The hypoxia-inducible factor-1 DNA recognition site is cAMP-responsive.
The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp hypoxia response element (HRE) known to be essential for oxygen-regulated erythropoietin gene expression. In electrophoretic mobility shift assays (EMSAs) HIF-1 DNA binding activity is only detectable in nuclear extracts of cells cultivated in a low oxygen atmosphere. In addition to HIF-1, a constitutive DNA binding activity also specifically binds the HIF-1 probe. Based on EMSAs using competitor oligonucleotides, specific antibodies and recombinant proteins, we previously reported that the constitutive HRE binding factor is composed of ATF-1 and CREB-1. Here we show that this site is functionally responsive to the cAMP agonist 8Br-cAMP in a dose-dependent manner under hypoxic but not under normoxic conditions. These results were confirmed by using the protein kinase A (PKA) activator Sp-cAMPS and the PKA inhibitor Rp-cAMPS: while Sp-cAMPS was synergistic with hypoxia on the HIF-1 DNA recognition site, the Rp-cAMPS isomer showed no effect. Our findings suggest that the PKA-signaling pathway is enhancing oxygen-dependent gene expression via the HRE.
Kvietikova
I
I
Institute of Physiology, University of Zürich-Irchel, Switzerland.
Wenger
R H
RH
Marti
H H
HH
Gassmann
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M
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D001665:Binding Sites; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004921:Erythropoietin / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D014157:Transcription Factors; D014162:Transfection
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D014157:Transcription Factors; D004921:Erythropoietin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases