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Abstract
Heme oxygenase-1 (HO-1) degrades heme and protects cells from oxidative challenge. This antioxidant activity is thought to result from the HO-1 enzymatic activity, manifested by a decrease in the concentration of the pro-oxidant substrate heme, and an increase in the antioxidant product bilirubin. Using a global transcriptional approach, and yeast as a model, we show that HO-1 affords cellular protection via up-regulation of transcripts encoding enzymes involved in cellular antioxidant defense, rather than via its oxygenase activity. Like mammalian cells, yeast responds to oxidative stress by expressing its HO-1 homolog and, compared with the wild type, heme oxygenase-null mutant cells have increased sensitivity toward oxidants that is rescued by overexpression of human HO-1 or its yeast homolog. Increased oxidant sensitivity of heme oxygenase-null mutant cells is explained by a decrease in the expression of the genes encoding γ-glutamylcysteine synthetase, glutathione peroxidase, catalase, and methionine sulfoxide reductase, because overexpression of any of these genes affords partial, and overexpression of all four genes provides complete, protection to the null mutant. Genes encoding antioxidant enzymes represent only a small portion of the 480 differentially expressed transcripts in heme oxygenase-null mutants. Transcriptional regulation may be explained by the nuclear localization of heme oxygenase observed in oxidant-challenged cells. Our results challenge the notion that HO-1 functions simply as a catabolic and antioxidant enzyme. They indicate much broader functions for HO-1, the unraveling of which may help explain the multiple biological responses reported in animals as a result of altered HO-1 expression.
Highlights
Heme oxygenase degrades heme to CO, Fe2ϩ, and biliverdin [1]
Expression of Hmx1p Is Induced in Response to Different Oxidants—As heme oxygenase-1 (HO-1) is induced in response to oxidant stress [4], we tested whether Hmx1p expression is induced in yeast cells, using an HA-tagged HMX1 strain and Western blotting with an anti-HA antibody [9]
Hmx1p in cells treated with H2O2 (4 mM, 6 h) was assessed by determining the respective density ratios of Hmx1p to either Nop2, Dpm1, or Kar2 from three separate experiments using ImageJ, with the value for the respective ratio for Hmx1-HAp set at 1. *, p Ͻ 0.05 compared with corresponding Hmx1-HAp (Wilcoxon Rank Sum Test)
Summary
mol/min/mgp wt wt HMX1 non-induced wt HMX1 induced hmx hmx HMX1 non-induced hmx HMX1 induced wt MXR1 induced. A ND, not detectable (detection limit, 0.1 nmol/min/mgp). NDa ND ND ND ND ND 1.3 Ϯ 0.2 as a positive and negative modulator of the transcription of aerobic and hypoxic genes, respectively [14]. It was recognized only recently that Hmx1p possesses classical heme oxygenase activity [15], raising the possibility that in addition to regulating cellular heme and iron levels, Hmx1p may share some of the additional activities of mammalian HO-1. We show that Hmx1p is induced in response to different stresses in addition to iron starvation, and that it protects yeast cells against oxidant challenge in a glutathione-dependent manner and via transcriptional regulation of genes encoding known enzymes involved in cellular antioxidant defense
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