The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1)DNA recognition site

Abstract

The hypoxia-inducible factor-I (HIF-1) was first described as a DNA binding activity that specifically recognizes an 8 bp motif known to be essential for hypoxia-inducible erythropoietin gene transcription.Subsequently HIF-1 activity has also been found in cell lines which do not express erythropoietin, suggesting that HIF-1 is part of a widespread oxygen sensing mechanism. In electrophoretic mobility shift assays 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 HIFI probe. Here we report that CRE and AP1 oligonucleotides efficiently competed for binding of the HIF1 probe to this constitutive factor, whereas HIF-1 activity itself remained unaffected. Monoclonal antibodies raised against the CRE binding factors ATF-1 and CREB-1 supershifted the constitutive factor, while Jun and Fos family members, which constitute the AP-1 factor, were immunologically undetectable. Recombinant ATF-1 and CREB-1 proteins bound HIF1 probes either as homodimers or as heterodimers, indicating a new binding specificity for ATF-1/CREB-1. Finally, reporter gene assays in HeLa cells treated with either a cAMP analogue or a phorbol ester suggest that the PKA, but not the PKC signalling pathway is involved in oxygen sensing.

Regulation of interleukin-8 expression by reduced oxygen pressure in human glioblastoma

“…These results extend previous observations that the AP-1 sites of the collagenase and the DTdiaphorase genes are involved in the response to anoxia in carcinoma cell lines of the cervix (HeLa) and the colon (HT29), respectively (Rupec and Baeuerle, 1995;Yao et al, 1994). IL-8 gene activation by anoxia does not appear to be regulated through the HIF-1 transcription complex, unlike the VEGF gene, since no consensus binding site for HIF-1 (Kvietikova et al, 1995) was found in the published IL-8 gene sequence. Binding to the AP-1 consensus sequence found in the VEGF gene is also increased in the response to anoxia in glioma cells (Ryuto et al, 1996).…”

Oxygen-regulated Transferrin Expression Is Mediated by Hypoxia-inducible Factor-1

“…Nuclear Extract Preparation-Nuclear extracts were prepared as described previously (24). Briefly, 1 ϫ 10 8 cells were washed twice with ice-cold phosphate-buffered saline and once with buffer A (10 mM TrisHCl (pH 7.8), 1.5 mM MgCl 2 , 10 mM KCl).…”

“…4. The EPOHBS oligonucleotides have been described previously (24). All oligonucleotides (Microsynth) were purified on 10% polyacrylamide gels prior to 5Ј end-labeling of the sense strand with [␥-32 P]ATP (Hartmann) using T4-polynucleotide kinase (Fermentas).…”

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

“…The DNA binding activity of HIF-1 was determined after Vuori et al (2004), Soitamo et al (2001), Kvietikova et al (1995) as an indicator for hypoxic signalling. Nuclear extraction protocols, developed for rainbow trout (Soitamo et al 2001) were not applicable to small tissue samples.…”

“…Electrophoresis was performed at 150 V, room temperature, 2 h in TBE buffer (89 mM Tris, 89 mM boric acid, 5 mM EDTA). Dried gels were autoradiographed (Kvietikova et al 1995). Calculation of the percentage intensity was based on densitometry of gel images, with the sum of all four bands taken as 100% value.…”