Hydrogen sulfide increases hypoxia-inducible factor-1 activity independently of von Hippel-Lindau tumor suppressor-1 in C. elegans

Abstract

Rapid alteration of gene expression in response to environmental changes is essential for normal development and behavior. The transcription factor hypoxia-inducible factor (HIF)-1 is well known to respond to alterations in oxygen availability. In nature, low oxygen environments are often found to contain high levels of hydrogen sulfide (H(2)S). Here, we show that Caenorhabditis elegans can have mutually exclusive responses to H(2)S and hypoxia, both involving HIF-1. Specifically, H(2)S results in HIF-1 activity throughout the hypodermis, whereas hypoxia causes HIF-1 activity in the gut as judged by a reporter for HIF-1 activity. C. elegans require hif-1 to survive in room air containing trace amounts of H(2)S. Exposure to H(2)S results in HIF-1 nuclear localization and transcription of HIF-1 targets. The effects of H(2)S on HIF-1 reporter activity are independent of von Hippel-Lindau tumor suppressor (VHL)-1, whereas VHL-1 is required for hypoxic regulation of HIF-1 reporter activity. Because H(2)S is naturally produced by animal cells, our results suggest that endogenous H(2)S may influence HIF-1 activity.

Figure 1.

HIF-1 protein levels increase when worms are exposed to H₂S. (A) A representative Western blot analysis shows HIF-1 protein increases when worms are exposed to 50 ppm H₂S or 0.5% O₂. The blots were stripped and reprobed with an anti-actin antibody to confirm equivalent amounts of total protein. The size markers are shown in kilodaltons. (B) Average increase in signal relative to control ± SD of three independent experiments. *p < 0.05 (Student's t test) compared with air-treated animals.

Figure 2.

H₂S exposure causes HIF-1 nuclear localization. Both H₂S and hypoxia (0.5% O₂) treatment induce HIF-1 nuclear localization throughout the animal. Images are stacks of all nuclei visible with DAPI staining. Animals were stained with anti-Ce-Hif-1 antibody (a, d, and g) and DAPI (b, e, and h) to visualize nuclei. Hypodermal nuclei (tailless arrows) and intestinal nuclei (tailed arrows) are clearly observed in the merged images (c, f, and i). (A) Anterior of the worm. (B) Posterior of the worm. Bars, 50 μm.

Figure 3.

H₂S exposure increases expression of NHR-57::GFP. A midfocal plane of a fluorescent confocal photomicrograph and Nomarski micrograph. Fluorescence is not apparent in untreated L4 nhr-57::gfp(iaIs07) transgenic animals. An nhr-57::gfp(iaIs07) animal exposed to hypoxia exhibits HIF-1 activity in the in the gut but absent in the hypodermis. At 1 h into H₂S treatment, fluorescence is apparent in the hypodermis throughout the animal but undetectable in the gut. Fluorescence becomes more intense until 3 h into exposure. nhr-57::gfp(iaIs07); hif-1(ia04) animals fail to show any fluorescence, regardless of H₂S treatment. Bar, 40 μm.

Figure 4.

H₂S exposure increases mRNA levels of HIF-1 target genes. Real-time reverse transcription-PCR was used to quantitate mRNA levels of HIF-1 target genes in wild-type or hif-1(ia04) worms exposed to H₂S or hypoxia. The cDNA of nhr-57 (A) or K10H10.2 (B) is shown as the average increase ± SD relative to sir-2.1 cDNA levels, which do not change during H₂S exposure.

Figure 5.

H₂S dependent HIF-1 activity is independent of VHL-1. nhr-57::gfp(iaIs07); egl-9(sa307) exhibits intense fluorescence is apparent throughout the animal, including in the hypodermis and the gut. 100 Untreated nhr-57::gfp(iaIs07); vhl-1(ok161) animals show basal GFP expression in the gut and H₂S treatment induces hypodermal expression. These results are representative of 100 of 100 worms examined for each panel. Bar, 40 μm.