The human placenta expresses multiple glucocorticoid receptor isoforms that are altered by fetal sex, growth restriction and maternal asthma

Abstract

Introduction

We have previously identified sex-specific differences in the fetal-placental response to cortisol. Our recent studies suggest that this differential response to cortisol is driven by differences in glucocorticoid receptor (GR) protein function rather than through changes in gene transcription or protein expression.

Methods

This study was designed to define whether the human placenta expresses different isoforms of the GR and whether expression was altered by fetal sex and maternal asthma. Asthma and non-asthma pregnant women were prospectively recruited at their first antenatal visit and placentae collected at delivery. Placental GR expression was examined in relation to maternal asthma, fetal sex and birthweight.

Results

Twelve specific bands for the GR were identified at molecular weights of 94, 91, 81, 74, 69, 68, 65, 60, 55, 50, 48 and 38 kDa. The 12 isoforms were localised to the placental trophoblast and expression varied in relation to cellular location in either the cytoplasm or nucleus, fetal sex, fetal size and the presence and absence of maternal asthma.

Conclusion

This is the first study to identify the presence of several protein isoforms of the GR in the human placenta. The data suggest glucocorticoid resistance observed in male placentae may be mediated through increased GRβ, GR A and GR P localisation to the nucleus. While female placentae may be more sensitive to cortisol in the presence of maternal asthma through a decrease in GRβ and an enhancement GRα activity via an interaction with GRα D3 and GRα C.

Introduction

Excess exposure to maternal glucocorticoids during pregnancy can significantly impact lifelong health [1]. There is some evidence to suggest that programming effects on fetal development are mediated by the placenta and induced in a sex-specific manner via changes in placental function [2]. We have previously identified sex-specific differences in the fetal-placental response to cortisol [3], [4], [5]. From this body of work, male fetuses appear to induce a state of glucocorticoid resistance in an environment of excess glucocorticoids while females appear hypersensitive to changes in glucocorticoid concentration [5]. Our recent studies suggest that this differential response to cortisol is driven by differences in glucocorticoid receptor (GR) protein function [4] rather than changes in GR gene transcription or GR protein expression [6].

GR is a ubiquitously expressed member of the nuclear receptor transcription factor super family. There is one human GR gene which is comprised of 8 translated exons and 9 untranslated first exons (exon 1) spanning 80 kB. The untranslated exon 1 of the GR gene can be spliced into 9 different promoter variants [7] that function in a tissue specific manner to regulate GR protein expression. Exons 2–9 can generate various isoforms of GR through alternative splicing [8], [9], [10] or through alternative initiation of translation [10], [11] resulting in the expression of GRα, GRβ, GRγ, GR-A and GR-P proteins. GRα is the functional isoform involved in transcriptional activation and transcriptional repression of multiple targets. Moreover, eight different GRα translational isoforms can originate from GRα mRNA. GRα-A and GRα-B encode for proteins of 94 and 91 kDa respectively. GRα C1–C3 (81–83 kDa) and GRα D1–D3 (50–55 kDa) proteins are also GR translational variants. It has been suggested that the translation of these isoforms function in a tissue specific manner [10]. All translational GRα variants have the ability to translocate into the nucleus to regulate transcriptional activities. GRβ inhibits activation of GRα through a dominant negative mechanism. Splice variants GRγ (95 KDa), GR-A (65 KDa) and GR-P (75 KDa) have low transactivation activities [12], [13].

We have previously shown that splice variants of GRα including GRβ, GR-P and GRγ mRNA are all detectable in the human placenta with GRα and promoter 1C showing the highest expression [14]. In pregnancies complicated by asthma we have additionally shown GR gene activity was positively correlated with cord blood cortisol compared to healthy controls [6]. Based on the evidence we have collected so far, we hypothesise that sex-specific differences in cortisol sensitivity in fetal-placental tissues are controlled by the interaction of the functional GRα with other GR isoforms. In the current study we aim to define the sex-specific expression, phosphorylation status and intracellular locality of the human GR isoforms in the term placentae of normal pregnancies and pregnancies complicated by maternal asthma.