Repression of Heme Oxygenase-1 by Hypoxia in Vascular Endothelial Cells Masaharu Nakayama,* Kazuhiro Takahashi,* Tomomi Kitamuro,* Ken-ichi Yasumoto,* Dai Katayose,† Kunio Shirato,† Yoshiaki Fujii-Kuriyama,‡ and Shigeki Shibahara* ,1 *Department of Molecular Biology and Applied Physiology and †First Department of Internal Medicine, Tohoku University School of Medicine, Miyagi, Japan; and ‡Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Miyagi, Japan Received April 7, 2000 Heme oxygenase 1 (HO-1), a rate-limiting enzyme in heme catabolism, has been reported to be induced by hypoxia. Unexpectedly, here we show that expression of HO-1 mRNA is repressed by hypoxia in primary cultures of human umbilical vein endothelial cells (HUVECs), but is increased by cobalt chloride (CoCl 2 ) that is known to mimic hypoxia. Under the culture conditions used, the DNA-binding and transactivation activities of hypoxia-inducible factor 1 were increased in HUVECs by hypoxia or CoCl 2 . Therefore, hypoxia and cobalt showed opposing effects on HO-1 mRNA expression, despite activation of hypoxia-inducible factor 1. The half-life of HO-1 mRNA was not changed by hypoxia, but was significantly prolonged by CoCl 2 . Hypoxia also represses HO-1 mRNA expression in hu- man coronary artery endothelial cells and astrocytes. The repression of HO-1 expression may represent the adaptation to hypoxia in certain cell types. © 2000 Academic Press Key Words: bilirubin; cobalt; desferrioxamine; endo- thelial cells; heme; hypoxia-inducible factor; oxygen sensor; stress response. Heme oxygenase is the rate-limiting enzyme in heme catabolism, which cleaves heme to release iron, carbon monoxide, and biliverdin at the expense of molecular oxygen and NADPH (1). Biliverdin is reduced to bili- rubin by biliverdin reductase (1). Carbon monoxide is an endogenous gas molecule that may activate guanylyl cyclase to regulate vascular tone. Biliverdin and bilirubin act as antioxidants (2). There are two well-characterized isozymes of heme oxygenase (3, 4). Heme oxygenase 1 (HO-1) is an inducible form and HO-2 is a constitutively expressed form. A number of reports have shown that HO-1 is induced by a variety of stimulants, such as its substrate heme, heat shock, heavy metals, nitric oxide donors, and UV light (3–10). HO-1 is therefore regarded as a stress-response protein that protects the cells against the oxidative stress. Hypoxia (low oxygen tension) has been shown to induce HO-1 expression in rat hearts (11) and in cul- tured rodent cells and human fibroblasts (12–15). The induction of HO-1 by hypoxia was shown to be medi- ated by hypoxia-inducible factor 1 (HIF1) in rodent cells (12–14). HIF1 is functionally activated by hypoxia and mediates transcriptional activation through bind- ing to the hypoxia response element (HRE) of target genes, including erythropoietin, vascular endothelial growth factor (VEGF), and glycolytic enzymes (16 –19). HIF1 is a basic helix-loop-helix-PAS (Per-Arnt-Sim) transcription factor, composed of HIF1 and HIF1 (20). HIF1 is also activated by the iron chelator, des- ferrioxamine, and transition metals, such as cobalt and nickel (21, 22). Desferrioxamine and cobaltous ions have been assumed to chelate and substitute for the central iron of the heme, respectively, thereby causing a conformational change of a hypothetical heme- containing oxygen sensor (22). The hypoxia-mediated induction of HO-1 was abro- gated in the HIF1-deficient mouse hepatoma cell line (12). On the other hand, Wood et al. reported that HO-1 was induced by hypoxia in HIF1-deficient Chinese hamster ovary cells (13), indicating that hypoxia- induced HO-1 expression is independent of HIF1. In this context, HLF, HIF1-like factor, forms complex with HIF1, and such a heterodimer also regulates gene expression in response to hypoxia through bind- ing to HRE of the target gene (23, 24). Taken together, these findings suggest that hypoxia induces HO-1 mRNA expression via functional activation of HIF1 or HLF. 1 To whom correspondence should be addressed at Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan. Fax: 81-22-717-8118. E-mail: shibahar@mail.cc. tohoku.ac.jp. Biochemical and Biophysical Research Communications 271, 665– 671 (2000) doi:10.1006/bbrc.2000.2683, available online at http://www.idealibrary.com on 665 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.