Hypoxic Regulation of Lactate Dehydrogenase A
INTERACTION BETWEEN HYPOXIA-INDUCIBLE FACTOR 1 AND cAMP RESPONSE ELEMENTS (*)
- From the Erythropoietin Group, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom
- § To whom correspondence should be addressed: Rm. 420, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United Kingdom. Tel.: 44-865-222381; Fax: 44-865-222500.
Abstract
The oxygen-regulated control system responsible for the induction of erythropoietin (Epo) by hypoxia is present in most (if not all) cells and operates on other genes, including those involved in energy metabolism. To understand the organization of cis-acting sequences that are responsible for oxygen-regulated gene expression, we have studied the 5′ flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH). Deletional and mutational analysis of the function of mouse LDH-reporter fusion gene constructs in transient transfection assays defined three domains, between −41 and −84 base pairs upstream of the transcription initiation site, which were crucial for oxygen-regulated expression. The most important of these, although not capable of driving hypoxic induction in isolation, had the consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-competed for the binding of HIF-1 with functionally active Epo and phosphoglycerate kinase-1 sequences. The second domain was positioned close to the HIF-1 site, in an analogous position to one of the critical regions in the Epo 3′ hypoxic enhancer. The third domain had the motif of a cAMP response element (CRE). Activation of cAMP by forskolin had no effect on the level of LDH mRNA in normoxia, but produced a magnified response to hypoxia that was dependent upon the integrity of the CRE, indicating an interaction between inducible factors binding the HIF-1 and CRE sites.
Footnotes
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↵¶ Rhodes Scholar.
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↵* This work was supported in part by the Wellcome Trust and the Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- Epo
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erythropoietin
- HIF-1
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hypoxia-inducible factor 1
- LDH
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lactate dehydrogenase
- bp
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base pair(s)
- CRE
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cAMP response element
- GH
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growth hormone
- H8
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N-(2-[methylamino] ethyl)-5-isoquinolinesulfonamide
- PGK
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phosphoglycerate kinase 1.
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- Received February 21, 1995.
- Revision received July 6, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
