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. 2013 Jul 19;288(29):20768-20775.
doi: 10.1074/jbc.M113.476903. Epub 2013 Jun 9.

Sirtuin-7 inhibits the activity of hypoxia-inducible factors

Maimon E Hubbi  1 , Hongxia Hu  2 , Kshitiz  3 , Daniele M Gilkes  1 , Gregg L Semenza  4
Affiliations

Sirtuin-7 inhibits the activity of hypoxia-inducible factors

Maimon E Hubbi et al. J Biol Chem. .

Abstract

Hypoxia-inducible factor (HIF) 1 and HIF-2 are heterodimeric proteins composed of an oxygen-regulated HIF-1α or HIF-2α subunit, respectively, and a constitutively expressed HIF-1β subunit, which mediate adaptive transcriptional responses to hypoxia. Here, we report that Sirt7 (sirtuin-7) negatively regulates HIF-1α and HIF-2α protein levels by a mechanism that is independent of prolyl hydroxylation and that does not involve proteasomal or lysosomal degradation. The effect of Sirt7 was maintained in the presence of the sirtuin inhibitor nicotinamide and upon deletion or mutation of its deacetylase domain, indicating a non-catalytic function. Knockdown of Sirt7 led to an increase in HIF-1α and HIF-2α protein levels and an increase in HIF-1 and HIF-2 transcriptional activity. Thus, we identify a novel molecular function of Sirt7 as a negative regulator of HIF signaling.

Keywords: Hypoxia; Hypoxia-inducible Factor (HIF); Sirtuins; Transcription.

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Figures

FIGURE 1.
FIGURE 1.
Sirt7 binds to HIF-1α and HIF-2α. A, 293T cells were cotransfected with FLAG epitope-tagged HIF-1α expression vector and expression vector encoding Myc epitope-tagged Sirt5, Sirt6, or Sirt7. 24 h post-transfection, cell lysates were immunoprecipitated (IP) with anti-Myc epitope antibody, and each Western blot (WB) was probed with the indicated antibodies. B, 293T cells were cotransfected with HIF-2α expression vector and expression vector encoding Myc-tagged Sirt5, Sirt6, or Sirt7. 24 h post-transfection, lysates were immunoprecipitated with anti-Myc antibody and probed with the indicated antibodies.
FIGURE 2.
FIGURE 2.
Sirt7 inhibits HIF transcriptional activity. A and B, Hep3B (A) or HeLa (B) cells were cotransfected with the indicated firefly luciferase reporter gene, which contained the ENO1 hypoxia response element upstream of a basal SV40 promoter (p2.1) or an intact promoter from the EPO, SOD2, or VEGFA gene; control Renilla luciferase reporter gene pSV-RL; HIF-1α expression vector; and either an empty vector (EV) or Sirt7 expression vector. 24 h post-transfection, cells were lysed, and the ratio of firefly to Renilla luciferase activity was determined. C and D, Hep3B (C) or HeLa (D) cells were cotransfected with the indicated luciferase promoter construct, HIF-2α expression vector, and either EV or Sirt7 expression vector. 24 h post-transfection, cells were lysed, and luciferase activity was determined. Results are shown as means ± S.E. #, p < 0.05; *, p < 0.01 versus HIF-1α or HIF-2α alone.
FIGURE 3.
FIGURE 3.
Sirt7 overexpression decreases HIF-1α and HIF-2α protein levels. Hep3B (A), HeLa (B), 293T (C), and MDA-MB-231 (D) cells were cotransfected with HIF-1α or HIF-2α expression vector and either EV or Sirt7 expression vector. 24 h post-transfection, cell lysates were subjected to Western blot assay (WB) with the indicated antibodies.
FIGURE 4.
FIGURE 4.
Effects of Sirt7 are independent of deacetylase activity. A and B, Hep3B cells were cotransfected with p2.1, pSV-RL, HIF-1α or HIF-2α expression vector, and either EV or expression vector encoding wild-type Sirt7 (WT) or catalytically inactive missense mutant Sirt7-S112A (M1) or Sirt7-H188Y (M2). 24 h post-transfection, cells were lysed, and the ratio of firefly to Renilla luciferase activity was determined. C, Hep3B cells were cotransfected with p2.1; pSV-RL; and EV or vector encoding wild-type Sirt7, Sirt7-S112A, or Sirt7-H188Y. 24 h post-transfection, cells were exposed to 1% O2 for an additional 24 h and lysed, and luciferase activity was determined. D and E, Hep3B cells were cotransfected with p2.1; pSV-RL; HIF-1α or HIF-2α expression vector; and either EV or expression vector encoding Sirt7 amino acids 1–90 (D90), 1–180 (D180), or 1–331 (D331). 24 h post-transfection, cells were lysed, and luciferase activity was determined. F, Hep3B cells were cotransfected with p2.1; pSV-RL; and either EV or vector encoding Sirt7 amino acids 1–90, 1–180, or 1–331. 24 h post-transfection, cells were exposed to 1% O2 for an additional 24 h and lysed, and luciferase activity was determined. G and H, Hep3B cells were cotransfected with p2.1, pSV-RL, HIF-1α or HIF-2α vector, and either EV or Sirt7 vector. 24 h post-transfection, cells were left untreated or treated with nicotinamide (20 mm) for an additional 24 h, after which cells were lysed, and luciferase activity was determined. I, Hep3B cells were cotransfected with p2.1, pSV-RL, and either EV or Sirt7 vector. 24 h post-transfection, cells were exposed to 1% O2 for an additional 24 h in the presence or absence of nicotinamide (20 mm) as indicated. J, Hep3B cells were cotransfected with vector encoding FLAG-HIF-1α or HIF-2α and either EV or vector encoding wild-type Sirt7, Sirt7-S112A, or Sirt7-H188Y. 24 h post-transfection, cell lysates were subjected to Western blotting (WB). K, Hep3B cells were cotransfected with vector encoding FLAG-HIF-1α or HIF-2α and either EV or vector encoding Sirt7 amino acids 1–90, 1–180, or 1–331. 24 h post-transfection, cell lysates were subjected to Western blotting. L, Hep3B cells were cotransfected with vector encoding FLAG-HIF-1α or HIF-2α and either EV or Sirt7 vector. Cells were left untreated or treated with nicotinamide (20 mm) for 24 h, after which cell lysates were subjected to Western blotting. Results are shown as means ± S.E. #, p < 0.05; *, p < 0.01 versus HIF-1α or HIF-2α alone.
FIGURE 5.
FIGURE 5.
Effects of Sirt7 are independent of proteasomal and lysosomal degradation. A, Hep3B cells were cotransfected with p2.1, pSV-RL, EV or Sirt7 expression vector, and vector encoding wild-type HIF-1α (WT) or triple-mutant HIF-1α (TM; P402A/P564A/N803A). 24 h post-transfection, cells were lysed, and luciferase activity was determined. B, Hep3B cells were cotransfected with p2.1, pSV-RL, EV or Sirt7 expression vector; and vector encoding wild-type or triple-mutant HIF-2α. 24 h post-transfection, cells were lysed, and luciferase activity was determined. C, Hep3B cells were cotransfected with either 1 μg of wild-type HIF-1α or HIF-2α vector or 100 ng of triple-mutant HIF-2α vector and either EV or Sirt7 vector. Total plasmid transfected was equalized with EV. 24 h post-transfection, cell lysates were analyzed by Western blotting (WB). D and E, Hep3B cells were cotransfected with p2.1, pSV-RL, HIF-1α (D) or HIF-2α (E) vector, and either EV or Sirt7 vector. 24 h post-transfection, cells were left untreated or treated with DMOG (1 mm) for an additional 24 h, after which cells were lysed, and luciferase activity was determined. F, Hep3B cells were cotransfected with HIF-1α or HIF-2α vector and either EV or Sirt7 vector. 24 h post-transfection, cells were treated with MG132 (20 μm) for an additional 8 h, after which cells were lysed, and Western blot assays were performed. G and H, Hep3B cells were cotransfected with p2.1, pSV-RL, expression vector encoding HIF-1α (G) or HIF-2α (H), and either EV or Sirt7 vector. 24 h post-transfection, cells were left untreated or treated with bafilomycin (5 nm) for an additional 24 h, after which cells were lysed, and luciferase activity was determined. I, Hep3B cells were cotransfected with HIF-1α or HIF-2α vector and either EV or Sirt7 vector. 24 h post-transfection, cells were treated with bafilomycin for an additional 8 h, after which Western blot assays were performed. Results are shown as means ± S.E. *, p < 0.01 for the indicated comparisons.
FIGURE 6.
FIGURE 6.
Knockdown of Sirt7 expression increases HIF-1α and HIF-2α protein levels and HIF transcriptional activity. A, Hep3B cells were cotransfected with p2.1, pSV-RL, HIF-1α expression vector; and either empty shRNA vector (−) or vector encoding one of five shRNA sequences _targeting Sirt7 mRNA (shSirt7; labeled A–E). 48 h post-transfection, cell lysates were subjected to Western blot assay. B, Hep3B cells were cotransfected with FLAG-HIF-1α vector and either empty shRNA vector or vector encoding shRNA sequences _targeting Sirt7, followed by Western blotting (WB). C, Hep3B cells were treated as described for A except that HIF-2α expression vector was used instead of HIF-1α. D, Hep3B cells were cotransfected with HIF-2α vector and either empty shRNA vector or vector encoding shRNA sequences _targeting Sirt7, followed by Western blotting. E and F, Hep3B (E) and HeLa (F) cells were cotransfected with p2.1, pSV-RL, and either empty shRNA vector or vector encoding shRNA sequences _targeting Sirt7. 24 h post-transfection, cells were exposed to 1% O2 for an additional 24 h and then lysed, and luciferase activity was determined. Results are shown as means ± S.E. #, p < 0.05; *, p < 0.01 versus HIF-1α, HIF-2α, or 1% O2 alone.
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References

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