The HIF-1 hypoxia-inducible factor modulates lifespan in C. elegans

Abstract

During normal development or during disease, animal cells experience hypoxic (low oxygen) conditions, and the hypoxia-inducible factor (HIF) transcription factors implement most of the critical changes in gene expression that enable animals to adapt to this stress. Here, we examine the roles of HIF-1 in post-mitotic aging. We examined the effects of HIF-1 over-expression and of hif-1 loss-of-function mutations on longevity in C. elegans, a powerful genetic system in which adult somatic cells are post-mitotic. We constructed transgenic lines that expressed varying levels of HIF-1 protein and discovered a positive correlation between HIF-1 expression levels and lifespan. The data further showed that HIF-1 acted in parallel to the SKN-1/NRF and DAF-16/FOXO transcription factors to promote longevity. HIF-1 over-expression also conferred increased resistance to heat and oxidative stress. We isolated and characterized additional hif-1 mutations, and we found that each of 3 loss-of-function mutations conferred increased longevity in normal lab culture conditions, but, unlike HIF-1 over-expression, a hif-1 deletion mutation did not extend the lifespan of daf-16 or skn-1 mutants. We conclude that HIF-1 over-expression and hif-1 loss-of-function mutations promote longevity by different pathways. These data establish HIF-1 as one of the key stress-responsive transcription factors that modulate longevity in C. elegans and advance our understanding of the regulatory networks that link oxygen homeostasis and aging.

Figure 1. Characterization of the hif-1 and hif-1(P621G) transgenes.

(A) Diagram of the minigenes that express epitope-tagged HIF-1. Genomic sequence including the hif-1 promoter sequence, exon 1, and intron 1, was fused to cDNA including exons 2–9 for the predominant hif-1 mRNA isoform (hif-1a). A myc epitope tag was inserted. A proline to glycine conversion at amino acid residue 621 prevents VHL-1-mediated degradation . (B) Representative protein blots show that epitope-tagged HIF-1 is stabilized by loss-of-function mutations in vhl-1 or egl-9. AHA-1 is ortholgous to HIFβ/ARNT , and the abundance of AHA-1 does not vary significantly in these strains. (C) Western blots show the transgene restores HIF-1-mediated gene expression. Pnhr-57:GFP is a reporter for HIF-1 activity . Its expression is dramatically reduced in hif-1(ia04) mutants and is restored by the iaIs28 integrated hif-1 transgene. (D) Oxygen-dependent degradation via the vhl-1/egl-9 pathway is abolished when the HIF-1 proline residue 621 is mutated to glycine.

Figure 2. HIF-1 over-expression extends longevity in a dose-dependent manner.

(A) Protein blots quantitate expression of HIF-1 and HIF-1(P621G) transgenes (tagged with the myc epitope). iaIs27 and iaIs28 contain integrated copies of the hif-1 minigene. iaIs32, iaIs33, and iaIs34 carry integrated copies of hif-1 (P621G). (B) Strains carrying hif-1 transgenes live longer (p<0.0001). Proportion alive is plotted over time. (C) The mean adult lifespans of strains over-expressing HIF-1 are positively correlated with the HIF-1 expression levels. Error bars represent the standard errors of the means.

Figure 3. HIF-1 over-expression extends the lifespan of daf-16-deficient and skn-1-deficient mutants.

(A, B) hif-1(P621G) transgenes (iaIs32 or iaIs34) extend the lifespan of animals carrying loss-of-function mutations in (A) daf-16 (p<0.0001) or (B) skn-1 (p<0.0001). (C) The longevity of daf-2(e1370) animals carrying a hif-1 (P621G) transgene is equivalent to that of daf-2(e1370) single mutants.

Figure 4. The daf-2 insulin-like receptor does not regulate expression of HIF-1 protein or its targets.

(A) A reduction of function mutation in daf-2 does not change the protein levels of epitope-tagged HIF-1 (iaIs28) or HIF-1(P621G) (iaIs32) in young adult animals (p>0.2 from three independent experiments). (B,C) The expression of two HIF-1 targets were assayed in wild-type animals and in animals carrying a loss-of-function mutation in daf-2. (B) Pnhr-57:GFP expression was assayed by protein blots and was not significantly different in these genetic backgrounds (p>0.2 from three independent experiments) (B) Quantitative RT-PCR revealed a marginal differences in the levels of K10H10.2 mRNA (p = 0.09).

Figure 5. hif-1 loss-of-function mutations extend the lifespan of wild-type animals, via pathway(s) that require daf-16 and skn-1.

(A,B) Characterization of hif-1 mutation alleles. (A) The hif-1 locus encodes 4 mRNA isoforms (www.wormbase.org). Exons are depicted as boxes, with coding sequences in black and untranslated regions in grey. The positions of two deletion alleles (ia04 and ok2564) and one nonsense mutation (ia07) are shown. (B) The hif-1 (ia07) point mutation introduces an early stop codon; (C) Protein blots show that all three mutations in hif-1 repress expression of the Pnhr-57::GFP reporter . Expression was assayed in vhl-1 (ok161) mutants, which are defective in oxygen-dependent degradation of HIF-1. (D) C. elegans carrying hif-1 loss-of-function mutations live longer than wild-type N2 (p<0.0001). (E) hif-1(ia04);daf-16(mu86) double mutant animals do not live longer than daf-16(mu86) single mutants. (F) daf-2 (e1370) single mutants and daf-2 (e1370); hif-1(ia04) double mutants have equivalent lifespans. (G) hif-1(ia04);skn-1(zu67) double mutant animals do not live longer than skn-1(zu67) single mutants.

Figure 6. hif-1 loss-of-function mutations and HIF-1 over-expression increase oxidative stress resistance and thermotolerance.

(A, B) Resistance to (A) t-butyl peroxide or (B) heat stress was assayed in hif-1 loss-of-function mutants and in animals over-expressing HIF-1. The daf-2 (e1370) and daf-16 (mu86) strains were included as a basis for comparison. Error bars represent the standard error of the means from at least 3 experiments. (*, p<0.0001 compared to wild type N2 worms using z-test).