Mitochondrial reactive oxygen species trigger hypoxia-induced transcription

Mitochondrial reactive oxygen species trigger hypoxia-induced transcription

Departments of ^*Medicine, ^†Pathology, ^‡Biochemistry and Molecular Biology, ^§Molecular Genetics and Cell Biology and The Howard Hughes Medical Institute, The University of Chicago, Chicago, IL 60637

Edited by Stuart H. Orkin, Harvard Medical School, Boston, MA, and approved August 4, 1998 (received for review March 16, 1998)

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 (ρ⁰ 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) ρ⁰ 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 ρ° 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.

Footnotes

↵ ¶ To whom reprint requests should be addressed at: Department of Medicine MC6026, The University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. e-mail: pschumac{at}medicine.bsd.uchicago.edu.
This paper was submitted directly (Track II) to the Proceedings Office.
ABBREVIATIONS:

EPO,

erythropoietin;

VEGF,

vascular endothelial growth factor;

ROS,

reactive oxygen species;

PDTC,

pyrrolidine dithiocarbamate;

HIF-1,

hypoxia inducible factor 1;

DCFH,

2′,7′-dichlorofluorescin;

DCF,

2′,7′-dichlorofluorescein;

DPI,

diphenylene iodonium;

DHR-123,

dihydrorhodamine 123;

N-MPG,

N-(2-mercaptopropionyl)-glycine