BMPR2 expression is suppressed by signaling through the estrogen receptor

Abstract

Background: Studies in multiple organ systems have shown cross-talk between signaling through the bone morphogenetic protein receptor type 2 (BMPR2) and estrogen pathways. In humans, pulmonary arterial hypertension (PAH) has a female predominance, and is associated with decreased BMPR2 expression. The goal of this study was to determine if estrogens suppress BMPR2 expression.

Methods: A variety of techniques were utilized across several model platforms to evaluate the relationship between estrogens and BMPR2 gene expression. We used quantitative RT-PCR, gel mobility shift, and luciferase activity assays in human samples, live mice, and cell culture.

Results: BMPR2 expression is reduced in lymphocytes from female patients compared with male patients, and in whole lungs from female mice compared with male mice. There is an evolutionarily conserved estrogen receptor binding site in the BMPR2 promoter, which binds estrogen receptor by gel-shift assay. Increased exogenous estrogen decreases BMPR2 expression in cell culture, particularly when induced to proliferate. Transfection of increasing quantities of estrogen receptor alpha correlates strongly with decreasing expression of BMPR2.

Conclusions: BMPR2 gene expression is reduced in females compared to males in live humans and in mice, likely through direct estrogen receptor alpha binding to the BMPR2 promoter. This reduced BMPR2 expression may contribute to the increased prevalence of PAH in females.

Figure 1

BMPR2 expression is lower in female humans and mice than males. (A) BMPR2 expression by quantitative RT-PCR, normalized to HPRT, is reduced in cultured human lymphocytes from females compared with males. (B) BMPR2 expression normalized to HPRT is lower in whole lung from ovariectomized female mice compared with males. (C) ERα and ERβ expression are both higher in whole lung from ovariectomized female mice compared with males. All comparisons were by unpaired Student's t-test.

Figure 2

The BMPR2 promoter contains a functional estrogen receptor binding site. (A) Human BMPR2 promoter sequence (top line) containing an estrogen receptor binding site (boxed), used for gel-shift assay below, is conserved across species. (B) Gel-shift assay indicating that the estrogen receptor binding site is functional. BMPR2 probe sequence binds a protein (lane 1) of identical size to that bound by the canonical estrogen receptor binding site in Vitellogenin (lane 5). Binding to both the BMPR2 probe sequence and the Vitellogenin probe sequence can be competed away using cold BMPR2 probe sequence (lanes 3 and 4). Protein binding is not found when protein is omitted (lane 6) or with use of scrambled probe sequence (lane 2).

Figure 3

Treatment with different estrogens reduces BMPR2 mRNA expression in multiple normal cell types. (A) BMPR2 expression by quantitative RT-PCR, normalized to HPRT, is reduced in cultured human lymphocytes treated with 1 μM E2 compared to untreated controls (P < 0.05). (B) BMPR2 expression by quantitative RT-PCR, normalized to HPRT, is reduced in cultured PMVECs treated with 1 μM E2 compared to untreated controls (P < 0.01). (C) BMPR2 expression by quantitative RT-PCR, normalized to HPRT, is reduced in cultured PMVECs treated with varying doses of E3 compared to untreated controls (P < 0.01 for each comparison). (D) Estrogen and induction of proliferation through PMA both independently inhibit BMPR2 expression, by densitometry of northern blot (three replicates per condition) at P < 0.05 by two-way ANOVA. The combination of proliferation and estrogen produces an approximately 4× inhibition of BMPR2 expression (P < 0.05 by Student's t test with adjustment for multiple comparisons).

Figure 4

In the presence of estrogen, addition of estrogen receptor to a cell line without endogenous expression reduces BMPR2 promoter-reporter activity, in a dose-response (P = 0.0013 by Pearson correlation).

Figure 5

While BMPR2 gene expression is reduced, canonical BMP pathway activity is increased by exposure to E2. (A) A BMP responsive plasmid (pBRE system) studies: pBRE luciferase activity, a measure of BMP activity, is increased with exposure to increasing doses of E2. Uniform exposure to 50 ng of BMP4 in concert with exposure to MPP or varying doses of E2 occurred. Exposure to MPP, an ERα antagonist, resulted in reduced luciferase expression, while E2 exposure increased luciferase expression. (B) Consistent with this finding, gene expression of the canonical BMP signaling target (ID1) is increased with exposure to 10 nm E2 for 16 h.