THE HYPOXIA-INDUCIBLE FACTOR-I DNA RECOGNITION SITE IS CAMP-RESPONSIVE

The hypoxia-inducible factor-1 (HIF-1) was first described as a DNA bi nding activity that specifically recognizes an 8 bp hypoxia response e lement (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 cell s cultivated in a low oxygen atmosphere. In addition to HIF-1, a const itutive 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 condition s. These results were confirmed by using the protein kinase A (PKA) ac tivator Sp-cAMPS and the PKA inhibitor Rp-cAMPS: while Sp-cAMPS was sy nergistic 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.