Estrogen-related receptor γ serves a role in blood pressure homeostasis during pregnancy

Abstract

Persistent hypoxia caused by shallow trophoblast invasion and poor placental perfusion may underlie the pathophysiology of preeclampsia, a leading cause of maternal and neonatal morbidity and mortality. Previously, we found that estrogen-related receptor γ (ERRγ) serves a critical and O2-dependent role in differentiation of human trophoblasts in culture and expression of tissue kallikrein and voltage-gated K(+) channels. In this study, we surprisingly observed that ERRγ expression was significantly increased in placentas from preeclamptic women compared with that in gestation-matched normotensive women. To further investigate a functional role for ERRγ during pregnancy, we analyzed ERRγ-deficient mice. Maternal systolic blood pressure was significantly reduced in pregnant ERRγ(+/-) females bred to ERRγ(+/-) males compared with that in wild-type (WT) mice and was markedly up-regulated by treatment of WT pregnant mice with the ERRγ agonist DY131. Placentas of ERRγ(+/-) mice manifested increased vascular endothelial growth factor A expression compared with that in WT mice. Notably, circulating levels of the antiangiogenic factor, soluble fms-like tyrosine kinase-1, were significantly reduced in ERRγ(+/-) pregnant mice as was serum aldosterone. These effects were associated with a decrease in maternal adrenal Cyp11b1 (steroid 11β-hydroxylase) and Cyp11b2 (aldosterone synthase) expression. In contrast, adrenal Cyp11b1 and Cyp11b2 mRNA were increased in pregnant WT mice treated with DY131. Moreover, chromatin immunoprecipitation and luciferase reporter assays identified Cyp11b2 as a transcriptional target of ERRγ. Collectively, these findings reveal a potential role of ERRγ in maternal blood pressure homeostasis during pregnancy and suggest that aberrant ERRγ expression may contribute to the pathogenesis of preeclampsia.

Figure 1.

ERRγ expression is increased in placentas of preeclamptic women. Placental tissues of preeclamptic and gestation-matched normotensive women near term were analyzed for ERRγ mRNA (A) and protein (B). An immunoblot of ERRγ protein in placental samples from 7 normotensive and 7 preeclamptic women (B) was analyzed by densitometric scanning and plotted relative to β-actin (C). *, P < .05.

Figure 2.

ERRγ serves an important role in BP homeostasis. Systolic BP was measured from 14.5 to 18.5 dpc in following groups: A, ERRγ^+/− females mated with ERRγ^+/− males (♀HET [heterozygous] × ♂HET) and WT females mated with WT males (♀WT × ♂WT); B, WT females treated with ERRγ agonist DY131 or vehicle (DMSO) from 10.5 to 17.5 dpc; C, ERRγ^+/− females mated with WT males (♀HET × ♂WT) and WT females mated with ERRγ^+/− males (♀WT × ♂HET); D, nonpregnant ERRγ^+/− and WT females mice compared with those of ERRγ^+/− females mated with ERRγ^+/− males (HET) and WT females mated with WT males (WT) at 18.5 dpc. Values are means ± SEM. *, P < .05; **, P < .01; ***, P < .001.

Figure 3.

ERRγ deficiency enhances VEGFA expression and placental angiogenesis and inhibits serum sFlt-1 levels during pregnancy. A, Placentas from WT females bred to WT males (WT, a and c) and ERRγ^−/− placentas from ERRγ^+/− females bred to ERRγ^+/− males (ERRγ^−/−, b, d, and e) were collected at 18.5 dpc and visualized with H&E (a and b) or by immunohistochemical analysis for endomucin (c and d; panel e is the negative control) (×400, data are representative of n = 4 placentas in each group). B and C, Endomucin (B) and VEGFA (C) mRNA was analyzed in WT placentas from WT females bred to WT males and in ERRγ^−/− (homozygous [HOMO]) placentas from ERRγ^+/− females bred to ERRγ^+/− males. D, sFlt-1 was assayed in serum harvested at 18.5 dpc from WT females bred to WT males (WT), from ERRγ^+/− females bred to ERRγ^+/− males (HET) and from a corresponding series of nonpregnant ERRγ^+/− or WT female mice (n = 6). E, Systolic BP was measured from 14.5 to 18.5 dpc in ERRγ^+/− and WT dams bred to ERRγ^+/− and WT males, respectively, after treatment with the eNOS inhibitor, l-NAME. Values are means ± SEM. **, P < .01; ***, P < .001.

Figure 4.

ERRγ^+/− pregnant mice manifest poor salt handling leading to hypotension. A and C, Urine was collected over a 24-hour period each day from 14.5 to 18.5 dpc. Total urine Na⁺ excretion per 24-hour period was assayed in ERRγ^+/− and WT dams that were untreated (nontreated, A) or administered high salt (2% salt, C). B, WT females mated with WT males (WT) or ERRγ^+/− females mated with ERRγ^+/− males (HET) mice were either untreated or given 2% salt from 10.5 to 18.5 dpc. Serum Na⁺ concentrations were assayed at 18.5 dpc (n = 4–9). D, Systolic BP was measured at 14.5 to 18.5 dpc in ERRγ^+/− females mated with ERRγ^+/− males (HET) and WT females mated with WT males (WT) with high-salt (2%) treatment. Values are the means ± SEM. *, P < .05; **, P < .01.

Figure 5.

ERRγ expressed in the mouse adrenal cortex modulates aldosterone production by regulating Cyp11b1 and Cyp11b2. A, Serum aldosterone was assayed in pregnant ERRγ^+/− and pregnant WT mice at 18.5 dpc and in nonpregnant WT or ERRγ^+/− female mice (n = 6–7). B–D, mRNA expression of ERRγ (B), Cyp11b1 (C) and Cyp11b2 (D) was analyzed in adrenals of ERRγ^+/− or WT pregnant mice at 18.5 dpc and of nonpregnant ERRγ^+/− females and WT female mice. E and F, Cyp11b1 (E) and Cyp11b2 (F) mRNA was analyzed in adrenals from 18.5 dpc WT dams injected daily with ERRγ agonist DY131 or vehicle. G, RNA isolated from zona glomerulosa-enriched and zona fasciculata/reticularis-enriched segments of adrenal glands of nonpregnant ERRγ^+/− and WT female mice was analyzed for ERRγ and Cyp11b2 by qRT-PCR. H, Adrenal glands from nonpregnant ERRγ^+/− and WT female mice were analyzed by immunohistochemistry for β-galactosidase (×400) (brown stain). *, P < .05; **, P < .01. Glo, zona glomerulosa; Fas, zona fasciculata; HET, heterozygous.

Figure 6.

ERRγ regulates BP homeostasis during pregnancy through actions in the maternal and fetal compartments. A, ChIP-quantitative PCR was performed with adrenals from ERRγ^+/− or WT nonpregnant female mice using primers that flank a putative ERRE at −242/−236; arrows denote positions of quantitative PCR primers (left panel). ERRγ binding was normalized to input and is expressed as fold increase over nonimmune IgG (right panel). Values are means ± SEM. *, P < .05. B, Luciferase assays were conducted in HEK293 cells transiently cotransfected with Cyp11b2-luciferase reporter plasmids, with or without mutation of putative ERRE (Cyp11b2m), along with either an ERRγ expression plasmid or control plasmid. Cells were assayed for firefly luciferase and Renilla luciferase activities 48 hours after transfection. Data are means ± SEM of normalized values from 3 independent experiments, each conducted in triplicate, and are expressed as fold induction of luciferase activity with ERRγ cotransfection over reporter alone. *, P < .05. C, Schematic representation of suggested mechanisms whereby ERRγ regulates BP during pregnancy through actions in mother and fetus.