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. 2009;4(5):e5539.
doi: 10.1371/journal.pone.0005539. Epub 2009 May 18.

Estradiol regulates expression of estrogen receptor ERalpha46 in human macrophages

Amy J Murphy  1 , Paul M Guyre, Charles R Wira, Patricia A Pioli
Affiliations

Estradiol regulates expression of estrogen receptor ERalpha46 in human macrophages

Amy J Murphy et al. PLoS One. 2009.

Abstract

Background: Monocytes and macrophages are key innate immune effector cells that produce cytokines and chemokines upon activation. We and others have shown that 17beta-estradiol (E2) has a direct role in the modulation of monocyte and macrophage immune function. However, relatively little is known about the ability of E2 to regulate isoform expression of estrogen receptors (ERs) in these cells.

Methodology/principal findings: In this study, we quantify expression of ERalpha and ERbeta in human monocytes and macrophages. We also show for the first time that the N-terminal truncated ERalpha variant, ERalpha46, is expressed in both cell types. Promoter utilization studies reveal that transcription of ERalpha in both cell types occurs from upstream promoters E and F. Treatment with E2 induces ERalpha expression in macrophages but has no effect on ERbeta levels in either cell type. During monocyte-to-macrophage differentiation, ERalpha is upregulated in a time-dependent manner. Previous studies by our group demonstrated that E2 treatment attenuates production of the chemokine CXCL8 in an ER-dependent manner. We now show that ERalpha expression levels parallel the ability of E2 to suppress CXCL8 production.

Conclusions/significance: This work demonstrates for the first time that human macrophages predominantly express the truncated ER variant ERalphap46, which is estradiol-inducible. This is mediated through usage of the ERalpha F promoter. Alternative promoter usage may account for tissue and cell type-specific differences in estradiol-induced effects on gene expression. These studies signify the importance of ERalpha expression and regulation in the ability of E2 to modulate innate immune responses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Estrogen receptors are differentially expressed in monocytes and macrophages.
A, total ERα and ERβ mRNA from M0 monocytes and macrophages from 3–4 individual donors were measured using TaqMan real-time PCR and values were normalized to β-actin. B and C, expression of ERα and ERβ protein was assessed by western blot analysis. Blots for GAPDH serve as a loading control. D, densitometry of total ERα protein expression normalized to GAPDH for three individual donors. E, the relative expression of ERα66 and ERα46 expressed as percent total ERα. *, p<0.05 vs ERβ. #, p<0.05 vs monocytes.
Figure 2
Figure 2. Estradiol induces ERα expression in macrophages and not monocytes.
Monocytes (M72) and macrophages were treated with 10−7 M E2 or ethanol control for 72 hrs and A, ERα and B, ERβ mRNA levels were measured. C, ERα and ERβ protein were also analyzed. D, quantification of macrophage ERα46 expression with and without E2 treatment from three different donors normalized to GAPDH expression. E, time course of ERα mRNA expression in macrophages treated with or without E2 normalized to β-actin and expressed as percent control. *, p<0.05 vs. control.
Figure 3
Figure 3. ERα expression increases during monocyte-to-macrophage differentiation.
A, monocytes were cultured for 0, 24, 48 or 72 hrs with 10−7 M E2 or ethanol control and ERα mRNA expression was measured and normalized to β-actin expression. B, ERα protein expression of monocytes (M72) cultured for 0 hrs and 72 hrs. C, comparison of ERα66 and ERα46 expression at 0 and 72 hrs expressed as percent total ERα. *, p<0.05 compared to monocytes. D, flow cytometric analysis of freshly isolated monocytes (M0), monocytes cultured for 72 hours (M72), and monocytes differentiated with GM-CSF for 7 days (macs). Cells were stained for surface expression of CD14, MHC II and CD16. E, change in mean fluorescence intensity in monocyte expression of CD14, MHC II and CD16 following 0, 24 hrs, 48 hrs and 72 hrs of culture.
Figure 4
Figure 4. ERα promoter utilization in monocytes and macrophages.
A, schematic representation of the ERα promoter. Boxes A–F represent up-stream exons and forward arrows denote transcription start sites. The inverted open triangle indicates the primary splice acceptor site and the inverted closed triangle represents the alternative splice acceptor site responsible for ERα46 message. Exons E and F splice into exon E1 before splicing to the ERα coding sequence. B, cells were treated as in Figure 2 and RT-PCR analysis was performed using forward primers specific for each upstream exon and a reverse primer specific for exon 2 (see Table 1) to determine ERα promoter usage. Reactions for β-actin were used as a control for cDNA integrity.
Figure 5
Figure 5. Estradiol induction of ERα promoter activity is mediated by a half ERE site.
RAW 264.7 cells were transiently transfected with the pGL4 luciferase expression vector containing either the wild-type ERα promoter F (pGL4-PF) or the half ERE mutated promoter F (pGL4-PF-mut ERE). Cells were treated with 10−7 M E2 for 48 hrs and then analyzed for luciferase activity. Results are expressed in relative light units normalized to vehicle-treated empty vector and are representative of at least three independent transfections. *p<0.05 vs. vehicle control.
Figure 6
Figure 6. ERα expression mirrors E2 modulation of LPS-induced CXCL8 production.
Monocytes were cultured for 24, 48 or 72 hrs. 10−7 M E2 or ethanol control was added during the final 24 hrs of culture. Subsequently, cells were stimulated in the presence or absence of LPS (10 ng/ml), for an additional 12 hrs. Culture supernatants were analyzed for CXCL8 by ELISA and reported as pg/ml. *, p<0.05 vs. control + LPS.
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