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

Abstract

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.

Figure 1

Increased PML immunoreactivity in preeclampsia. Sections of cotyledon from normal and preeclamptic placentae were reacted with anti-PML antibodies and localized with Alexa-fluor goat anti-rabbit Fab’2 secondary. Representative sections of PML immunoreactivity within the central cotyledon (panel A), a paracentral cotyledon (panel B) and a peripheral cotyledon (panel C) are shown (600x). All images were captured with camera settings identical to the non-immune IgG threshold exposure level (IgG).

Figure 2

Nuclear PML protein expression in normal and preeclamptic placentae. A. Representative immunoblot of PML expression in normal and preeclamptic placentae. Nuclear lysates were separated by SDS-PAGE, transferred to nitrocellulose and probed with anti-PML. Blots were stripped and reprobed with antibodies to β-actin to control for loading consistency. B. Increased PML expression in preeclamptic placental lysates. Normalized PML/β-actin expression in uncomplicated pregnancies was set = 1.0 (n = 6 for each group except cotyledon 3 where n = 5). Normalized PML expression from all 3 sampled regions of the preeclamptic placenta within each region (left panel) and pooled normative values of overall PML protein expression in preeclamptic versus normal samples (right panel) are shown.

Figure 3

Expression of PML mRNA is decreased in preeclamptic placentae. Total RNA was extracted from villus samples of normal and preeclamptic placentae and relative gene expression evaluated by real time RT-PCR for PML, PGF, GCM-1, and sflt-1. Relative gene expression in the preeclamptic placentae was normalized to levels in the pool of normal placentae. All findings were significantly different from normals (set to 100%).

Figure 4

Induction of nuclear PML expression in primary endothelial cells but not trophoblast in vitro. A. Immunofluorescence reactivity of PML in cells cultured in either standard conditions (21%O₂), 200U interferon-β, or 1%O₂. Cells were fixed, permeablized and reacted with antibodies to PML (100x magnification). B. Nuclear protein extracts from parallel cultures were subjected to immunoblotting with antibodies to PML as in Figure 2. Results are representative of three (HUVEC) or two (trophoblast) independent experiments with identical results.