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Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway

Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 31, Issue of August 2, pp. 27975–27981, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway* Received for publication, April 29, 2002 Published, JBC Papers in Press, May 24, 2002, DOI 10.1074/jbc.M204152200 Caroline Treins‡§, Sophie Giorgetti-Peraldi‡¶, Joseph Murdaca‡, Gregg L. Semenza, and Emmanuel Van Obberghen‡ From the ‡INSERM U145, Institut Fe ´de ´ratif de Recherche 50, Faculte´de Me ´decine, Avenue de Valombrose, 06107 Nice Cedex 2, France and The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914 Hypoxia-inducible factor 1 (HIF-1) is a transcription insulin receptor, insulin receptor substrates 1 and 2 are in- volved mainly in the activation of the PI3K pathway, whereas factor involved in normal mammalian development and in the pathogenesis of several disease states. It consists Shc participates in the activation of the Ras/MAPK cascade. , which is degraded during nor- of two subunits, HIF-1 The Ras/MAPK and PI3K pathways have been implicated in , which is constitutively expressed. moxia, and HIF-1 insulin-induced gene transcription (3, 4). The activated MAPK TCF Activated HIF-1 induces the expression of genes in- phosphorylates transcription factors such as p62 involved volved in angiogenesis, erythropoiesis, and glucose me- in the transcription of genes that are implicated in proliferation tabolism. We have previously reported that insulin stim- and differentiation in response to insulin (5). In contrast, insu- ulates vascular endothelial growth factor (VEGF) lin regulates the expression of genes involved in glucose me- expression (1). In this study, we show that insulin acti- tabolism through a PI3K-dependent pathway. Thus, insulin vates HIF-1, leading to VEGF expression in retinal epi- inhibits the transcription of genes encoding PEPCK, the rate- protein expression thelial cells. Insulin activates HIF-1 limiting enzyme in gluconeogenesis, and glucose-6-phospha- in a dose-dependent manner with a maximum reached tase through a PI3K pathway (6, 7). Furthermore, a PI3K-de- is correlated with within 6 h. The expression of HIF-1 pendent pathway is involved in the regulation of gene the activation of HIF-1 DNA binding activity and the expression of lipogenic enzymes by insulin such as FAS (fatty- transactivation of a HIF-1-dependent reporter gene. In- acid synthase) (8). Finally, insulin also regulates the expres- mRNA transcrip- sulin does not appear to affect HIF-1 sion of genes implicated in the angiogenic response such as protein expression through a tion but regulates HIF-1 erythropoietin (EPO) and vascular endothelial growth factor translation-dependent pathway. The expression of an (VEGF), but the molecular details of this action are lacking active form of protein kinase B and treatment of cells (9, 10). with specific inhibitors of phosphatidylinositol 3-kinase VEGF is a key angiogenic factor involved in a wide variety of (PI3K), MAPK, and target of rapamycin (TOR) show that biological processes including embryonic development, wound mainly PI3K and to a lesser extent TOR are required for expression. HIF-1 activity and insulin-induced HIF-1 healing, tumor progression, and metastasis. VEGF has VEGF expression are also dependent on PI3K- and TOR- emerged as a major mediator of intraocular neovascularization dependent signaling. In conclusion, we show here that and as such plays a key role in the etiology of diabetic retinop- insulin regulates HIF-1 action through a PI3K/TOR- athy (11). Indeed, it has been observed that intraocular VEGF dependent pathway, resulting in increased VEGF levels are increased in diabetic patients suffering from prolif- expression. erative retinopathy (12). VEGF expression is mainly regulated by tissue oxygen content (13, 14) but also by growth factors and cytokines, including platelet-derived growth factor, epidermal Insulin controls glucose and lipid metabolism, regulates pro- growth factor, insulin, insulin-like growth factor-I, tumor ne- tein synthesis, and promotes cell growth and differentiation. crosis factor , and transforming growth factor  (15–20). Hy- Following ligand binding, the insulin receptor tyrosine kinase poxia stimulates VEGF expression through at least three is activated, leading to receptor autophosphorylation and the mechanisms including increased gene transcription, regulation subsequent phosphorylation of intracellular proteins including at the translational level, and mRNA stabilization (14, 21). The insulin receptor substrates 1 and 2 and Shc. These initial transcriptional regulation of VEGF is mediated by the tran- events stimulate multiple signaling cascades that mediate cel- scription factor hypoxia-inducible factor 1 (HIF-1) (22–24). lular responses to the hormone (2). Among the substrates of the HIF-1 is a basic helix-loop-helix transcription factor, which is composed of two subunits, HIF-1 and HIF-1. HIF-1, also * This work was supported in part by INSERM, the Association pour la Recherche contre le Cancer (Grant 5492), the University of Nice- Sophia Antipolis, Aventis Pharma Deutschland GmbH Grant 99206 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; (Frankfurt, Germany), and the European Community (QLG1-CT-1999- ARPE, arising retinal pigment epithelial; EPO, erythropoietin; HIF-1, 00674). The costs of publication of this article were defrayed in part by hypoxia-inducible factor 1; MAPK, mitogen-activated protein kinase; the payment of page charges. This article must therefore be hereby MEK, mitogen-activated protein kinase/extracellular signal-regulated marked “advertisement” in accordance with 18 U.S.C. Section 1734 kinase kinase; PKB, protein kinase B; CREB, cAMP-response element- solely to indicate this fact. binding protein; E3, ubiquitin-protein isopeptide ligase; PEPCK, phos- § Recipient of a fellowship from the Ministe ` re de l’Enseignement phoenolpyruvate carboxykinase; TOR, target of rapamycin; VEGF, vas- Supe ´ rieur et de la Recherche (France). cular endothelial growth factor; CoCl , cobalt chloride; 4E-BP1, To whom correspondence should be addressed. Tel.: 33-4-93-81-54- eukaryotic translation initiation factor 4E-binding protein; PKB-myr, 47; Fax: 33-4-93-81-54-32; E-mail: [email protected]. constitutively active form of PKB. This paper is available on line at http://www.jbc.org 27975 This is an Open Access article under the CC BY license. 27976 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway Western Blot Analysis—Serum-starved cells were treated with li- known as the arylhydrocarbon nuclear translocator, is consti- gands, chilled to 4 °C, and washed with ice-cold phosphate-buffered tutively expressed, whereas HIF-1 expression is increased M NaCl, 3 mM KCl, 6 mM Na saline (140 m HPO ,1mM KH PO , pH 7.4) 2 4 2 4 upon hypoxia. In normoxia, HIF-1 is rapidly ubiquitinated by M Hepes, 150 mM NaCl, 10 mM and solubilized with lysis buffer (50 m the von Hippel-Lindau tumor suppressor E3 ligase complex EDTA, 10 mM Na P O , 100 mM NaF, 2 mM vanadate, 1 mM 4-(2- 4 2 7 and subjected to proteasomal degradation (25). Under hypoxic g/ml aprotinin, aminoethyl)benzenesulfonyl fluoride hydrochloride, 10 conditions, HIF-1 is not degraded and accumulates to form an 10 g/ml leupeptin, pH 7.4, 1% Triton X-100) for 20 min at 4 °C. The proteins were separated by SDS-PAGE and transferred by electro- active complex with HIF-1. HIF-1 regulates the transcription blotting to nitrocellulose membranes (Hybond-C, Amersham Bio- of numerous genes involved in vascular development (VEGF, sciences). The membranes were soaked first in blocking buffer (20 mM EPO, and heme oxygenase 1), in glucose and energy metabo- Tris, pH 7.4, 137 mM NaCl, 0.1% (v/v) Tween 20) containing 5% (w/v) lism (glucose transporters and glycolytic enzymes), in iron me- bovine serum albumin or nonfat milk and second in blocking buffer tabolism (transferrin), and in cell proliferation and viability containing antibodies. After the washes, the proteins were detected (insulin-like growth factor-2 and insulin-like growth factor- using horseradish peroxidase-linked secondary antibodies and en- hanced chemiluminescence according to the manufacturer’s instruc- binding protein-1). It has been shown that insulin increases tions (Amersham Biosciences). VEGF expression through a PI3K-dependent pathway in fibro- Nuclear Extract Preparation and Electrophoretic Mobility Shift As- blasts overexpressing insulin receptors (18). However, the say—The nuclear extracts were prepared as described previously (1). identity and regulation of the transcription factor involved in Sense and antisense oligonucleotides corresponding to the HIF-1 bind- this process remain unknown. ing site in the human EPO gene, 5-GATCGCCCTACGTGCTGTCTCA- Here we report that insulin stimulates HIF-1 subunit ac- 3, were used (31). The oligonucleotides were annealed, and the double- stranded oligonucleotide (10 pmol) was labeled with T4 polynucleotide cumulation, HIF-1 activation, and VEGF expression. Our re- kinase and [- P]dATP. The probe was purified with the Probequant sults show that insulin regulates HIF-1 expression through a kit. translation-dependent pathway. Moreover, insulin-induced Binding reactions were performed as described previously (32). The HIF-1 regulation and VEGF expression require a PI3K/TOR- g of nuclear extract and 0.1 g of denatured reactions contained 10 dependent pathway. salmon sperm DNA (Sigma) in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 1 mM MgCl ,1mM EDTA, 5 mM dithiothreitol, and 5% (v/v) EXPERIMENTAL PROCEDURES glycerol. After preincubation for 5 min at room temperature, the probe Materials—cDNA for VEGF was obtained from J. Plouet (Tou- (2.5  10 cpm) was added, and the incubation was continued for an louse, France). Insulin was a kind gift from Novo-Nordisk (Copenhagen, additional 15 min, after which the reaction mixtures were loaded onto Denmark). The antibody to HIF-1 (clone H167) was purchased from 5% nondenaturing polyacrylamide gels. Electrophoresis was performed M Tris-HCl, 22.25 mM boric Novus Biologicals, Inc. (Littleton, CO). The antibody to Shc was ob- at 185 V in 0.25 Tris borate EDTA (22.25 m acid, and 1.25 mM EDTA) at 4 °C. The gels were vacuum-dried, and tained from BD Transduction Laboratories (Franklin Lakes, NJ). The radioactivity was determined on a Storm 840. antibody to phospho-PKB (Ser-473) was purchased from New England HIF-1 DNA Binding Assay—The nuclear extracts were prepared Biolabs (Beverly, MA). The antibody to MEK-1 is directed against the using a nuclear extract kit (Active Motif Europe, Rixensart, Belgium) 17-amino acid amino-terminal of MEK-1 (26). according to manufacturer’s instructions. HIF-1 binding to the hypoxia All of the chemical reagents were purchased from Sigma. U0126 was response element was assessed using Trans-AM HIF transcription fac- purchased from Promega Inc. (Madison, WI). Oligonucleotides and cul- tor assay kit (Active Motif Europe). In this assay, an oligonucleotide ture media were purchased from Invitrogen. containing the HIF-1 binding site from the EPO gene is attached to a Cell Culture—Arising retinal pigment epithelia cell line-19 (ARPE- 96-well plate. The active form of HIF-1 contained in cell extracts spe- 19, ATCC CRL-2302) was grown in F12/Dulbecco’s modified Eagle’s cifically binds to this oligonucleotide and can be revealed by incubation medium containing 10% (v/v) fetal calf serum (Invitrogen) at 37 °C with with antibodies using enzyme-linked immunosorbent assay technology 5% CO . with absorbance reading. 10 g of nuclear extract were analyzed for DNA Plasmids—pGL2 basic P12 VEGF promoter corresponds to HIF-1 binding to the oligonucleotide according to the manufacturer’s nucleotides from 1005 to 906 of the human VEGF promoter cloned instructions. The specificity for this assay was monitored by competi- upstream of a luciferase-coding sequence (27). The construct PKB-myr tion with free wild type or mutated oligonucleotide according to the (constitutively active form of PKB) was a gift from B. Hemmings (F. manufacturer’s instructions. Miescher Institute, Basel, Switzerland) (28). pCEP-MEK* is a consti- Luciferase Assays—To assay the transcriptional activity of HIF-1, we tutively active form of MEK that was obtained by deleting the region used the pGL2 basic P12 VEGF promoter vector, which contains a encompassing amino acids 32–52 and by mutating the two serine res- HIF-1 binding site (from 975 to 968) downstream from the luciferase idues 218 and 222 to aspartic acid (29). gene (27). ARPE cells in 12-wells plates were transiently co-transfected Transfection—ARPE-19 cells were transiently transfected using the with the reporter plasmid and with Rous sarcoma virus--galactosidase FuGENE 6 transfection reagent (Roche Diagnostics) according to man- as a control for transfection efficiency. The cells were stimulated for ufacturer’s instructions. Sixteen hours after the addition of DNA, F12/ 16 h, and luciferase assays were performed as described in the protocols Dulbecco’s modified Eagle’s medium with 10% (v/v) fetal calf serum was and applications guide (Promega). The luciferase activity was measured changed, and before being stimulated, the cells were incubated for an using a chemiluminometer Wallac 1420 (PerkinElmer Life Sciences). additional 16 h with F12/Dulbecco’s modified Eagle’s medium with 0.2% The -galactosidase activity was performed as described in the Pro- (w/v) bovine serum albumin. mega’s protocols and applications guide. Cells lysates were incubated RNA Isolation and Northern Blot Analysis—Trizol reagent (Invitro- with a 2 assay buffer (200 mM sodium phosphate buffer, pH 7.8, 2 mM gen) was used to extract total cellular RNA from confluent cells grown MgCl in 100-mm tissue culture plates according to the manufacturer’s in- , 100 mM -mercaptoethanol, 1.33 mg/ml o-nitrophenyl -D-ga- structions. Cells were serum-deprived overnight in medium containing lactopyranoside). The absorbance at 420 nm was measured with a 0.2% (w/v) bovine serum albumin, and cells were pretreated with or not spectrophotometer Wallac 1420. with inhibitors for 30 min and stimulated for the indicated lengths of time. RNA was extracted, and 10 g of total RNA were denatured in RESULTS formamide and formaldehyde and separated by electrophoresis in form- Insulin Stimulates HIF-1 Accumulation in Human Retinal aldehyde-containing agarose gels. RNA was transferred to Hybond-N Epithelial Cells—We have previously shown that insulin in- membranes (Amersham Biosciences) and cross-linked to the membrane duces VEGF expression in cell culture and in intact animals by UV radiation. Human VEGF cDNA fragment or a PCR product encoding amino acids 330 –528 of human HIF-1 was used as a probe (1). To study the effect of the hormone on the transcription (30). The probes were labeled with [- P]dCTP by random priming factor HIF-1, we first investigated the insulin effect on HIF-1 using the Rediprime kit (Amersham Biosciences) and purified with the protein expression. ARPE-19 cells were treated for 4 h with Probequant kit (Amersham Biosciences). The hybridizations were per- insulin or with cobalt chloride (CoCl ) as a positive control. formed at 42 °C in NorthernMax hybridization buffer (Ambion, Inc, Whole cell lysates and nuclear extracts were analyzed by West- Austin, TX). The membranes were washed in 1 SSC, 0.5% (w/v) SDS, ern blotting using an antibody to HIF-1 (Fig. 1, A and B). The and radioactivity was quantitated using a Storm 840 (Amersham Biosciences). divalent metal CoCl is known to induce HIF-1 expression, 2 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway 27977 tide to assess HIF-1 DNA binding activity (Fig. 2B). HIF-1 DNA binding activity was stimulated after4hof insulin or CoCl treatment. The specificity of HIF-1 binding was tested by competition with free oligonucleotides. The binding was spe- cific, because it was abolished in the presence of an excess of wild type oligonucleotide, whereas the mutated oligonucleotide had no effect. We next measured the transcriptional activity of HIF-1 using a SV40 promoter-luciferase unit downstream of a 99-bp hy- poxia response element (pGL2 basic P12 VEGF promoter) rel- ative to co-transfected Rous sarcoma virus--galactosidase (Fig. 2C). After 16 h of insulin or CoCl treatment, the lucifer- ase activity in cell extracts was determined and normalized to the -galactosidase activity. Insulin and CoCl induced a sta- tistically significant 1.55- and 3.5-fold increase (0.0864 and 0.375, respectively) in luciferase activity, respectively. There- fore, the accumulation of HIF-1 subunit induced by insulin can be correlated with the activation of the transcription factor HIF-1. Insulin Induces HIF-1 through a Translation-dependent Pathway—To obtain a better understanding of the processes involved in HIF-1 accumulation in response to insulin treat- ment, we investigated the effect of insulin on the amount of HIF-1 mRNA. ARPE-19 cells were stimulated with insulin or CoCl for 6 h, RNA was extracted, and Northern blot analysis was performed using a HIF-1 cDNA probe (Fig. 3A). We found that insulin or CoCl treatment did not modify HIF-1 mRNA expression, suggesting that insulin does not regulate HIF-1 mRNA transcription. HIF-1 has been shown to be degraded through the proteasome pathway during normoxia. Therefore, to study the effect of insulin on HIF-1 degradation, we looked at the effect of a specific inhibitor of proteasome, MG132 (Fig. 3B). ARPE-19 cells were incubated with insulin in the absence or presence of MG132. After 4 h, insulin was removed, and the FIG.1. Insulin stimulates HIF-1 accumulation in ARPE-19 level of HIF-1 protein was evaluated by Western blot using a cells. ARPE-19 cells were stimulated with insulin (100 nM) or CoCl HIF-1 antibody. As expected, in the absence of MG132, insu- (200 M) for 4 h. Whole cell lysates (A) or nuclear extracts (B) were lin induced an accumulation of HIF-1. Within 15 min after the prepared and analyzed by Western blotting using antibody to HIF-1. Expression of HIF-1 was normalized using a Western blot with anti- removal of insulin, a decrease in HIF-1 protein could be seen, bodies to Shc or a nuclear protein CREB. ARPE-19 cells were stimu- and HIF-1 was undetectable 30 min after withdrawal of the lated with different concentrations of insulin for4h(C) or with insulin hormone. We observed that the inhibition of the proteasome by (100 nM) for the indicated time (D). Whole cell lysates were prepared MG132 led to a high level of expression of HIF-1, even in the and analyzed by Western blotting using antibodies to HIF-1 or Shc. absence of insulin stimulation. This elevated the expression of HIF-1 in the presence of the proteasome inhibitor, preventing HIF-1 DNA binding activity, and transactivation of HIF-1 tar- us from detecting any effect of insulin on HIF-1 degradation. get genes (33–36). Indeed, the CoCl treatment of cells led to an To analyze the insulin effect on HIF-1 synthesis, we per- accumulation of HIF-1 in ARPE-19 cells. Insulin induces formed a time course of HIF-1 disappearance in the presence HIF-1 expression in both whole cell lysates and in nuclear of the protein translation inhibitor, cycloheximide (Fig. 3C). To extracts. As observed in Fig. 1, C and D, HIF-1 expression was this end, ARPE-19 cells were treated with insulin or CoCl for induced by 0.1 nM insulin, and the maximal induction was seen 4 h, and cycloheximide was added for 15– 60 min. In cells at 100 nM. The expression of HIF-1 was transiently detectable exposed to CoCl , HIF-1 level remained constant over 60 min within 1 h and maximal within 6 h and then returned to basal despite the lack of ongoing protein synthesis. This observation levels within 24 h of treatment. These results show that the is consistent with previous studies showing that CoCl had no incubation of ARPE-19 cells with insulin results in a time- and effect on HIF-1 synthesis but blocked its degradation. In concentration-dependent elevation of HIF-1 protein levels. ARPE-19 cells treated with insulin, the addition of cyclohexi- Insulin Activates the Transcription Factor HIF-1—We next mide led to a decrease in HIF-1 expression starting at 15 min. determined whether insulin-induced HIF-1 accumulation was After 60 min, HIF-1 was no longer detectable. Together, these correlated with an activation of the transcription factor HIF-1. results suggest that insulin increases HIF-1 protein levels To do this, we measured the ability of HIF-1 to bind to DNA through a translation-dependent pathway. and to transactivate a HIF-1-dependent reporter gene. Constitutively Active PKB Induces HIF-1 Expression—We ARPE-19 cells were treated for 4 h with insulin or CoCl , and investigated the role of MEK- and PKB-dependent pathways in the nuclear extracts were isolated. A double-stranded oligonu- the regulation of HIF-1 in ARPE-19 cells. To this end, we cleotide containing the HIF-1 binding site present in the EPO gene was used as a probe in an electrophoretic mobility shift transfected ARPE-19 cells with pcDNA as a control with a constitutively active form of MEK (MEK*) or with a PKB-myr. assay (Fig. 2A). Both insulin and CoCl induced a shift of the The transfected cells were treated or not treated with insulin labeled probe. This binding was verified by an enzyme-linked immunosorbent assay test using an immobilized oligonucleo- (100 nM), and whole cell lysates were prepared and analyzed by 27978 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway FIG.2. Insulin activates the transcription factor HIF-1. A, ARPE-19 cells were stimulated for 4 h with insulin (100 nM) or CoCl (200 M). Nuclear extracts were prepared and analyzed by electrophoretic mobility shift assay (EMSA). 10 g of aliquots of protein were incubated with radiolabeled oligonucleotide containing the HIF-1 binding site from the EPO gene. B, ARPE-19 cells were stimulated for 4 h with insulin (Ins) (100 nM) or CoCl (200 M). Nuclear extracts were prepared, and HIF-1 binding to hypoxia response element (HRE) oligonucleotide was quantified with Trans-AM HIF-1 transcription factor assay kit. The specificity of binding was assessed by competition with free wild type (wt) or mutated (mut) oligonucleotide. The results are presented as fold stimulation, which was calculated as the ratio of the different samples to the control (untreated cells). Results shown represent the mean  S.E. of four independent experiments performed in duplicate. C, ARPE-19 cells were transfected with the pGL2 basic P12 VEGF-luciferase reporter construct and an expression vector encoding for the -galactosidase gene under the control of the Rous sarcoma virus promoter. ARPE-19 cells were serum-starved and incubated with insulin (100 nM) or CoCl (200 M) for 16 h. Luciferase activity was measured. Results are expressed as a ratio of luciferase activity over -galactosidase activity. Results shown represent the mean S.E. of 20 independent experiments performed in triplicate. Western blotting using antibody to HIF-1 (Fig. 4). The expres- insulin increased HIF-1 expression. This increase was not sion of MEK* did not affect the amount of HIF-1 protein com- affected by pretreatment with the MEK inhibitor U0126. pared with control conditions. However, the expression of PKB- Rather, the inhibition of PI3K activation by LY249002 totally myr is sufficient to increase the HIF-1 protein level in basal blocked the expression of HIF-1 in response to insulin in both conditions. In addition, when PKB-myr is expressed, insulin total cell lysates and in nuclear extracts. In addition, the treat- treatment does not further increase the level of HIF-1 protein. ment with rapamycin decreased HIF-1 protein after insulin Insulin Stimulates HIF-1 Accumulation and VEGF Expres- stimulation by half the level. We confirmed that insulin-in- sion through a PI3K/TOR-dependent pathway—To further duced activation of PI3K, MAPK, and TOR was blocked by evaluate the contribution of the PI3K pathway to the regula- treatment with the respective inhibitor (data not shown). tion of HIF-1 protein levels, we used the pharmacological To determine whether these inhibitors blocked HIF-1 activ- inhibitors of PI3K and of a downstream effector, TOR. ity, we measured the DNA binding activity of HIF-1 using an ARPE-19 cells were treated for 4 h with CoCl or with insulin enzyme-linked immunosorbent assay test. ARPE-19 cells were in the absence or presence of the inhibitors of PI3K stimulated with insulin in the absence or presence of (LY294002), TOR (rapamycin), or MEK (U0126). Whole cell LY294002, rapamycin, or U0126 (Fig. 5B). Insulin induced a lysates and nuclear extracts were prepared and analyzed by 3-fold increase in DNA binding activity of HIF-1. The inhibition Western blotting using antibodies to HIF-1, Shc, or CREB of PI3K by LY294002 totally blocked HIF-1 activation, whereas (Fig. 5A). In agreement with the results obtained in Fig. 4, the inhibition of TOR by rapamycin induced a 40% decrease in Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway 27979 FIG.4. Constitutively active PKB induces HIF-1 accumula- tion in ARPE-19 cells. ARPE-19 cells were transfected with pcDNA (control), a constitutively active form of MEK (MEK*), or PKB-myr. ARPE-19 cells were stimulated for 4 h with insulin (100 nM). Whole cell lysates were prepared and analyzed by Western blotting using antibod- ies to HIF-1, Shc, phosphorylated PKB, or MEK. FIG.3. Insulin induces HIF-1 through a translation-depend- ent pathway. A, ARPE-19 cells were stimulated with insulin (100 nM) or CoCl (200 M) for 6 h. RNA was extracted and analyzed by Northern blotting using a probe corresponding to the HIF-1 cDNA. The blot was subsequently probed with 18 S rRNA as a control. B, ARPE-19 cells were treated or not for 4 h with the proteasome inhibitor MG132 (10 mM) and stimulated with insulin (100 nM). After 4 h, insulin was removed, and cells were incubated in Dulbecco’s modified Eagle’s me- dium containing 0.2% bovine serum albumin (BSA) for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1 or Shc. C, HIF-1 expression was induced by the exposure of ARPE-19 cells to insulin (100 nM) or CoCl (200 M) for 4 h. Cycloheximide (CHX) was added to a final concentra- tion of 10 g/ml, and the cells were harvested after being incubated for the indicated time in the presence of CHX and the inducer. Whole cell lysates were prepared and analyzed by Western blotting using antibod- ies to HIF-1 or Shc. insulin-induced HIF-1 activation. As expected, MEK does not appear to be involved, because the inhibition of MEK by U0126 had no effect on HIF-1 activity in response to insulin. Thus, the extent of inhibition of HIF-1 expression by the inhibitors FIG.5. Insulin activates HIF-1 through a PI3K/TOR-dependent tightly correlated with their ability to block insulin-induced pathway. A, ARPE-19 cells were stimulated for 4 h with CoCl (200 DNA binding activity of HIF-1. M) or with insulin (100 nM) with or without pretreatment for 30 min To examine whether this correlation could be extended to the with LY294002 (50 M), U0126 (10 M), or rapamycin (50 nM). Whole HIF-1 activation and VEGF mRNA expression in response to cell lysates or nuclear extracts were prepared and analyzed by Western blotting using antibodies to HIF-1, Shc, or CREB. B, ARPE-19 cells insulin, we analyzed the VEGF mRNA expression profile after were stimulated for 4 h with insulin (100 nM) with or without pretreat- treatment with these inhibitors. ARPE-19 cells were treated ment for 30 min with LY294002 (LY) (50 M), U0126 (10 M), or with CoCl or with insulin in the absence or presence of rapamycin (Rapa) (50 nM). Nuclear extracts were prepared, and HIF-1 LY294002, U0126, or rapamycin. RNA was extracted, and binding was quantified with Trans-AM HIF-1 transcription factor assay kit. The results are presented as fold stimulation, which was calculated Northern blot analyses were performed using a VEGF cDNA as the ratio of the different samples to the control cells. Results shown probe (Fig. 6). Insulin and CoCl induced a 3- and 5-fold in- represent the mean  S.E. of two independent experiments performed crease in VEGF mRNA expression, respectively. The inhibition in duplicate. of PI3K blocked the insulin-induced VEGF mRNA expression. Moreover, rapamycin treatment resulted in a 40% inhibition. In contrast, blocking MEK did not affect the ability of insulin to be dependent on the PI3K/TOR pathway. This accumulation induce VEGF mRNA expression in these cells. In summary, the leads to HIF-1 transcriptional activity and VEGF mRNA insulin-induced accumulation of the HIF-1 subunit appears to expression. 27980 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway tion. Nevertheless, we cannot exclude the possibility that insu- lin regulates the translation of a protein, which inhibits HIF-1 degradation. It is of interest to note that a recent study shows that heregulin, which activates the tyrosine kinase receptor HER2, stimulates HIF-1 synthesis (30), similar to our results concerning insulin action. In ARPE-19 cells, we found that the insulin effect on HIF-1 expression, HIF-1 activation, and VEGF expression are de- pendent on the PI3KPKBTOR pathway. In contrast, the MEK pathway does not appear to be required for insulin action on HIF-1. Both the MAPK and PI3K pathways have been impli- cated in HIF-1 regulation. The p42 and p44 MAPKs activate HIF-1 by promoting the phosphorylation of HIF-1 in response to hypoxia and its accumulation in response to advanced gly- cation end products or mersalyl (1, 44 – 46). PI3K-dependent pathways have been implicated in HIF-1 and VEGF expression in transformed cells (47–50). Moreover, the activation of PKB or inactivation of the tumor suppressor gene encoding phos- phatase and tensin homolog deleted on chromosome 10, which dephosphorylates the PI3K reaction products phosphatidyl- FIG.6. Insulin stimulates VEGF mRNA expression through a inositol 3,4-biphosphate and phosphatidylinositol 3,4,5- PI3K/TOR-dependent pathway. ARPE-19 cells were stimulated for triphosphate, increases HIF-1 protein levels and HIF-1-de- 6 h with CoCl (200 M), or with insulin (100 nM) with or without pretreatment for 30 min with LY294002 (LY) (50 M), U0126 (10 M), or pendent reporter gene expression (30, 48, 50, 51). rapamycin (Rapa) (50 nM). RNA was extracted and analyzed by North- TOR seems to be involved only partly in the insulin action on ern blotting using a VEGF cDNA probe. The blot was subsequently HIF-1 activity, because the inhibition of TOR does not com- probed with a probe for 18 S rRNA as a control. pletely abolish HIF-1 expression and HIF-1 activation. These results suggest that at least the two following pathways are involved in insulin-induced HIF-1 regulation, a PKB-depend- DISCUSSION ent/TOR-independent pathway and a PKB/TOR-dependent Diabetic retinopathy is the major cause of blindness in West- pathway. The PKB-dependent/TOR-independent pathway re- ern countries. VEGF is involved in the pathogenesis of both mains unknown, because a direct phosphorylation of HIF-1 by background and proliferative retinopathy. Intraocular VEGF is PKB has been excluded (51). However, PKB could be involved increased in eyes from patients with blood-retinal barrier in the insulin regulation of HIF-1 translation. It has been breakdown and neovascularization. Clinical studies have dem- previously shown that insulin stimulates protein synthesis by onstrated that long term insulin therapy reduces the risk of the activation of eIF2B (eukaryotic translation initiation factor diabetic retinopathy progression. However, it has also been 2B), an essential translation initiation factor, through a PI3K/ observed that intensive insulin therapy leads to a transient PKB/glycogen synthase kinase-3 pathway (52, 53). For the worsening of retinopathy characterized by a blood-retinal bar- PKB/TOR-dependent pathway, it has been shown that TOR rier breakdown (37–39). It has been proposed that the worsen- activity positively regulates translation. Insulin induces the ing of retinopathy could be attributed to chronic hyperinsuline- phosphorylation of 4E-BP1 through a PI3KPKBTOR pathway mia induced by insulin treatment. Indeed, it has been shown (54). The phosphorylation of 4E-BP1 results in a decrease in its that insulin stimulates VEGF expression, which in turn would binding affinity for eukaryotic translation initiation factor 4E, stimulate neovascularization (40, 41). However, the molecular an essential translation initiation factor. The subsequent dis- mechanisms involved in insulin-induced VEGF expression re- sociation of eIF-4E (eukaryotic translation initiation factor) main unknown. In this study, we show that insulin stimulates from 4E-BP1 promotes cap structure-dependent translation VEGF expression through the activation of the transcription initiation (55, 56). We could hypothesize that insulin-activated factor HIF-1. This activation is regulated by a PI3K-dependent TOR by phosphorylation of 4E-BP1 dissociates eukaryotic signaling pathway involving TOR. Moreover, in contrast to translation initiation factor 4E from 4E-BP1 and stimulates hypoxia, which is a major activator of HIF-1, insulin does not the translation initiation of the HIF-1 mRNA. regulate HIF-1 through the inhibition of its degradation but In diabetes, several factors could be involved in the worsen- through a translation-dependent mechanism. ing of the diabetic retinopathy including (i) advanced glycation In ARPE-19 cells, insulin stimulates the accumulation of the end products generated during hyperglycemia, (ii) hypoxia re- regulated subunit HIF-1. An increase in HIF-1 expression is sulting from microvascular retinal occlusion, and (iii) hyperin- directly correlated with the activity of the transcription factor sulinemia stimulating VEGF expression through the up-regu- HIF-1. Indeed, we show that insulin induces increased HIF-1 lation of HIF-1 expression (1, 57). Moreover, the co-treatment protein levels, augmented HIF-1 DNA binding activity, and of retinal epithelial cells with both insulin and advanced gly- stimulation of HIF-1-mediated reporter gene transcription. In cation end products increases VEGF expression (1). The ad- normoxic conditions, HIF-1 is maintained at low levels by a vanced glycation end products and insulin activate HIF-1 degradation process involving the ubiquitin-proteasome sys- tem (42, 43). Hypoxia rapidly increases the amount of HIF-1 through distinct pathways, because advanced glycation end- induced HIF-1 activation is dependent on MAPK, whereas by inhibiting its proteasome-dependent degradation. Surpris- ingly, insulin does not seem to act on HIF-1 degradation. A insulin-induced HIF-1 activation is dependent on PI3K. Hy- poxia blocks HIF-1 degradation, whereas growth factors act- comparison of the half-life of HIF-1 after the removal of insu- lin or in presence of both insulin and cycloheximide, a transla- ing through tyrosine kinase receptors would increase its syn- tion inhibitor, shows that insulin does not stabilize the HIF-1 thesis. The combination of these different signals enhances the protein. Furthermore, insulin does not affect the transcription activation of the transcription factor leading to increased of HIF-1 mRNA, suggesting that it regulates HIF-1 transla- VEGF gene expression. The result would be an amplification of Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway 27981 P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Science 292, 468 – 472 the angiogenic signal leading to further progression of diabetic 26. Frodin, M., Peraldi, P., and Van Obberghen, E. (1994) J. Biol. Chem. 269, retinopathy. 6207– 6214 27. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., In conclusion, we have shown that insulin activates the and Semenza, G. L. (1996) Mol. Cell. Biol. 16, 4604 – 4613 transcription factor HIF-1 through a PI3KPKBTOR-depend- 28. Andjelkovic, M., Alessi, D. R., Meier, R., Fernandez, A., Lamb, N. J., Frech, M., ent pathway. Ours results suggest that insulin similar to Cron, P., Cohen, P., Lucocq, J. M., and Hemmings, B. A. (1997) J. Biol. Chem. 272, 31515–31524 heregulin regulates HIF-1 synthesis. It remains to estab- 29. Tanti, J. F., Gremeaux, T., Grillo, S., Calleja, V., Klippel, A., Williams, L. T., lished whether such an effect on HIF-1 synthesis is a general Van Obberghen, E., and Le Marchand-Brustel, Y. (1996) J. Biol. Chem. 271, feature of receptor tyrosine kinases. 25227–25232 30. Laughner, E., Taghavi, P., Chiles, K., Mahon, P. C., and Semenza, G. L. (2001) Mol. Cell. Biol. 21, 3995– 4004 Acknowledgments—We thank P. Peraldi for critical reading of this 31. Wang, G. L., and Semenza, G. L. (1995) J. Biol. Chem. 270, 1230 –1237 paper. We also thank J. Plouet (Toulouse, France) for VEGF cDNA 32. Semenza, G. L., and Wang, G. L. (1992) Mol. Cell. Biol. 12, 5447–5454 and B. Hemmings, S. Tartare-Deckert, and M. Montminy for the gift of 33. Wang, G. L., and Semenza, G. L. (1993) Blood 82, 3610 –3615 antibodies. 34. Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5510 –5514 REFERENCES 35. Jiang, B. H., Zheng, J. Z., Leung, S. W., Roe, R., and Semenza, G. L. (1997) J. Biol. Chem. 272, 19253–19260 1. Treins, C., Giorgetti-Peraldi, S., Murdaca, J., and Van Obberghen, E. (2001) J. Biol. Chem. 276, 43836 – 43841 36. Pugh, C. W., O’Rourke, J. F., Nagao, M., Gleadle, J. M., and Ratcliffe, P. J. (1997) J. Biol. Chem. 272, 11205–11214 2. Withers, D. J., and White, M. (2000) Endocrinology 141, 1917–1921 3. Saltiel, A. R., and Pessin, J. E. (2002) Trends Cell Biol. 12, 65–71 37. Agardh, C. D., Eckert, B., and Agardh, E. (1992) J. Diabetes Complications 6, 96 –100 4. Van Obberghen, E., Baron, V., Delahaye, L., Emanuelli, B., Filippa, N., Giorgetti-Peraldi, S., Lebrun, P., Mothe-Satney, I., Peraldi, P., Rocchi, S., 38. Brinchmann-Hansen, O., Dahl-Jorgensen, K., Sandvik, L., and Hanssen, K. F. (1992) Br. Med. J. 304, 19 –22 Sawka-Verhelle, D., Tartare-Deckert, S., and Giudicelli, J. (2001) Eur. J. Clin. Invest. 31, 966 –977 39. Moskalets, E., Galstyan, G., Starostina, E., Antsiferov, M., and Chantelau, E. (1994) J Diabetes Complications 8, 45–50 5. Saltiel, A. R., and Kahn, C. R. (2001) Nature 414, 799 – 806 6. Sutherland, C., O’Brien, R. M., and Granner, D. K. (1995) J. Biol. Chem. 270, 40. Poulaki, V., Qin, W., Joussen, A. M., Hurlbut, P., Weigand, S. J., Rudge, J., Yancopoulos, G. D., and Adamis, A. P. (2002) J. Clin. Invest. 109, 805– 815 15501–15506 7. Dickens, M., Svitek, C. A., Culbert, A. A., O’Brien, R. M., and Tavare, J. M. 41. Zelzer, E., Levy, Y., Kahana, C., Shilo, B. Z., Rubinstein, M., and Cohen, B. (1998) J. Biol. Chem. 273, 20144 –20149 (1998) EMBO J. 17, 5085–5094 8. Wang, D., and Sul, H. S. (1998) J. Biol. Chem. 273, 25420 –25426 42. Salceda, S., and Caro, J. (1997) J. Biol. Chem. 272, 22642–22647 9. Masuda, S., Chikuma, M., and Sasaki, R. (1997) Brain Res. 746, 63–70 43. Huang, L. E., Gu, J., Schau, M., and Bunn, H. F. (1998) Proc. Natl. Acad. Sci. 10. Lu, M., Amano, S., Miyamoto, K., Garland, R., Keough, K., Qin, W., and U. S. A. 95, 7987–7992 Adamis, A. P. (1999) Invest. Ophthalmol. Visual Sci. 40, 3281–3286 44. Richard, D. E., Berra, E., Gothie, E., Roux, D., and Pouyssegur, J. (1999) 11. Duh, E., and Aiello, L. P. (1999) Diabetes 48, 1899 –1906 J. Biol. Chem. 274, 32631–32637 12. Funatsu, H., Yamashita, H., Noma, H., Shimizu, E., Yamashita, T., and Hori, 45. Minet, E., Arnould, T., Michel, G., Roland, I., Mottet, D., Raes, M., Remacle, J., S. (2001) Jpn. J. Ophthalmol. 45, 577–584 and Michiels, C. (2000) FEBS Lett. 468, 53–58 13. Ikeda, E., Achen, M. G., Breier, G., and Risau, W. (1995) J. Biol. Chem. 270, 46. Agani, F., and Semenza, G. L. (1998) Mol. Pharmacol. 54, 749 –754 19761–19766 47. Jiang, B. H., Zheng, J. Z., Aoki, M., and Vogt, P. K. (2000) Proc. Natl. Acad. Sci. 14. Levy, A. P., Levy, N. S., Wegner, S., and Goldberg, M. A. (1995) J. Biol. Chem. U. S. A. 97, 1749 –1753 270, 13333–13340 48. Zhong, H., Chiles, K., Feldser, D., Laughner, E., Hanrahan, C., Georgescu, 15. Wang, D., Huang, H. J., Kazlauskas, A., and Cavenee, W. K. (1999) Cancer M. M., Simons, J. W., and Semenza, G. L. (2000) Cancer Res. 60, 1541–1545 Res. 59, 1464 –1472 49. Blancher, C., Moore, J. W., Robertson, N., and Harris, A. L. (2001) Cancer Res. 16. Maity, A., Pore, N., Lee, J., Solomon, D., and O’Rourke, D. M. (2000) Cancer 61, 7349 –7355 Res. 60, 5879 –5886 50. Jiang, B. H., Jiang, G., Zheng, J. Z., Lu, Z., Hunter, T., and Vogt, P. K. (2001) 17. Goldman, C. K., Kim, J., Wong, W. L., King, V., Brock, T., and Gillespie, G. Y. Cell Growth Differ. 12, 363–369 (1993) Mol. Biol. Cell 4, 121–133 51. Zundel, W., Schindler, C., Haas-Kogan, D., Koong, A., Kaper, F., Chen, E., 18. Miele, C., Rochford, J. J., Filippa, N., Giorgetti-Peraldi, S., and Van Gottschalk, A. R., Ryan, H. E., Johnson, R. S., Jefferson, A. B., Stokoe, D., Obberghen, E. (2000) J. Biol. Chem. 275, 21695–21702 and Giaccia, A. J. (2000) Genes Dev. 14, 391–396 19. Punglia, R. S., Lu, M., Hsu, J., Kuroki, M., Tolentino, M. J., Keough, K., Levy, 52. Welsh, G. I., Stokes, C. M., Wang, X., Sakaue, H., Ogawa, W., Kasuga, M., and A. P., Levy, N. S., Goldberg, M. A., D’Amato, R. J., and Adamis, A. P. (1997) Proud, C. G. (1997) FEBS Lett. 410, 418 – 422 Diabetes 46, 1619 –1626 53. Welsh, G. I., Miller, C. M., Loughlin, A. J., Price, N. T., and Proud, C. G. (1998) 20. Ryuto, M., Ono, M., Izumi, H., Yoshida, S., Weich, H. A., Kohno, K., and FEBS Lett. 421, 125–130 Kuwano, M. (1996) J. Biol. Chem. 271, 28220 –28228 54. Takata, M., Ogawa, W., Kitamura, T., Hino, Y., Kuroda, S., Kotani, K., Klip, 21. Stein, I., Neeman, M., Shweiki, D., Itin, A., and Keshet, E. (1995) Mol. Cell. A., Gingras, A. C., Sonenberg, N., and Kasuga, M. (1999) J. Biol. Chem. 274, Biol. 15, 5363–5368 20611–20618 22. Wang, G. L., and Semenza, G. L. (1993) J. Biol. Chem. 268, 21513–21518 55. Sonenberg, N., and Gingras, A. C. (1998) Curr. Opin. Cell Biol. 10, 268 –275 23. Semenza, G. L. (1998) Curr. Opin. Genet. Dev. 8, 588 –594 56. Lawrence, J. C., Fadden, P., Haystead, T. A., and Lin, T. A. (1997) Adv. 24. Semenza, G. L. (2000) J. Appl. Physiol. 88, 1474 –1480 Enzyme Regul. 37, 239 –267 25. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., 57. Pe’er, J., Folberg, R., Itin, A., Gnessin, H., Hemo, I., and Keshet, E. (1998) Kriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, Ophthalmology 105, 412– 416 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 31, Issue of August 2, pp. 27975–27981, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Insulin Stimulates Hypoxia-inducible Factor 1 through a Phosphatidylinositol 3-Kinase/Target of Rapamycin-dependent Signaling Pathway* Received for publication, April 29, 2002 Published, JBC Papers in Press, May 24, 2002, DOI 10.1074/jbc.M204152200 Caroline Treins‡§, Sophie Giorgetti-Peraldi‡¶, Joseph Murdaca‡, Gregg L. Semenza, and Emmanuel Van Obberghen‡ From the ‡INSERM U145, Institut Fe ´de ´ratif de Recherche 50, Faculte´de Me ´decine, Avenue de Valombrose, 06107 Nice Cedex 2, France and The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-3914 Hypoxia-inducible factor 1 (HIF-1) is a transcription insulin receptor, insulin receptor substrates 1 and 2 are in- volved mainly in the activation of the PI3K pathway, whereas factor involved in normal mammalian development and in the pathogenesis of several disease states. It consists Shc participates in the activation of the Ras/MAPK cascade. , which is degraded during nor- of two subunits, HIF-1 The Ras/MAPK and PI3K pathways have been implicated in , which is constitutively expressed. moxia, and HIF-1 insulin-induced gene transcription (3, 4). The activated MAPK TCF Activated HIF-1 induces the expression of genes in- phosphorylates transcription factors such as p62 involved volved in angiogenesis, erythropoiesis, and glucose me- in the transcription of genes that are implicated in proliferation tabolism. We have previously reported that insulin stim- and differentiation in response to insulin (5). In contrast, insu- ulates vascular endothelial growth factor (VEGF) lin regulates the expression of genes involved in glucose me- expression (1). In this study, we show that insulin acti- tabolism through a PI3K-dependent pathway. Thus, insulin vates HIF-1, leading to VEGF expression in retinal epi- inhibits the transcription of genes encoding PEPCK, the rate- protein expression thelial cells. Insulin activates HIF-1 limiting enzyme in gluconeogenesis, and glucose-6-phospha- in a dose-dependent manner with a maximum reached tase through a PI3K pathway (6, 7). Furthermore, a PI3K-de- is correlated with within 6 h. The expression of HIF-1 pendent pathway is involved in the regulation of gene the activation of HIF-1 DNA binding activity and the expression of lipogenic enzymes by insulin such as FAS (fatty- transactivation of a HIF-1-dependent reporter gene. In- acid synthase) (8). Finally, insulin also regulates the expres- mRNA transcrip- sulin does not appear to affect HIF-1 sion of genes implicated in the angiogenic response such as protein expression through a tion but regulates HIF-1 erythropoietin (EPO) and vascular endothelial growth factor translation-dependent pathway. The expression of an (VEGF), but the molecular details of this action are lacking active form of protein kinase B and treatment of cells (9, 10). with specific inhibitors of phosphatidylinositol 3-kinase VEGF is a key angiogenic factor involved in a wide variety of (PI3K), MAPK, and target of rapamycin (TOR) show that biological processes including embryonic development, wound mainly PI3K and to a lesser extent TOR are required for expression. HIF-1 activity and insulin-induced HIF-1 healing, tumor progression, and metastasis. VEGF has VEGF expression are also dependent on PI3K- and TOR- emerged as a major mediator of intraocular neovascularization dependent signaling. In conclusion, we show here that and as such plays a key role in the etiology of diabetic retinop- insulin regulates HIF-1 action through a PI3K/TOR- athy (11). Indeed, it has been observed that intraocular VEGF dependent pathway, resulting in increased VEGF levels are increased in diabetic patients suffering from prolif- expression. erative retinopathy (12). VEGF expression is mainly regulated by tissue oxygen content (13, 14) but also by growth factors and cytokines, including platelet-derived growth factor, epidermal Insulin controls glucose and lipid metabolism, regulates pro- growth factor, insulin, insulin-like growth factor-I, tumor ne- tein synthesis, and promotes cell growth and differentiation. crosis factor , and transforming growth factor  (15–20). Hy- Following ligand binding, the insulin receptor tyrosine kinase poxia stimulates VEGF expression through at least three is activated, leading to receptor autophosphorylation and the mechanisms including increased gene transcription, regulation subsequent phosphorylation of intracellular proteins including at the translational level, and mRNA stabilization (14, 21). The insulin receptor substrates 1 and 2 and Shc. These initial transcriptional regulation of VEGF is mediated by the tran- events stimulate multiple signaling cascades that mediate cel- scription factor hypoxia-inducible factor 1 (HIF-1) (22–24). lular responses to the hormone (2). Among the substrates of the HIF-1 is a basic helix-loop-helix transcription factor, which is composed of two subunits, HIF-1 and HIF-1. HIF-1, also * This work was supported in part by INSERM, the Association pour la Recherche contre le Cancer (Grant 5492), the University of Nice- Sophia Antipolis, Aventis Pharma Deutschland GmbH Grant 99206 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; (Frankfurt, Germany), and the European Community (QLG1-CT-1999- ARPE, arising retinal pigment epithelial; EPO, erythropoietin; HIF-1, 00674). The costs of publication of this article were defrayed in part by hypoxia-inducible factor 1; MAPK, mitogen-activated protein kinase; the payment of page charges. This article must therefore be hereby MEK, mitogen-activated protein kinase/extracellular signal-regulated marked “advertisement” in accordance with 18 U.S.C. Section 1734 kinase kinase; PKB, protein kinase B; CREB, cAMP-response element- solely to indicate this fact. binding protein; E3, ubiquitin-protein isopeptide ligase; PEPCK, phos- § Recipient of a fellowship from the Ministe ` re de l’Enseignement phoenolpyruvate carboxykinase; TOR, target of rapamycin; VEGF, vas- Supe ´ rieur et de la Recherche (France). cular endothelial growth factor; CoCl , cobalt chloride; 4E-BP1, To whom correspondence should be addressed. Tel.: 33-4-93-81-54- eukaryotic translation initiation factor 4E-binding protein; PKB-myr, 47; Fax: 33-4-93-81-54-32; E-mail: [email protected]. constitutively active form of PKB. This paper is available on line at http://www.jbc.org 27975 This is an Open Access article under the CC BY license. 27976 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway Western Blot Analysis—Serum-starved cells were treated with li- known as the arylhydrocarbon nuclear translocator, is consti- gands, chilled to 4 °C, and washed with ice-cold phosphate-buffered tutively expressed, whereas HIF-1 expression is increased M NaCl, 3 mM KCl, 6 mM Na saline (140 m HPO ,1mM KH PO , pH 7.4) 2 4 2 4 upon hypoxia. In normoxia, HIF-1 is rapidly ubiquitinated by M Hepes, 150 mM NaCl, 10 mM and solubilized with lysis buffer (50 m the von Hippel-Lindau tumor suppressor E3 ligase complex EDTA, 10 mM Na P O , 100 mM NaF, 2 mM vanadate, 1 mM 4-(2- 4 2 7 and subjected to proteasomal degradation (25). Under hypoxic g/ml aprotinin, aminoethyl)benzenesulfonyl fluoride hydrochloride, 10 conditions, HIF-1 is not degraded and accumulates to form an 10 g/ml leupeptin, pH 7.4, 1% Triton X-100) for 20 min at 4 °C. The proteins were separated by SDS-PAGE and transferred by electro- active complex with HIF-1. HIF-1 regulates the transcription blotting to nitrocellulose membranes (Hybond-C, Amersham Bio- of numerous genes involved in vascular development (VEGF, sciences). The membranes were soaked first in blocking buffer (20 mM EPO, and heme oxygenase 1), in glucose and energy metabo- Tris, pH 7.4, 137 mM NaCl, 0.1% (v/v) Tween 20) containing 5% (w/v) lism (glucose transporters and glycolytic enzymes), in iron me- bovine serum albumin or nonfat milk and second in blocking buffer tabolism (transferrin), and in cell proliferation and viability containing antibodies. After the washes, the proteins were detected (insulin-like growth factor-2 and insulin-like growth factor- using horseradish peroxidase-linked secondary antibodies and en- hanced chemiluminescence according to the manufacturer’s instruc- binding protein-1). It has been shown that insulin increases tions (Amersham Biosciences). VEGF expression through a PI3K-dependent pathway in fibro- Nuclear Extract Preparation and Electrophoretic Mobility Shift As- blasts overexpressing insulin receptors (18). However, the say—The nuclear extracts were prepared as described previously (1). identity and regulation of the transcription factor involved in Sense and antisense oligonucleotides corresponding to the HIF-1 bind- this process remain unknown. ing site in the human EPO gene, 5-GATCGCCCTACGTGCTGTCTCA- Here we report that insulin stimulates HIF-1 subunit ac- 3, were used (31). The oligonucleotides were annealed, and the double- stranded oligonucleotide (10 pmol) was labeled with T4 polynucleotide cumulation, HIF-1 activation, and VEGF expression. Our re- kinase and [- P]dATP. The probe was purified with the Probequant sults show that insulin regulates HIF-1 expression through a kit. translation-dependent pathway. Moreover, insulin-induced Binding reactions were performed as described previously (32). The HIF-1 regulation and VEGF expression require a PI3K/TOR- g of nuclear extract and 0.1 g of denatured reactions contained 10 dependent pathway. salmon sperm DNA (Sigma) in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 50 mM NaCl, 1 mM MgCl ,1mM EDTA, 5 mM dithiothreitol, and 5% (v/v) EXPERIMENTAL PROCEDURES glycerol. After preincubation for 5 min at room temperature, the probe Materials—cDNA for VEGF was obtained from J. Plouet (Tou- (2.5  10 cpm) was added, and the incubation was continued for an louse, France). Insulin was a kind gift from Novo-Nordisk (Copenhagen, additional 15 min, after which the reaction mixtures were loaded onto Denmark). The antibody to HIF-1 (clone H167) was purchased from 5% nondenaturing polyacrylamide gels. Electrophoresis was performed M Tris-HCl, 22.25 mM boric Novus Biologicals, Inc. (Littleton, CO). The antibody to Shc was ob- at 185 V in 0.25 Tris borate EDTA (22.25 m acid, and 1.25 mM EDTA) at 4 °C. The gels were vacuum-dried, and tained from BD Transduction Laboratories (Franklin Lakes, NJ). The radioactivity was determined on a Storm 840. antibody to phospho-PKB (Ser-473) was purchased from New England HIF-1 DNA Binding Assay—The nuclear extracts were prepared Biolabs (Beverly, MA). The antibody to MEK-1 is directed against the using a nuclear extract kit (Active Motif Europe, Rixensart, Belgium) 17-amino acid amino-terminal of MEK-1 (26). according to manufacturer’s instructions. HIF-1 binding to the hypoxia All of the chemical reagents were purchased from Sigma. U0126 was response element was assessed using Trans-AM HIF transcription fac- purchased from Promega Inc. (Madison, WI). Oligonucleotides and cul- tor assay kit (Active Motif Europe). In this assay, an oligonucleotide ture media were purchased from Invitrogen. containing the HIF-1 binding site from the EPO gene is attached to a Cell Culture—Arising retinal pigment epithelia cell line-19 (ARPE- 96-well plate. The active form of HIF-1 contained in cell extracts spe- 19, ATCC CRL-2302) was grown in F12/Dulbecco’s modified Eagle’s cifically binds to this oligonucleotide and can be revealed by incubation medium containing 10% (v/v) fetal calf serum (Invitrogen) at 37 °C with with antibodies using enzyme-linked immunosorbent assay technology 5% CO . with absorbance reading. 10 g of nuclear extract were analyzed for DNA Plasmids—pGL2 basic P12 VEGF promoter corresponds to HIF-1 binding to the oligonucleotide according to the manufacturer’s nucleotides from 1005 to 906 of the human VEGF promoter cloned instructions. The specificity for this assay was monitored by competi- upstream of a luciferase-coding sequence (27). The construct PKB-myr tion with free wild type or mutated oligonucleotide according to the (constitutively active form of PKB) was a gift from B. Hemmings (F. manufacturer’s instructions. Miescher Institute, Basel, Switzerland) (28). pCEP-MEK* is a consti- Luciferase Assays—To assay the transcriptional activity of HIF-1, we tutively active form of MEK that was obtained by deleting the region used the pGL2 basic P12 VEGF promoter vector, which contains a encompassing amino acids 32–52 and by mutating the two serine res- HIF-1 binding site (from 975 to 968) downstream from the luciferase idues 218 and 222 to aspartic acid (29). gene (27). ARPE cells in 12-wells plates were transiently co-transfected Transfection—ARPE-19 cells were transiently transfected using the with the reporter plasmid and with Rous sarcoma virus--galactosidase FuGENE 6 transfection reagent (Roche Diagnostics) according to man- as a control for transfection efficiency. The cells were stimulated for ufacturer’s instructions. Sixteen hours after the addition of DNA, F12/ 16 h, and luciferase assays were performed as described in the protocols Dulbecco’s modified Eagle’s medium with 10% (v/v) fetal calf serum was and applications guide (Promega). The luciferase activity was measured changed, and before being stimulated, the cells were incubated for an using a chemiluminometer Wallac 1420 (PerkinElmer Life Sciences). additional 16 h with F12/Dulbecco’s modified Eagle’s medium with 0.2% The -galactosidase activity was performed as described in the Pro- (w/v) bovine serum albumin. mega’s protocols and applications guide. Cells lysates were incubated RNA Isolation and Northern Blot Analysis—Trizol reagent (Invitro- with a 2 assay buffer (200 mM sodium phosphate buffer, pH 7.8, 2 mM gen) was used to extract total cellular RNA from confluent cells grown MgCl in 100-mm tissue culture plates according to the manufacturer’s in- , 100 mM -mercaptoethanol, 1.33 mg/ml o-nitrophenyl -D-ga- structions. Cells were serum-deprived overnight in medium containing lactopyranoside). The absorbance at 420 nm was measured with a 0.2% (w/v) bovine serum albumin, and cells were pretreated with or not spectrophotometer Wallac 1420. with inhibitors for 30 min and stimulated for the indicated lengths of time. RNA was extracted, and 10 g of total RNA were denatured in RESULTS formamide and formaldehyde and separated by electrophoresis in form- Insulin Stimulates HIF-1 Accumulation in Human Retinal aldehyde-containing agarose gels. RNA was transferred to Hybond-N Epithelial Cells—We have previously shown that insulin in- membranes (Amersham Biosciences) and cross-linked to the membrane duces VEGF expression in cell culture and in intact animals by UV radiation. Human VEGF cDNA fragment or a PCR product encoding amino acids 330 –528 of human HIF-1 was used as a probe (1). To study the effect of the hormone on the transcription (30). The probes were labeled with [- P]dCTP by random priming factor HIF-1, we first investigated the insulin effect on HIF-1 using the Rediprime kit (Amersham Biosciences) and purified with the protein expression. ARPE-19 cells were treated for 4 h with Probequant kit (Amersham Biosciences). The hybridizations were per- insulin or with cobalt chloride (CoCl ) as a positive control. formed at 42 °C in NorthernMax hybridization buffer (Ambion, Inc, Whole cell lysates and nuclear extracts were analyzed by West- Austin, TX). The membranes were washed in 1 SSC, 0.5% (w/v) SDS, ern blotting using an antibody to HIF-1 (Fig. 1, A and B). The and radioactivity was quantitated using a Storm 840 (Amersham Biosciences). divalent metal CoCl is known to induce HIF-1 expression, 2 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway 27977 tide to assess HIF-1 DNA binding activity (Fig. 2B). HIF-1 DNA binding activity was stimulated after4hof insulin or CoCl treatment. The specificity of HIF-1 binding was tested by competition with free oligonucleotides. The binding was spe- cific, because it was abolished in the presence of an excess of wild type oligonucleotide, whereas the mutated oligonucleotide had no effect. We next measured the transcriptional activity of HIF-1 using a SV40 promoter-luciferase unit downstream of a 99-bp hy- poxia response element (pGL2 basic P12 VEGF promoter) rel- ative to co-transfected Rous sarcoma virus--galactosidase (Fig. 2C). After 16 h of insulin or CoCl treatment, the lucifer- ase activity in cell extracts was determined and normalized to the -galactosidase activity. Insulin and CoCl induced a sta- tistically significant 1.55- and 3.5-fold increase (0.0864 and 0.375, respectively) in luciferase activity, respectively. There- fore, the accumulation of HIF-1 subunit induced by insulin can be correlated with the activation of the transcription factor HIF-1. Insulin Induces HIF-1 through a Translation-dependent Pathway—To obtain a better understanding of the processes involved in HIF-1 accumulation in response to insulin treat- ment, we investigated the effect of insulin on the amount of HIF-1 mRNA. ARPE-19 cells were stimulated with insulin or CoCl for 6 h, RNA was extracted, and Northern blot analysis was performed using a HIF-1 cDNA probe (Fig. 3A). We found that insulin or CoCl treatment did not modify HIF-1 mRNA expression, suggesting that insulin does not regulate HIF-1 mRNA transcription. HIF-1 has been shown to be degraded through the proteasome pathway during normoxia. Therefore, to study the effect of insulin on HIF-1 degradation, we looked at the effect of a specific inhibitor of proteasome, MG132 (Fig. 3B). ARPE-19 cells were incubated with insulin in the absence or presence of MG132. After 4 h, insulin was removed, and the FIG.1. Insulin stimulates HIF-1 accumulation in ARPE-19 level of HIF-1 protein was evaluated by Western blot using a cells. ARPE-19 cells were stimulated with insulin (100 nM) or CoCl HIF-1 antibody. As expected, in the absence of MG132, insu- (200 M) for 4 h. Whole cell lysates (A) or nuclear extracts (B) were lin induced an accumulation of HIF-1. Within 15 min after the prepared and analyzed by Western blotting using antibody to HIF-1. Expression of HIF-1 was normalized using a Western blot with anti- removal of insulin, a decrease in HIF-1 protein could be seen, bodies to Shc or a nuclear protein CREB. ARPE-19 cells were stimu- and HIF-1 was undetectable 30 min after withdrawal of the lated with different concentrations of insulin for4h(C) or with insulin hormone. We observed that the inhibition of the proteasome by (100 nM) for the indicated time (D). Whole cell lysates were prepared MG132 led to a high level of expression of HIF-1, even in the and analyzed by Western blotting using antibodies to HIF-1 or Shc. absence of insulin stimulation. This elevated the expression of HIF-1 in the presence of the proteasome inhibitor, preventing HIF-1 DNA binding activity, and transactivation of HIF-1 tar- us from detecting any effect of insulin on HIF-1 degradation. get genes (33–36). Indeed, the CoCl treatment of cells led to an To analyze the insulin effect on HIF-1 synthesis, we per- accumulation of HIF-1 in ARPE-19 cells. Insulin induces formed a time course of HIF-1 disappearance in the presence HIF-1 expression in both whole cell lysates and in nuclear of the protein translation inhibitor, cycloheximide (Fig. 3C). To extracts. As observed in Fig. 1, C and D, HIF-1 expression was this end, ARPE-19 cells were treated with insulin or CoCl for induced by 0.1 nM insulin, and the maximal induction was seen 4 h, and cycloheximide was added for 15– 60 min. In cells at 100 nM. The expression of HIF-1 was transiently detectable exposed to CoCl , HIF-1 level remained constant over 60 min within 1 h and maximal within 6 h and then returned to basal despite the lack of ongoing protein synthesis. This observation levels within 24 h of treatment. These results show that the is consistent with previous studies showing that CoCl had no incubation of ARPE-19 cells with insulin results in a time- and effect on HIF-1 synthesis but blocked its degradation. In concentration-dependent elevation of HIF-1 protein levels. ARPE-19 cells treated with insulin, the addition of cyclohexi- Insulin Activates the Transcription Factor HIF-1—We next mide led to a decrease in HIF-1 expression starting at 15 min. determined whether insulin-induced HIF-1 accumulation was After 60 min, HIF-1 was no longer detectable. Together, these correlated with an activation of the transcription factor HIF-1. results suggest that insulin increases HIF-1 protein levels To do this, we measured the ability of HIF-1 to bind to DNA through a translation-dependent pathway. and to transactivate a HIF-1-dependent reporter gene. Constitutively Active PKB Induces HIF-1 Expression—We ARPE-19 cells were treated for 4 h with insulin or CoCl , and investigated the role of MEK- and PKB-dependent pathways in the nuclear extracts were isolated. A double-stranded oligonu- the regulation of HIF-1 in ARPE-19 cells. To this end, we cleotide containing the HIF-1 binding site present in the EPO gene was used as a probe in an electrophoretic mobility shift transfected ARPE-19 cells with pcDNA as a control with a constitutively active form of MEK (MEK*) or with a PKB-myr. assay (Fig. 2A). Both insulin and CoCl induced a shift of the The transfected cells were treated or not treated with insulin labeled probe. This binding was verified by an enzyme-linked immunosorbent assay test using an immobilized oligonucleo- (100 nM), and whole cell lysates were prepared and analyzed by 27978 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway FIG.2. Insulin activates the transcription factor HIF-1. A, ARPE-19 cells were stimulated for 4 h with insulin (100 nM) or CoCl (200 M). Nuclear extracts were prepared and analyzed by electrophoretic mobility shift assay (EMSA). 10 g of aliquots of protein were incubated with radiolabeled oligonucleotide containing the HIF-1 binding site from the EPO gene. B, ARPE-19 cells were stimulated for 4 h with insulin (Ins) (100 nM) or CoCl (200 M). Nuclear extracts were prepared, and HIF-1 binding to hypoxia response element (HRE) oligonucleotide was quantified with Trans-AM HIF-1 transcription factor assay kit. The specificity of binding was assessed by competition with free wild type (wt) or mutated (mut) oligonucleotide. The results are presented as fold stimulation, which was calculated as the ratio of the different samples to the control (untreated cells). Results shown represent the mean  S.E. of four independent experiments performed in duplicate. C, ARPE-19 cells were transfected with the pGL2 basic P12 VEGF-luciferase reporter construct and an expression vector encoding for the -galactosidase gene under the control of the Rous sarcoma virus promoter. ARPE-19 cells were serum-starved and incubated with insulin (100 nM) or CoCl (200 M) for 16 h. Luciferase activity was measured. Results are expressed as a ratio of luciferase activity over -galactosidase activity. Results shown represent the mean S.E. of 20 independent experiments performed in triplicate. Western blotting using antibody to HIF-1 (Fig. 4). The expres- insulin increased HIF-1 expression. This increase was not sion of MEK* did not affect the amount of HIF-1 protein com- affected by pretreatment with the MEK inhibitor U0126. pared with control conditions. However, the expression of PKB- Rather, the inhibition of PI3K activation by LY249002 totally myr is sufficient to increase the HIF-1 protein level in basal blocked the expression of HIF-1 in response to insulin in both conditions. In addition, when PKB-myr is expressed, insulin total cell lysates and in nuclear extracts. In addition, the treat- treatment does not further increase the level of HIF-1 protein. ment with rapamycin decreased HIF-1 protein after insulin Insulin Stimulates HIF-1 Accumulation and VEGF Expres- stimulation by half the level. We confirmed that insulin-in- sion through a PI3K/TOR-dependent pathway—To further duced activation of PI3K, MAPK, and TOR was blocked by evaluate the contribution of the PI3K pathway to the regula- treatment with the respective inhibitor (data not shown). tion of HIF-1 protein levels, we used the pharmacological To determine whether these inhibitors blocked HIF-1 activ- inhibitors of PI3K and of a downstream effector, TOR. ity, we measured the DNA binding activity of HIF-1 using an ARPE-19 cells were treated for 4 h with CoCl or with insulin enzyme-linked immunosorbent assay test. ARPE-19 cells were in the absence or presence of the inhibitors of PI3K stimulated with insulin in the absence or presence of (LY294002), TOR (rapamycin), or MEK (U0126). Whole cell LY294002, rapamycin, or U0126 (Fig. 5B). Insulin induced a lysates and nuclear extracts were prepared and analyzed by 3-fold increase in DNA binding activity of HIF-1. The inhibition Western blotting using antibodies to HIF-1, Shc, or CREB of PI3K by LY294002 totally blocked HIF-1 activation, whereas (Fig. 5A). In agreement with the results obtained in Fig. 4, the inhibition of TOR by rapamycin induced a 40% decrease in Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway 27979 FIG.4. Constitutively active PKB induces HIF-1 accumula- tion in ARPE-19 cells. ARPE-19 cells were transfected with pcDNA (control), a constitutively active form of MEK (MEK*), or PKB-myr. ARPE-19 cells were stimulated for 4 h with insulin (100 nM). Whole cell lysates were prepared and analyzed by Western blotting using antibod- ies to HIF-1, Shc, phosphorylated PKB, or MEK. FIG.3. Insulin induces HIF-1 through a translation-depend- ent pathway. A, ARPE-19 cells were stimulated with insulin (100 nM) or CoCl (200 M) for 6 h. RNA was extracted and analyzed by Northern blotting using a probe corresponding to the HIF-1 cDNA. The blot was subsequently probed with 18 S rRNA as a control. B, ARPE-19 cells were treated or not for 4 h with the proteasome inhibitor MG132 (10 mM) and stimulated with insulin (100 nM). After 4 h, insulin was removed, and cells were incubated in Dulbecco’s modified Eagle’s me- dium containing 0.2% bovine serum albumin (BSA) for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1 or Shc. C, HIF-1 expression was induced by the exposure of ARPE-19 cells to insulin (100 nM) or CoCl (200 M) for 4 h. Cycloheximide (CHX) was added to a final concentra- tion of 10 g/ml, and the cells were harvested after being incubated for the indicated time in the presence of CHX and the inducer. Whole cell lysates were prepared and analyzed by Western blotting using antibod- ies to HIF-1 or Shc. insulin-induced HIF-1 activation. As expected, MEK does not appear to be involved, because the inhibition of MEK by U0126 had no effect on HIF-1 activity in response to insulin. Thus, the extent of inhibition of HIF-1 expression by the inhibitors FIG.5. Insulin activates HIF-1 through a PI3K/TOR-dependent tightly correlated with their ability to block insulin-induced pathway. A, ARPE-19 cells were stimulated for 4 h with CoCl (200 DNA binding activity of HIF-1. M) or with insulin (100 nM) with or without pretreatment for 30 min To examine whether this correlation could be extended to the with LY294002 (50 M), U0126 (10 M), or rapamycin (50 nM). Whole HIF-1 activation and VEGF mRNA expression in response to cell lysates or nuclear extracts were prepared and analyzed by Western blotting using antibodies to HIF-1, Shc, or CREB. B, ARPE-19 cells insulin, we analyzed the VEGF mRNA expression profile after were stimulated for 4 h with insulin (100 nM) with or without pretreat- treatment with these inhibitors. ARPE-19 cells were treated ment for 30 min with LY294002 (LY) (50 M), U0126 (10 M), or with CoCl or with insulin in the absence or presence of rapamycin (Rapa) (50 nM). Nuclear extracts were prepared, and HIF-1 LY294002, U0126, or rapamycin. RNA was extracted, and binding was quantified with Trans-AM HIF-1 transcription factor assay kit. The results are presented as fold stimulation, which was calculated Northern blot analyses were performed using a VEGF cDNA as the ratio of the different samples to the control cells. Results shown probe (Fig. 6). Insulin and CoCl induced a 3- and 5-fold in- represent the mean  S.E. of two independent experiments performed crease in VEGF mRNA expression, respectively. The inhibition in duplicate. of PI3K blocked the insulin-induced VEGF mRNA expression. Moreover, rapamycin treatment resulted in a 40% inhibition. In contrast, blocking MEK did not affect the ability of insulin to be dependent on the PI3K/TOR pathway. This accumulation induce VEGF mRNA expression in these cells. In summary, the leads to HIF-1 transcriptional activity and VEGF mRNA insulin-induced accumulation of the HIF-1 subunit appears to expression. 27980 Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway tion. Nevertheless, we cannot exclude the possibility that insu- lin regulates the translation of a protein, which inhibits HIF-1 degradation. It is of interest to note that a recent study shows that heregulin, which activates the tyrosine kinase receptor HER2, stimulates HIF-1 synthesis (30), similar to our results concerning insulin action. In ARPE-19 cells, we found that the insulin effect on HIF-1 expression, HIF-1 activation, and VEGF expression are de- pendent on the PI3KPKBTOR pathway. In contrast, the MEK pathway does not appear to be required for insulin action on HIF-1. Both the MAPK and PI3K pathways have been impli- cated in HIF-1 regulation. The p42 and p44 MAPKs activate HIF-1 by promoting the phosphorylation of HIF-1 in response to hypoxia and its accumulation in response to advanced gly- cation end products or mersalyl (1, 44 – 46). PI3K-dependent pathways have been implicated in HIF-1 and VEGF expression in transformed cells (47–50). Moreover, the activation of PKB or inactivation of the tumor suppressor gene encoding phos- phatase and tensin homolog deleted on chromosome 10, which dephosphorylates the PI3K reaction products phosphatidyl- FIG.6. Insulin stimulates VEGF mRNA expression through a inositol 3,4-biphosphate and phosphatidylinositol 3,4,5- PI3K/TOR-dependent pathway. ARPE-19 cells were stimulated for triphosphate, increases HIF-1 protein levels and HIF-1-de- 6 h with CoCl (200 M), or with insulin (100 nM) with or without pretreatment for 30 min with LY294002 (LY) (50 M), U0126 (10 M), or pendent reporter gene expression (30, 48, 50, 51). rapamycin (Rapa) (50 nM). RNA was extracted and analyzed by North- TOR seems to be involved only partly in the insulin action on ern blotting using a VEGF cDNA probe. The blot was subsequently HIF-1 activity, because the inhibition of TOR does not com- probed with a probe for 18 S rRNA as a control. pletely abolish HIF-1 expression and HIF-1 activation. These results suggest that at least the two following pathways are involved in insulin-induced HIF-1 regulation, a PKB-depend- DISCUSSION ent/TOR-independent pathway and a PKB/TOR-dependent Diabetic retinopathy is the major cause of blindness in West- pathway. The PKB-dependent/TOR-independent pathway re- ern countries. VEGF is involved in the pathogenesis of both mains unknown, because a direct phosphorylation of HIF-1 by background and proliferative retinopathy. Intraocular VEGF is PKB has been excluded (51). However, PKB could be involved increased in eyes from patients with blood-retinal barrier in the insulin regulation of HIF-1 translation. It has been breakdown and neovascularization. Clinical studies have dem- previously shown that insulin stimulates protein synthesis by onstrated that long term insulin therapy reduces the risk of the activation of eIF2B (eukaryotic translation initiation factor diabetic retinopathy progression. However, it has also been 2B), an essential translation initiation factor, through a PI3K/ observed that intensive insulin therapy leads to a transient PKB/glycogen synthase kinase-3 pathway (52, 53). For the worsening of retinopathy characterized by a blood-retinal bar- PKB/TOR-dependent pathway, it has been shown that TOR rier breakdown (37–39). It has been proposed that the worsen- activity positively regulates translation. Insulin induces the ing of retinopathy could be attributed to chronic hyperinsuline- phosphorylation of 4E-BP1 through a PI3KPKBTOR pathway mia induced by insulin treatment. Indeed, it has been shown (54). The phosphorylation of 4E-BP1 results in a decrease in its that insulin stimulates VEGF expression, which in turn would binding affinity for eukaryotic translation initiation factor 4E, stimulate neovascularization (40, 41). However, the molecular an essential translation initiation factor. The subsequent dis- mechanisms involved in insulin-induced VEGF expression re- sociation of eIF-4E (eukaryotic translation initiation factor) main unknown. In this study, we show that insulin stimulates from 4E-BP1 promotes cap structure-dependent translation VEGF expression through the activation of the transcription initiation (55, 56). We could hypothesize that insulin-activated factor HIF-1. This activation is regulated by a PI3K-dependent TOR by phosphorylation of 4E-BP1 dissociates eukaryotic signaling pathway involving TOR. Moreover, in contrast to translation initiation factor 4E from 4E-BP1 and stimulates hypoxia, which is a major activator of HIF-1, insulin does not the translation initiation of the HIF-1 mRNA. regulate HIF-1 through the inhibition of its degradation but In diabetes, several factors could be involved in the worsen- through a translation-dependent mechanism. ing of the diabetic retinopathy including (i) advanced glycation In ARPE-19 cells, insulin stimulates the accumulation of the end products generated during hyperglycemia, (ii) hypoxia re- regulated subunit HIF-1. An increase in HIF-1 expression is sulting from microvascular retinal occlusion, and (iii) hyperin- directly correlated with the activity of the transcription factor sulinemia stimulating VEGF expression through the up-regu- HIF-1. Indeed, we show that insulin induces increased HIF-1 lation of HIF-1 expression (1, 57). Moreover, the co-treatment protein levels, augmented HIF-1 DNA binding activity, and of retinal epithelial cells with both insulin and advanced gly- stimulation of HIF-1-mediated reporter gene transcription. In cation end products increases VEGF expression (1). The ad- normoxic conditions, HIF-1 is maintained at low levels by a vanced glycation end products and insulin activate HIF-1 degradation process involving the ubiquitin-proteasome sys- tem (42, 43). Hypoxia rapidly increases the amount of HIF-1 through distinct pathways, because advanced glycation end- induced HIF-1 activation is dependent on MAPK, whereas by inhibiting its proteasome-dependent degradation. Surpris- ingly, insulin does not seem to act on HIF-1 degradation. A insulin-induced HIF-1 activation is dependent on PI3K. Hy- poxia blocks HIF-1 degradation, whereas growth factors act- comparison of the half-life of HIF-1 after the removal of insu- lin or in presence of both insulin and cycloheximide, a transla- ing through tyrosine kinase receptors would increase its syn- tion inhibitor, shows that insulin does not stabilize the HIF-1 thesis. The combination of these different signals enhances the protein. Furthermore, insulin does not affect the transcription activation of the transcription factor leading to increased of HIF-1 mRNA, suggesting that it regulates HIF-1 transla- VEGF gene expression. The result would be an amplification of Insulin Stimulates HIF-1 via PI3K/TOR-dependent Pathway 27981 P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Science 292, 468 – 472 the angiogenic signal leading to further progression of diabetic 26. Frodin, M., Peraldi, P., and Van Obberghen, E. (1994) J. Biol. Chem. 269, retinopathy. 6207– 6214 27. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., In conclusion, we have shown that insulin activates the and Semenza, G. L. (1996) Mol. Cell. Biol. 16, 4604 – 4613 transcription factor HIF-1 through a PI3KPKBTOR-depend- 28. Andjelkovic, M., Alessi, D. R., Meier, R., Fernandez, A., Lamb, N. J., Frech, M., ent pathway. Ours results suggest that insulin similar to Cron, P., Cohen, P., Lucocq, J. M., and Hemmings, B. A. (1997) J. Biol. Chem. 272, 31515–31524 heregulin regulates HIF-1 synthesis. It remains to estab- 29. Tanti, J. F., Gremeaux, T., Grillo, S., Calleja, V., Klippel, A., Williams, L. T., lished whether such an effect on HIF-1 synthesis is a general Van Obberghen, E., and Le Marchand-Brustel, Y. (1996) J. Biol. Chem. 271, feature of receptor tyrosine kinases. 25227–25232 30. Laughner, E., Taghavi, P., Chiles, K., Mahon, P. C., and Semenza, G. L. (2001) Mol. Cell. Biol. 21, 3995– 4004 Acknowledgments—We thank P. Peraldi for critical reading of this 31. Wang, G. L., and Semenza, G. L. (1995) J. Biol. Chem. 270, 1230 –1237 paper. We also thank J. Plouet (Toulouse, France) for VEGF cDNA 32. Semenza, G. L., and Wang, G. L. (1992) Mol. Cell. Biol. 12, 5447–5454 and B. Hemmings, S. Tartare-Deckert, and M. Montminy for the gift of 33. Wang, G. L., and Semenza, G. L. (1993) Blood 82, 3610 –3615 antibodies. 34. Wang, G. L., Jiang, B. H., Rue, E. A., and Semenza, G. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5510 –5514 REFERENCES 35. Jiang, B. H., Zheng, J. Z., Leung, S. W., Roe, R., and Semenza, G. L. (1997) J. Biol. Chem. 272, 19253–19260 1. Treins, C., Giorgetti-Peraldi, S., Murdaca, J., and Van Obberghen, E. (2001) J. Biol. Chem. 276, 43836 – 43841 36. Pugh, C. W., O’Rourke, J. F., Nagao, M., Gleadle, J. M., and Ratcliffe, P. J. (1997) J. Biol. Chem. 272, 11205–11214 2. Withers, D. J., and White, M. (2000) Endocrinology 141, 1917–1921 3. Saltiel, A. R., and Pessin, J. E. (2002) Trends Cell Biol. 12, 65–71 37. Agardh, C. D., Eckert, B., and Agardh, E. (1992) J. Diabetes Complications 6, 96 –100 4. Van Obberghen, E., Baron, V., Delahaye, L., Emanuelli, B., Filippa, N., Giorgetti-Peraldi, S., Lebrun, P., Mothe-Satney, I., Peraldi, P., Rocchi, S., 38. Brinchmann-Hansen, O., Dahl-Jorgensen, K., Sandvik, L., and Hanssen, K. F. (1992) Br. Med. J. 304, 19 –22 Sawka-Verhelle, D., Tartare-Deckert, S., and Giudicelli, J. (2001) Eur. J. Clin. Invest. 31, 966 –977 39. Moskalets, E., Galstyan, G., Starostina, E., Antsiferov, M., and Chantelau, E. (1994) J Diabetes Complications 8, 45–50 5. Saltiel, A. R., and Kahn, C. R. (2001) Nature 414, 799 – 806 6. Sutherland, C., O’Brien, R. M., and Granner, D. K. (1995) J. Biol. Chem. 270, 40. Poulaki, V., Qin, W., Joussen, A. M., Hurlbut, P., Weigand, S. J., Rudge, J., Yancopoulos, G. D., and Adamis, A. P. (2002) J. Clin. Invest. 109, 805– 815 15501–15506 7. Dickens, M., Svitek, C. A., Culbert, A. A., O’Brien, R. M., and Tavare, J. M. 41. Zelzer, E., Levy, Y., Kahana, C., Shilo, B. Z., Rubinstein, M., and Cohen, B. (1998) J. Biol. Chem. 273, 20144 –20149 (1998) EMBO J. 17, 5085–5094 8. Wang, D., and Sul, H. S. (1998) J. Biol. Chem. 273, 25420 –25426 42. Salceda, S., and Caro, J. (1997) J. Biol. Chem. 272, 22642–22647 9. Masuda, S., Chikuma, M., and Sasaki, R. (1997) Brain Res. 746, 63–70 43. Huang, L. E., Gu, J., Schau, M., and Bunn, H. F. (1998) Proc. Natl. Acad. Sci. 10. Lu, M., Amano, S., Miyamoto, K., Garland, R., Keough, K., Qin, W., and U. S. A. 95, 7987–7992 Adamis, A. P. (1999) Invest. Ophthalmol. Visual Sci. 40, 3281–3286 44. Richard, D. E., Berra, E., Gothie, E., Roux, D., and Pouyssegur, J. (1999) 11. Duh, E., and Aiello, L. P. (1999) Diabetes 48, 1899 –1906 J. Biol. Chem. 274, 32631–32637 12. Funatsu, H., Yamashita, H., Noma, H., Shimizu, E., Yamashita, T., and Hori, 45. Minet, E., Arnould, T., Michel, G., Roland, I., Mottet, D., Raes, M., Remacle, J., S. (2001) Jpn. J. Ophthalmol. 45, 577–584 and Michiels, C. (2000) FEBS Lett. 468, 53–58 13. Ikeda, E., Achen, M. G., Breier, G., and Risau, W. (1995) J. Biol. Chem. 270, 46. Agani, F., and Semenza, G. L. (1998) Mol. Pharmacol. 54, 749 –754 19761–19766 47. Jiang, B. H., Zheng, J. Z., Aoki, M., and Vogt, P. K. (2000) Proc. Natl. Acad. Sci. 14. Levy, A. P., Levy, N. S., Wegner, S., and Goldberg, M. A. (1995) J. Biol. Chem. U. S. A. 97, 1749 –1753 270, 13333–13340 48. Zhong, H., Chiles, K., Feldser, D., Laughner, E., Hanrahan, C., Georgescu, 15. Wang, D., Huang, H. J., Kazlauskas, A., and Cavenee, W. K. (1999) Cancer M. M., Simons, J. W., and Semenza, G. L. (2000) Cancer Res. 60, 1541–1545 Res. 59, 1464 –1472 49. Blancher, C., Moore, J. W., Robertson, N., and Harris, A. L. (2001) Cancer Res. 16. Maity, A., Pore, N., Lee, J., Solomon, D., and O’Rourke, D. M. (2000) Cancer 61, 7349 –7355 Res. 60, 5879 –5886 50. Jiang, B. H., Jiang, G., Zheng, J. Z., Lu, Z., Hunter, T., and Vogt, P. K. (2001) 17. Goldman, C. K., Kim, J., Wong, W. L., King, V., Brock, T., and Gillespie, G. Y. Cell Growth Differ. 12, 363–369 (1993) Mol. Biol. Cell 4, 121–133 51. Zundel, W., Schindler, C., Haas-Kogan, D., Koong, A., Kaper, F., Chen, E., 18. Miele, C., Rochford, J. J., Filippa, N., Giorgetti-Peraldi, S., and Van Gottschalk, A. R., Ryan, H. E., Johnson, R. S., Jefferson, A. B., Stokoe, D., Obberghen, E. (2000) J. Biol. Chem. 275, 21695–21702 and Giaccia, A. J. (2000) Genes Dev. 14, 391–396 19. Punglia, R. S., Lu, M., Hsu, J., Kuroki, M., Tolentino, M. J., Keough, K., Levy, 52. Welsh, G. I., Stokes, C. M., Wang, X., Sakaue, H., Ogawa, W., Kasuga, M., and A. P., Levy, N. S., Goldberg, M. A., D’Amato, R. J., and Adamis, A. P. (1997) Proud, C. G. (1997) FEBS Lett. 410, 418 – 422 Diabetes 46, 1619 –1626 53. Welsh, G. I., Miller, C. M., Loughlin, A. J., Price, N. T., and Proud, C. G. (1998) 20. Ryuto, M., Ono, M., Izumi, H., Yoshida, S., Weich, H. A., Kohno, K., and FEBS Lett. 421, 125–130 Kuwano, M. (1996) J. Biol. Chem. 271, 28220 –28228 54. Takata, M., Ogawa, W., Kitamura, T., Hino, Y., Kuroda, S., Kotani, K., Klip, 21. Stein, I., Neeman, M., Shweiki, D., Itin, A., and Keshet, E. (1995) Mol. Cell. A., Gingras, A. C., Sonenberg, N., and Kasuga, M. (1999) J. Biol. Chem. 274, Biol. 15, 5363–5368 20611–20618 22. Wang, G. L., and Semenza, G. L. (1993) J. Biol. Chem. 268, 21513–21518 55. Sonenberg, N., and Gingras, A. C. (1998) Curr. Opin. Cell Biol. 10, 268 –275 23. Semenza, G. L. (1998) Curr. Opin. Genet. Dev. 8, 588 –594 56. Lawrence, J. C., Fadden, P., Haystead, T. A., and Lin, T. A. (1997) Adv. 24. Semenza, G. L. (2000) J. Appl. Physiol. 88, 1474 –1480 Enzyme Regul. 37, 239 –267 25. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., 57. Pe’er, J., Folberg, R., Itin, A., Gnessin, H., Hemo, I., and Keshet, E. (1998) Kriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, Ophthalmology 105, 412– 416

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Published: Aug 1, 2002

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