HIF-2α, but not HIF-1α, mediates hypoxia-induced up-regulation of Flt-1 gene expression in placental trophoblasts

Abstract

Placental hypoxia and elevated levels of circulating soluble Fms-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic factor, are closely related to the pathogenesis of preeclampsia. Although sFlt-1 secretion from the placental trophoblasts is increased under hypoxic conditions, the underlying molecular mechanism remains unclear. Previously, an authentic hypoxia response element in the Flt-1 gene promoter was shown to be a potential binding site for hypoxia-inducible factors (HIFs). Here, we investigated the roles of HIF-1α and HIF-2α in Flt-1 gene expression in trophoblast-derived choriocarcinoma cell lines and cytotrophoblasts exposed to hypoxic conditions. In the cell lines, increased expression of sFlt-1 splice variants and nuclear accumulation of HIF-1α and HIF-2α were observed after hypoxic stimulation. A specific small interfering RNA or an inhibitor molecule targeting HIF-2α decreased hypoxia-induced up-regulation of Flt-1 gene expression. Moreover, in cytotrophoblasts, increased sFlt-1 mRNA expression and elevated sFlt-1 production were induced by hypoxic stimulation. Notably, hypoxia-induced elevation of sFlt-1 secretion from the cytotrophoblasts was inhibited by silencing the HIF-2α, but not HIF-1α mRNA. These findings suggest that hypoxia-induced activation of HIF-2α is essential for the increased production of sFlt-1 proteins in trophoblasts. Targeting the HIF-2α may be a novel strategy for the treatment of preeclampsia.

Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells

“…During this process, the HIF-α/HIF-1β heterodimer binds the consensus HRE sequence 5′-(A/G)CGTG-3′ of the target genes [24]. We recently discovered that HIF-2α, not HIF-1α, mediates the hypoxia-induced up-regulation of FLT1 expression in choriocarcinoma cell lines (BeWo, JAR, and JEG-3) and primary trophoblasts [21]. The HRE motif contains one CpG dinucleotide, and it has been confirmed that CpG methylation within this sequence prevents the Fig.…”

“…RNA was extracted and mRNA expression was assessed using qRT-PCR as described previously [21]. All data were normalized to β-actin expression or GAPDH expression.…”

“…The cell lysates were subjected to immunoprecipitation using Dynabeads Protein G (Invitrogen) bound to antihuman FLT1 monoclonal antibody reacted with the first Ig-like domain of FLT1 (KM1730) as described previously [21]. The precipitated proteins were subjected to Western blotting as described above.…”

“…Secreted sFLT1 isoforms within the conditioned medium were concentrated using a previously described pulldown method with Heparin-Sepharose beads [21].…”

“…Recently, we reported that hypoxia-inducible factor-2α (HIF-2α) mediates the hypoxia-induced up-regulation of FLT1 gene expression in the BeWo, JAR and JEG-3 three choriocarcinoma cell lines [21]. Under hypoxia, HIF-2α forms a heterodimer with HIF-1β, binds to the hypoxia response element (HRE) of target genes, and induces hypoxia-related gene expression [24].…”