Dysregulation of promyelocytic leukemia (PML) protein expression in preeclamptic placentae.

Promyelocytic leukemia (PML) protein is a nucleoprotein that can regulate a variety of cellular stress responses. The aim of this study was to determine qualitative and quantitative changes in PML expression in preeclamptic placentae. Immunoblot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunohistochemistry techniques were used to determine PML gene expression and localization in normal (n = 6) and preeclamptic (n = 6) placentae and primary cells. Promyelocytic leukemia protein was immunolocalized within nuclei of villus mesenchyme, but largely absent in trophoblast nuclei, with a trend for increased PML reactivity in preeclamptic placenta. Immunoblot analyses of nuclear extracts confirmed relative increases (approximately 3-fold) of PML expression in preeclamptic placentae (P < .05). Conversely, less PML messenger RNA (mRNA; approximately 2-fold) was detected in preeclamptic versus normal placental samples. In vitro, PML expression could be increased by hypoxia in cultured endothelial cells but not trophoblast. Increased PML protein expression in preeclamptic villi suggests it could contribute to decreased vascularity and placental growth and/or function.

Glial cell missing 1 regulates placental growth factor (PGF) gene transcription in human trophoblast.

Placental growth factor (PGF, previously known as PlGF) is prominently expressed by trophoblasts in human placenta, whereas most nontrophoblast cells express low levels of PGF mRNA under normal physiological conditions. We have shown that hypoxia decreases PGF expression in the trophoblast, but little is known about transcriptional regulation of PGF gene expression. We sought to determine promoter regions of the human PGF gene that contribute to its restricted high constitutive expression in the trophoblast. Overlapping putative promoter regions of human PGF gene encompassing 2-1.5 kb were cloned into reporter vectors and co-transfected into trophoblast and nontrophoblast cell lines. Promoter activity generated by a 2-1.5-kb clone was significantly higher in trophoblasts than in nontrophoblasts. Selective deletion mutants showed that a clone encompassing the PGF (2-828/++34) region generated promoter activity similar to the 2-1.5-kb region in the trophoblast. However, deletion of another 131 bp from this subclone (2-698/++34) resulted in significantly less promoter activity in the trophoblast. The (2-828/2-698) region significantly enhanced activity of a minimal promoter construct in trophoblast but not in nontrophoblast cells, suggesting that this region contributes to regulating PGF transcription in the trophoblast. Site-directed mutagenesis of a glial cell missing 1 (GCM1) motif in the 131-bp region significantly decreased enhancer activity in the trophoblast. Furthermore, overexpression of GCM1 significantly increased PGF 2-1.5-kb promoter activity and PGF mRNA expression in trophoblast and nontrophoblast cells. Forced overexpression of GCM1 restored PGF expression in the hypoxic trophoblast. These data support a functional role for GCM1 contributing to constitutively high trophoblast PGF expression and is the first direct evidence of an oxygen-responsive, trophoblast-specific transcription factor contributing to the regulation of PGF expression.