Hypoxia-inducible factor-1β is essential for upregulation of the hypoxia-induced FLT1 gene in placental trophoblasts

Abstract

Placental hypoxia and increased levels of maternal blood anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT1), are associated with the pathogenesis of preeclampsia. We have demonstrated that hypoxia-inducible factor (HIF)-2α mediates the upregulation of the hypoxia-induced FLT1 gene in trophoblasts and their cell lines. Here, we investigated the involvement of HIF-1β, which acts as a dimerisation partner for HIF-α, in the upregulation of the FLT1 gene via hypoxia. We confirmed the interactions between HIF-1β and HIF-2α in the nuclei of BeWo, JAR and JEG-3 cells under hypoxia via co-immunoprecipitation. We found that hypoxia-induced upregulation of the FLT1 gene in BeWo cells and secretion of sFLT1 in human primary trophoblasts were significantly reduced by siRNAs targeting HIF-1β. Moreover, the upregulation of the FLT1 gene in BeWo cells induced by dimethyloxalylglycine (DMOG) was also inhibited by silencing either HIF-2α or HIF-1β mRNA. It was recently shown that DNA demethylation increases both basal and hypoxia-induced expression levels of the FLT1 gene in three trophoblast-derived cell lines. In the demethylated BeWo cells, siRNAs targeting HIF-2α and HIF-1β suppressed the further increase in the expression levels of the FLT1 gene due to hypoxia or treatment with DMOG. However, luciferase reporter assays and bisulfite sequencing revealed that a hypoxia response element (–966 to –962) of the FLT1 gene is not involved in hypoxia or DMOG-induced upregulation of the FLT1 gene. These findings suggest that HIF-1β is essential for the elevated production of sFLT1 in the hypoxic trophoblasts and that the HIF-2α/HIF-1β complex may be a crucial therapeutic target for preeclampsia.

Overexpression of Human sFLT1 in the Spongiotrophoblast Is Sufficient to Induce Placental Dysfunction and Fetal Growth Restriction in Transgenic Mice

“…The mice with placenta-specific hsFLT1 overexpression also showed signs of increased inflammation, as the expression of TNFα was upregulated by placental hsFLT1. The pro-inflammatory cytokine TNFα is expressed and secreted physiologically from syncytiotrophoblast cells in the placenta [42], although upregulated placental TNFα expression in pregnant women is associated with FGR [43]. Here, in this study, the Tnfα mRNA level was upregulated in FGR placentas at 14.5 dpc, resulting in elevated protein levels at 18.5 dpc due to placental hsFLT1 overexpression.…”

Epigenetic drug screening for trophoblast syncytialization reveals a novel role for MLL1 in regulating fetoplacental growth

“…Numerous studies have confirmed a close relationship between tissue hypoxia and the occurrence of syncytialization dysregulation-related diseases, such as PE and IUGR [42][43][44][45]. According to the RNA-seq data from Ren et al [46,47], KEGG analyses indicated a significant activation of the HIF-1 signaling pathway in early, late, and all stages of PE placenta samples (Fig.…”

CRISPR/Cas9-mediated mutations in both a cAMP response element and an ETS-binding site suppress FLT1 gene expression

“…Various 5′-deletion constructs of pFLT1-Luc (−1629/+8) [21], which is a promoter-less firefly luciferase vector pGL4.10 (Promega, Madison, WI, USA) containing the 5'-untranslated region and exon 1 of the FLT１ gene (GenBank accession no. JX512442, positions 2374 to 4010), were prepared using a kilosequence deletion kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions.…”

“…Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) with a luminometer (Gene Light 210A; Microtech Nichion, Chiba, Japan) as previously described [21]. The firefly luciferase activity of the reporter plasmid was normalized to the Renilla luciferase activity.…”

“…In vitro studies have shown that sFLT1 production increases under hypoxic conditions in human primary placental trophoblasts [17][18][19]. We have recently found that hypoxia-inducible factor-2α (HIF-2α) and HIF-1β, but not HIF-1α, mediate hypoxia-induced up-regulation of FLT1 expression not only in human trophoblast-derived cell lines but also in human primary trophoblasts in vitro [20,21]. However, the basal transcriptional machinery of FLT1 in trophoblasts has not been fully elucidated.…”