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First published online 8 November 2005
doi: 10.1242/jcs.02648

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The insulin-PI3K/TOR pathway induces a HIF-dependent transcriptional response in Drosophila by promoting nuclear localization of HIF-/Sima

¹ Instituto Leloir and IIB, FCEyN-Universidad de Buenos Aires, CONICET, Patricias Argentinas 435, Buenos Aires (1405), Argentina
² The Burnham Institute, La Jolla, CA 92037, USA

View larger version (24K): [in a new window]   Fig. 1. Hypoxic induction of an HRE-response in a stably transfected Drosophila S2 cell line. (A) Schematic representation of the HIF-responsive firefly luciferase reporter gene used in this study (HRE-Luc), in which transcription depends on a dimerized 51 bp sequence from the murine lactate dehydrogenase enhancer that contains two HIF responsive elements (HREs) and one cyclic AMP responsive element (CRE). (B) Effect of hypoxia on HRE-Luc activity in S2 cells. Cells were exposed to different oxygen levels for 20 hours or stimulated with 100 µM DFO. Luciferase activity was determined and normalized to protein concentration in the extracts. Induction of the reporter augmented when oxygen levels decrease. (C) Variations in dLDH mRNA levels. To measure the effect of hypoxia on endogenous dLDH gene expression, cells were exposed to 2% O2 for 16 hours. Total RNA was then prepared and analyzed by northern blot. LDH mRNA is strongly induced by hypoxia (upper panel). 18S rRNA that was used as loading control remains constant (lower panel). (D) Role of Sima and Tango in HRE-Luc induction. Cells were incubated with dsRNA directed against GFP (control), sima or tango and 3 days later, the culture was treated or not with DFO (100 µM) or was exposed to 1% O2 for 20 hours. Cells were then lysed and luciferase activity was determined. Hypoxia or DFO-dependent induction of the reporter was specifically abrogated by sima or tango RNAi treatment. (E) Effect of exogenous Sima. Cells were transiently transfected with an empty vector (pAC) or with a plasmid expressing Sima protein (pAC-Sima), together with 100 ng of a vector encoding Renilla luciferase (pRL). Twenty-four hours after transfection, cells were lysed and firefly or Renilla luciferase activity was determined. Induction of the HRE-Luc reporter was proportional to the transfected amount of sima-encoding plasmid. Normalized luciferase activity (firefly/Renilla ratio) is expressed as fold-induction with respect to the activity upon transfection of an empty vector. In all experiments involving luciferase determinations, data represent the mean±s.e.m. of three independent experiments performed in triplicate and expressed as fold-induction with respect to untreated cells.

View larger version (17K): [in a new window]   Fig. 2. Insulin induces an HRE-response in S2 cells. (A) Effect of insulin on HRE-Luc reporter induction. S2 cells stably transfected with HRE-Luc were cultured for 3 days and then exposed or not to hypoxia (1% O2), DFO (100 µM) or insulin (50 µg/ml) for 20 hours. Luciferase activity was determined and normalized to protein concentration. HRE-Luc was induced by insulin, and attained levels similar to those upon exposure to hypoxia and higher than those observed upon DFO treatment. (B) Treatment with different doses of insulin. Cells were stimulated with 1-50 µg/ml insulin for 20 hours, lysed and luciferase activity was determined and normalized to protein concentration. HRE-Luc was induced as a function of insulin concentration. (C) Endogenous dLDH mRNA levels upon insulin treatment. To assess the effect of insulin on the expression of the endogenous dLDH gene, cells were treated for 4, 8, or 16 hours with insulin (10 µg/ml) and then total RNA was prepared and analyzed by northern blot using a specific dLDH probe (upper panel) or 18S rRNA as a control (lower panel); expression of dLDH mRNA increased as a function of the time of exposure to insulin. (D) Role of Sima and Tango in insulin-dependent induction of HRE-Luc. Cells were incubated in the absence (control) or presence of dsRNA directed against GFP, sima or tango and 3 days later, they were treated or not with 10 µg/ml insulin for 20 hours. Cells were then lysed and luciferase activity determined. Induction of the HRE reporter by insulin was totally abrogated by sima or tango dsRNA.

View larger version (20K): [in a new window]   Fig. 3. HRE-Luc induction in S2 cells is mediated by the activation of the PI3K-AKT pathway. (A) Effect of a PI3K inhibitor on the insulin-dependent HRE response. To assess the effect of PI3K inhibition on the insulin-stimulated HRE response, cells were treated for 20 hours with insulin (10 µg/ml) in the presence of LY294002 () or the vehicle (DMSO) (). LY294002 was added 1 hour prior to insulin stimulation and luciferase activity was determined and normalized to protein concentration. LY294002 provoked a dose-dependent inhibition of the HRE response. (B) Effect of LY294002 on dLDH mRNA induction. To study the effect of PI3K inhibition on dLDH mRNA increase after insulin stimulation, S2 cells were treated for 16 hours with insulin (10 µg/ml) in the presence or not of 10 µM LY294002. Total RNA was prepared and analyzed by northern blot using a specific dLDH probe (upper panel) or 18S rRNA as a control (lower panel). Treatment with LY294002 provoked a clear reduction of dLDH mRNA levels. (C) Role of dAKT and dPDK1 in HRE-Luc induction by insulin. Cells were incubated in the presence or absence of dsRNA directed against GFP, dAKT or dPDK1. After 3 days, the cells were treated or not with insulin 10 µg/ml for 20 hours, lysed and luciferase activity was determined. dAKT and dPDK1 dsRNAs caused strong inhibition of HRE-Luc stimulation by insulin. (D) Effect of exogenous AKT on HRE-Luc expression. Cells were transiently transfected with a plasmid expressing AKT (pAC-AKT, 250 ng), together with 100 ng of a plasmid expressing Renilla luciferase (pRL). Twenty-four hours after transfection, cells were lysed and luciferase activity was determined. Results are shown as the ratio of firefly to Renilla luciferase activities; transfection of AKT led to strong induction of the HRE reporter. (E) Effect of dPTEN silencing. Cells were incubated in the presence or absence of dsRNA directed against GFP or dPTEN, lysed and luciferase activity was determined. dPTEN dsRNA treatment provoked strong induction of the HRE response.

View larger version (46K): [in a new window]   Fig. 4. PTEN2L117 homozygous embryos show constitutive induction of the transgenic HRE reporter LDH-LacZ. (A) Normoxic wild-type embryos. Drosophila wild-type embryos maintained in normoxia do not express the hypoxia-inducible reporter LDH-LacZ, as revealed by X-gal staining. (B) Hypoxic wild-type embryos. Embryos were exposed to 3% O2 for 4 hours, fixed and stained with X-gal. Induction of the LDH-LacZ reporter was observed in an expression pattern that is coincident with some of the tracheal branches (arrow) (Lavista-Llanos et al., 2002). (C) Effect of PTEN loss-of-function on HRE induction in vivo. In PTEN2L117homozygous embryos the LDH-LacZ reporter was induced in normoxia (arrow) in a pattern that paralleled induction of the reporter in hypoxic wild-type embryos (compare with B).

View larger version (21K): [in a new window]   Fig. 5. Insulin treatment provokes an increase in Sima protein levels; involvement of TOR in the HRE response. (A) Sima protein levels upon insulin treatment. S2 cells were treated with insulin (10 µg/ml) (I) or exposed to hypoxia (2% O2) for 16 hours (H) and Sima protein levels in nuclear extracts were analyzed by western blot (upper panel), hsp70 (lower panel) was used as a loading control. Sima protein levels were strongly upregulated upon insulin treatment, attaining levels similar to those caused by exposure to 2% O2 during 16 hours (results of a representative experiment (n=3) are shown). (B) sima mRNA upon insulin treatment. Total RNA from cells treated or not with insulin (10 µg/ml) was analysed by northern blot using a specific sima probe (upper panel), S18 rRNA was used as a loading control (lower panel). sima mRNA levels did not change upon insulin treatment. (C) Effect of TOR on insulin-dependent induction of HRE-Luc. Cells were treated or not with insulin (10 µg/ml) for 20 hours in the presence of rapamycin (200 nM). Luciferase activity was determined and normalized to protein concentration; rapamycin provoked clear inhibition of the of HRE-Luc induction. (D) Silencing of dTOR, dRheb or dS6K. Cells were incubated in the presence of dsRNA directed against GFP, dTOR, dRheb or dS6K and 3 days later the cells were treated or not with 10 µg/ml insulin for 20 hours, then lysed and luciferase activity was determined; dTOR, dRheb and dS6K dsRNAs provoked strong inhibition of HRE induction.

View larger version (33K): [in a new window]   Fig. 6. Hypoxia or activation of the PI3K-AKT pathway promote nuclear localization of Sima in vivo. (A) Definition of three categories of Sima subcellular localization. Sima and a ß-galactosidase (ß-gal) variant bearing a nuclear localization signal were simultaneously expressed in UAS transgenic embryos under control of an engrailed-Gal4 driver. Embryos were fixed, subjected to double -Sima (red)/-ßgal (green) imunofluorescence and observed at the confocal microscope. Three arbitrary categories of subcellular localization were defined: `Cytoplasmic', where more than 90% of the cells in the embryo exhibited cytoplasmic localization; `Nuclear', where more than 90% of the cells showed nuclear localization and `Ubiquitous', where less than 90% of the cells showed cytoplasmic or nuclear localization. (B) Sima subcellular localization throughout development at different oxygen concentrations (white, Cytoplasmic; gray, Ubiquitous; black, Nuclear). Overnight egg-laying plates were exposed to different oxygen concentrations for 4 hours and then fixed and immunostained. In normoxia, at embryonic stages 11-14, Sima protein was exclusively cytoplasmic, whereas later in embryogenesis Sima became gradually more nuclear. As oxygen concentration decreased Sima protein became progressively more nuclear. (C) Effect of the activation of the PI3K-AKT pathway on Sima subcellular localization (white, Cytoplasmic; gray, Ubiquitous; black, Nuclear). Overnight egg-laying plates with normoxic embryos of the indicated genotype were fixed and immunostained. engrailed-driven overexpression of dAKT led to an increase in Sima nuclear localization at stages 15-16. Introduction of an EP element overexpressing dPDK1 along with dAKT enhanced Sima nuclear localization in comparison with embryos overexpressing dAKT alone. dPTEN2L117 homozygous mutant embryos also showed increased nuclear localization; confirming that loss of dPTEN was indeed the cause of increased nuclear localization of Sima, ectopic expression of dPTEN in the dPTEN2L117 homozygous mutant background rescued wild-type localization of Sima.

View larger version (9K): [in a new window]   Fig. 7. Model of insulin signaling through HIF/Sima. The activated InR can signal through AKT to prevent TSC1-TSC2 from inhibiting TOR, thereby activating S6K leading to growth stimulation. In addition, TOR can activate Sima/HIF-1 that induces transcription of several target genes, including scylla/RTP801 that in turn stimulates TSC1-TSC2, resulting in a negative feedback loop that reduces TOR activity. Activated AKT can stimulate nuclear localization of Sima/HIF-1 through an unknown mechanism (dashed line), contributing to the negative regulation of TOR. Low oxygen levels can inhibit growth by reducing the activity of Hph/Fatiga, which provokes stabilization of HIF-/Sima and, in consequence, induction of scylla/RTP801, thereby reducing TOR activity.