Evidence for the functional activity of hypoxia-inducible transcription factors overexpressed in preeclamptic placentae

Abstract

Placentas from women with preeclampsia overexpress the hypoxia-inducible transcription factor proteins, HIF-1α and -2α (Rajakumar 2001, Biol Reprod 64; p499–506 and p1019–1020). As a first step in evaluating whether HIF-α overexpressed in preeclamptic placentae is capable of transactivation, we tested its ability to bind to the DNA hypoxia response element (HRE). Six pairs of normal and preeclamptic placentae obtained by cesarean section were investigated. Three biopsy sites per placenta were analyzed. We first confirmed HIF-1α protein overexpression in the preeclamptic placentae using Western analysis. The ratios of the arbitrary densitometry units for HIF-1α protein from the preeclamptic and normal placentae (PE/NP) in the three biopsy sites were: 1.9±0.3, 1.7±0.2 and 1.8±0.2, each p<0.05 vs 1.0. (A ratio of >1.0 indicates that HIF-1α protein expression in placentas of women with PE exceeds that in placentas of NP women.) Conventional methods for extracting nuclear proteins and subsequent analysis by electrophoretic mobility shift assay were not suited for the frozen, archived samples (data not shown). Therefore, we employed DNA affinity chromatography using a biotinylated oligonucleotide representing the HRE of the erythropoietin gene coupled to streptavidin-coated Dynabeads. The HRE-bound proteins were then characterized by Western blot analysis. The PE/NP ratios of HRE-bound HIF-1α in the three biopsy sites from the six pairs of normal and preeclamptic placentae were 1.7±0.2, 2.1±0.4 and 2.4±0.5, each p<0.05 vs 1.0. Having established DNA-binding potential at least in vitro, we subsequently analyzed three proteins that have been shown to be regulated by HIF-α as downstream, molecular markers of HIF-1α activity in vivo. VEGF receptor Flt-1 and Flk-1 play key roles in angiogenesis. Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis. All three genes contain functional HRE in their promoter sequences. Total proteins were extracted from the same biopsy samples that were used for total and HRE-bound HIF-1α. Using specific antibodies we performed Western analysis and the levels of these three proteins were quantitated. The Flt-1 and tyrosine hydroxylase proteins were significantly higher, and Flk-1 significantly lower in placentae from preeclamptic compared to normal pregnancies. In summary, HIF-1α protein overexpressed in preeclamptic placentae is capable of binding to its DNA recognition sequence in vitro, and modulates gene expression in vivo.