Mitochondrial reactive oxygen species trigger hypoxia-induced transcription

Abstract

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1. 5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (rho0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) rho0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in rho degrees cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Figure 1

(A) Cellular O₂ uptake rates in wild-type and ρ⁰-Hep3B cells. (B) Southern blot analysis of undigested total cellular DNA from wild-type and ρ⁰-Hep3B cells. Hybridization was performed with a cytochrome oxidase subunit II probe, spanning bps 7757–8195, generated by reverse transcription–PCR. (C) EPO secretion from wild-type (wt) and ρ⁰-Hep3B cells exposed to normoxia (21% O₂/5% CO₂/74% N₂), hypoxia (1.5% O₂/5% CO₂/93.5% N₂), or CoCl₂ (100 μM) during normoxia for 24 hr (n = 3, mean ± SD). EPO was measured by radioimmunoassay (46). (D) Northern blot analysis of RNA from wild-type and ρ⁰-Hep3B cells during normoxia (21% O₂/5% CO₂/74% N₂), hypoxia (1.5% O₂/5% CO₂/93.5% N₂), or CoCl₂ (100 μM) during normoxia for 24 hr. ALDA, aldolase; PGK-1, phosphoglycerate kinase. (E) HIF-1 DNA binding in nuclear extracts from wild-type and ρ⁰-Hep3B cells. Both cell types were exposed to normoxia (21% O₂/5% CO₂/74% N₂), hypoxia (1.5% O₂/5% CO₂/93.5% N₂) or CoCl₂ (100 μM) during normoxia for 4 hr. C, constitutive; NS, nonspecific.

Figure 2

Effect of hypoxia and CoCl₂ on ROS generation. Graphs are averages from three experiments. (A) DCF fluorescence in wild-type cells at different levels of O₂. Graded hypoxia was produced in a flow-through chamber perfused with solutions equilibrated with different gas mixtures. Duration of hypoxia was 2 hr, beginning 30 min after baseline normoxic (21% O₂/5% CO₂/74% N₂) measurements. Recovery to normoxia was initiated at 150 min. (B) DCF fluorescence in wild-type cells during hypoxia (2% O₂/5% CO₂/93% N₂) and ebselen (25 μM). (C) DCF fluorescence in wild-type cells during hypoxia (2% O₂/5% CO₂/93% N₂) and PDTC (100 μM). (D) DCF fluorescence in ρ⁰-Hep3B cells during hypoxia (1% O₂/5% CO₂/94% N₂). (E) DCF fluorescence in wild-type cells during normoxic CoCl₂ (100 μM). (F) DCF fluorescence in wild-type cells during normoxic CoCl₂ (100 μM) and ebselen (25 μM). (G) Effect of PDTC (100 μM) on DCF fluorescence during CoCl₂ treatment in wild-type cells. (H) Effect of CoCl₂ on DCF fluorescence in ρ⁰ cells.

Figure 3

(A) Mitochondrial electron transport and ROS generation. Antimycin A and myxothiazol alter ROS generation by changing the lifetime of ubisemiquinone. Sites of inhibition are indicated with boxes. (B) DCF fluorescence in wild-type Hep3B cells during hypoxia (2% O₂/5% CO₂/93% N₂) and DPI (3 μM). (C) DCF fluorescence in wild-type cells during hypoxia (2% O₂/5% CO₂/93% N₂) and myxothiazol (100 ng/ml). (D) DCF fluorescence in wild-type cells during hypoxia (2% O₂/5% CO₂/93% N₂) and rotenone (3 μg/ml). (E) DCF fluorescence in wild-type cells during hypoxia (2% O₂/5% CO₂/93% N₂) and antimycin A (3 μg/ml). Graphs are representative examples.

Figure 4

(A) Northern blot analysis of RNA isolated from wild-type Hep3B cells during hypoxia (1.5% O₂/5% CO₂/93.5% N₂) or normoxic CoCl₂ in the presence of DPI (3 μM), myxothiazol (100 ng/ml), rotenone (3 μg/ml), ebselen (25 μM), or PDTC (100 μM) for 16 hr. (B) HIF-1 DNA binding in nuclear extracts from wild-type cells during hypoxia (1.5% O₂/5% CO₂/93.5% N₂) or normoxic CoCl₂ in the presence of DPI (3 μM), myxothiazol (100 ng/ml), rotenone (3 μg/ml), ebselen (25 μM), or PDTC (100 μM) for 4 hr. (C) Northern analysis of RNA from ρ⁰-Hep3B cells during CoCl₂ (100 μM) in the presence of ebselen (25 μM) or PDTC (100 μM) for 16 hr. (D) HIF-1 DNA binding in nuclear extracts from wild-type or ρ⁰ cells during CoCl₂ in the presence of ebselen (25 μM) or PDTC (100 μM) for 4 hr. C, constitutive; NS, nonspecific.

Figure 5

(A) ATP levels (Boehringer Mannheim Bioluminescence Kit) in wild-type and ρ⁰-Hep3B cells during 21% O₂, 1.5% O₂, 0% O₂, and antimycin (5 μg/ml) at 21% O₂ for 1 hr. (B) HIF-1 DNA-binding activity in nuclear extracts from wild-type cells during hypoxia (1.5% O₂/5% CO₂/93.5% N₂) in the presence of antimycin (5 μg/ml). (C) HIF-1 DNA binding in nuclear extracts from ρ⁰ cells during CoCl₂ (100 μM), 1.5% O₂, 0.5% O₂, and 0% O₂. C, constitutive; NS, nonspecific.

Figure 6

Intracellular DCF fluorescence in Hep3B cells after incubation at 21%, 5%, or 1.5% O₂ for 5 hr. Antimycin A (A.A, 5 μg/ml) was used to inhibit electron transport at site III. Cells treated with CoCl₂ were maintained under 21% O₂. (n = three experiments per group; means ± 1 SD.)