig. 10). was different to that of wild-type HIF1. Confocal immunoflu- We observed that rapamycin robustly inhibited the hypoxia- orescence studies showed that both wild-type and HIF1a(F99A) induced activation of HIF in cells without TSC2 by 70%. This mutant was mainly nuclear (Fig. 8), as observed by co-localiza- rapamycin-sensitive level of HIF transcriptional activity during tion with the nuclear stain, DAPI. We next examined if the hypoxia was caused by the loss of TSC2 because transient HIF1(F99A) mutant was deficient at forming transcriptional expression of TSC2 also potently blocked the activity of HIF by complexes. To examine this possibility, we measured the inter- 70%. This result demonstrates that the loss of function of TSC2 action of p300 with both the HIF1(F99A) mutant and wild- potently drives HIF-mediated transcription during conditions type HIF1 (Fig. 9). We observed a significant loss of p300 bind- of low oxygen and is sensitive to treatments with rapamycin. ing to the HIF1(F99A) mutant, which suggests that mTOR It is known that HIF-mediated gene expression is regulated enhances HIF1 transcription through assembly of the HIF1 through PI3K- and mTOR-dependent mechanisms (35–37). transcriptional machinery. To investigate PI3K and mTOR induced activation of HIF in We wanted to examine the levels of HIF transcription within more detail, we treated HEK293 cells with insulin for 18 h in the cells lacking TSC2. It is known that the loss of function of TSC2 presence and absence of LY294002 (to inhibit PI3K) and rapa- potently enhances cell signaling through mTOR (8). We trans- mycin (to inhibit mTOR) during hypoxia. We investigated the fected TSC2 MEFS with the HIF reporter construct to activity of HIF (Fig. 11A) and the amount of VEGF-A protein measure the relative levels of HIF transcriptional activity within that was secreted by these cells (Fig. 11B). Insulin increased the 20540 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 28 •JULY 13, 2007 Regulation of HIF1 by mTOR sufficient to activate PI3K/mTOR signaling after 18 h of treat- ment. Treatments with either LY294002 or rapamycin were sufficient to block insulin-induced phosphorylation of S6K1 showing that we were inhibiting PI3K and mTOR-mediated signaling in these cells. It is important to note that LY294002 also inhibits mTOR. Therefore, the difference of inhibition we observe with rapamycin and LY294002 represents the level of HIF transcriptional activity (Fig. 11A) and VEGF-A secretion (Fig. 11B) that is dependent on PI3K. Upon insulin stimulation, we observed a marked increased in the HIF transcriptional activity within cells during hypoxia that was completely blocked by treatments with LY294002 and significantly impaired by rapamycin (Fig. 11A). Similarly, LY294002 blocked FIGURE 10. Hypoxia-induced HIF transcription in TSC2-null cells are insulin-induced VEGF-A secretion during hypoxia while highly sensitive to rapamycin. Serum-starved TSC2 MEFS were tran- VEGF-A secretion was significantly repressed by rapamycin siently transfected with a HIF-inducible luciferase reporter and either empty pRK7 or pRK7/FLAG-TSC2 (where indicated). These cells were serum-starved (Fig. 11B). These experiment shows that mTOR is necessary for and were transferred to low oxygen (1%) or maintained at 21% O , where the maximal activation of HIF-mediated transcription upon indicated, for 18 h in the presence or absence of 50 nM rapamycin. Lysates prepared were analyzed for luciferase fluorescence and the HIF transcrip- insulin stimulation. tional activity from the TSC2 cells during hypoxia was standardized to 100%. n  6. *, p and **, p  0.05 relative to activity of the TSC2 control. DISCUSSION Protein levels of FLAG-TSC2 were determined by Western blot analysis after immunoprecipitation with anti-FLAG antibodies. The asterisk indicates a non- We show that mTOR positively enhances the level of HIF- specific band that migrates above FLAG-TSC2. mediated transcription. Rheb-specific activation of mTOR enhanced the transcriptional activity of HIF during condi- tions that favored HIF1 stabilization, i.e. during hypoxia (Fig. 2A) and treatments with DMOG (Fig. 1A). This enhanced HIF activity was blocked by treatments with rapa- mycin showing that Rheb-induced HIF activity was caused by the heightened activity of mTOR. A potential function of mTOR would be to regulate the expression levels of HIF1. Dogma has it that stability is the rate-limiting factor that determines the protein levels of HIF1. However, it is important to appreciate that the regulation of HIF1 expres- sion is multifaceted with additional inputs that function at the level of transcription and translation (30, 35–37). Indeed, it is known that the translation of HIF1 can be modulated by mTOR through cap-dependent mechanisms that is driven by eIF4E and repressed by the translation repressor, 4E-BP1 (30). The reduction of endogenous HIF1 that we observe after rapamycin treatment in the presence of DMOG or hypoxia conditions (Figs. 1B and 2B, respectively), could be accountable by the reduced rates of HIF1 protein synthesis. For added complexity, the translation of HIF1 can also be maintained through an internal ribosomal entry site (IRES). IRES-mediated translation does not require eIF4E and so FIGURE 11. Insulin-induced HIF activation and VEGF-A secretion is partially confers a rapamycin-insensitive mechanism to promote blocked by rapamycin. A, serum-starved HEK293 cells transfected with a HIF- inducible luciferase reporter were transferred to low oxygen (1%) or maintained HIF1 translation during times when mTOR signaling is at 21% O . Where indicated, cells where stimulated with 100 nM insulin in the switched off (38). Given that rapamycin did not reduce the presence or absence of either 50 nM rapamycin or 50M LY294002. After 18 h, the cells were harvested, and the lysates were analyzed for luciferase fluorescence. levels of HIF1 protein in cells over-expressing Rheb (Figs. 1B The HIF1 transcriptional activity from hypoxic unstimulated cells was standard- and 2B), the reduction of Rheb-induced HIF transcriptional ized to 100%. B, levels of VEGF-A secretion within the cell media was measured as activity by rapamycin was not caused by a loss of the HIF1 pg/mg cell protein . The level of VEGF-A secretion from hypoxic-unstimulated cells was standardized to 100%. For both graphs; n  6. *, p and **, p  0.05 protein. This rules out the possibility that mTOR modulates the relative to the levels of HIF activity or VEGF-A secretion obtained from stimulated stability of HIF1 and is supported by the observation that cells during hypoxia. C, S6K1 phosphorylation was analyzed by a mobility shift on SDS-PAGE. p70 and p80 isoforms of S6K1 are marked. rapamycin treatment did not alter the rates of protein break- down of HIF1 (Fig. 6). Furthermore, rapamycin potently phosphorylation of S6K1 (as observed by a mobility shift of the impaired the activity of HIF in VHL-null cells (Fig. 7). VHL is p70 and p85 isoforms of S6K1 to the higher phosphorylated required for the ubiquitin-mediated degradation of the -sub- bands) (Fig. 11C), showing that treatment with 100 nM insulin is unit of HIF. Therefore, our data suggests that mTOR promotes JULY 13, 2007• VOLUME 282 • NUMBER 28 JOURNAL OF BIOLOGICAL CHEMISTRY 20541 Regulation of HIF1 by mTOR the transcriptional activity of HIF1 and does not involve VHL- with each of these hamartomas syndromes are known to have mediated degradation of HIF1. high levels of mTOR activation (see review in Ref. 8). Interest- The mechanism by how mTOR modulates HIF within cells ingly, we observed high levels of HIF activity in cells lacking has remained elusive to date. In this manuscript, we have iden- TSC2, which was reversed when we added back TSC2 or inhib- tified a potential FVMVL TOS motif within a previously unde- ited mTOR with rapamycin (Fig. 9). Our work suggests that the fined region of HIF1. This motif is similar to well known TOS high degree of vascularization observed in tumors arising from motifs found in both 4E-BP1 and S6K1, which are FEMDI and these syndromes could be the direct consequence of high levels FD(L/I)DL, respectively. Raptor is thought to bind to the TOS of mTOR activity. For instance, aberrant signaling through motif within mTOR substrates. This interaction recruits these mTOR would enhance the activity of HIF and encourage the substrates to mTOR for their optimal phosphorylation. For process of angiogenesis during hypoxia. instance, mutation of the TOS motif by alanine substitution of It is known that a negative feedback loop, which is activated the phenylalanine rendered both 4E-BP1 and S6K1 unrespon- by HIF, inhibits the mTOR pathway and HIF function. This sive to mTOR (14, 15). We show that raptor interacts with feedback loop is regulated by Redd1/2 (Regulated in Develop- HIF1 (Fig. 4, B and C) and this interaction is inhibited when ment and DNA damage responses), which are also referred to as the phenylalanine within the TOS motif is mutated to an ala- RTP801/801L and are transcriptionally up-regulated by HIF nine (Fig. 4D). We observed a lower level of raptor interaction (40). The inhibition of mTOR by either Redd1 or Redd2 with HIF1 when compared with 4E-BP1 (Fig. 4D) and this requires TSC2 (41) and suggests that Redd1/2 activates might be caused by the differences in the cellular distribution of TSC1/2. The mechanism by how Redd1/2 signals through both HIF1 and 4E-BP1, i.e. 4E-BP1 is cytoplasmic while HIF1 TSC2 is currently undefined. This negative feedback loop is mainly nuclear. We show that mutation of the TOS motif makes physiological sense, as it would be unfavorable for cellu- renders HIF1 transcriptionally inactive and this TOS mutant lar HIF responses to be maintained for long periods of time. In is unresponsive to enhanced mTOR signaling when Rheb is our experiments, we override this Redd1/2 negative feedback overexpressed (Fig. 5A). The HIF1 TOS mutant was predom- loop by overexpressing Rheb, which is sufficient to potently inantly nuclear (Fig. 8). These data suggest that raptor interac- enhance mTOR signaling during long term conditions of tion with HIF1 does not influence the nuclear translocation of hypoxia (Fig. 2B). Cells lacking functional TSC1/TSC2 would HIF, a process that requires the interaction of HIF with also lack this hypoxic induced negative feedback loop through HIF1. Interestingly, mutation of the TOS motif within HIF1, Redd1/2. The inability of Redd1/2 to activate the TSC1/TSC2 transforms HIF1 into a dominant negative mutant that inhib- heterodimer in TSC2-null cells might be the reason why we its endogenous HIF activity (Fig. 5A). It is possible that the observe high levels of HIF activity that is potently suppressed by HIF1(F99A) mutant sequesters the HIF subunit to form treatments with rapamycin (Fig. 9). This study suggests that inactive heterodimers, and thus competes with the interaction inhibition of mTOR might be a suitable strategy to treat har- of HIF with endogenous HIF1. Mutation of the TOS motif matomas syndromes to repress mTOR-mediated angiogenesis significantly impaired the interaction of HIF1 with the co- through HIF. activator CBP/p300 (Fig. 9) and supports our view that the HIF1(F99A) mutant is unable to form functional transcription Acknowledgments—We thank Prof. Grahame Hardie and the Divi- complexes. sion of Molecular Physiology for their support. HIF1 is a reported phosphoprotein. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Jul 13, 2007

References