Transforming growth factor beta1 targets estrogen receptor signaling in bronchial epithelial cells

Abstract

Background: Sex differences in idiopathic pulmonary fibrosis (IPF) suggest a protective role for estrogen (E2); however, mechanistic studies in animal models have produced mixed results. Reports using cell lines have investigated molecular interactions between transforming growth factor beta1 (TGF-β1) and estrogen receptor (ESR) pathways in breast, prostate, and skin cells, but no such interactions have been described in human lung cells. To address this gap in the literature, we investigated a role for E2 in modulating TGF-β1-induced signaling mechanisms and identified novel pathways impacted by estrogen in bronchial epithelial cells.

Methods: We investigated a role for E2 in modulating TGF-β1-induced epithelial to mesenchymal transition (EMT) in bronchial epithelial cells (BEAS-2Bs) and characterized the effect of TGF-β1 on ESR mRNA and protein expression in BEAS-2Bs. We also quantified mRNA expression of ESRs in lung tissue from individuals with IPF and identified potential downstream targets of E2 signaling in BEAS-2Bs using RNA-Seq and gene set enrichment analysis.

Results: E2 negligibly modulated TGF-β1-induced EMT; however, we report the novel observation that TGF-β1 repressed ESR expression, most notably estrogen receptor alpha (ESR1). Results of the RNA-Seq analysis showed that TGF-β1 and E2 inversely modulated the expression of several genes involved in processes such as extracellular matrix (ECM) turnover, airway smooth muscle cell contraction, and calcium flux regulation. We also report that E2 specifically modulated the expression of genes involved in chromatin remodeling pathways and that this regulation was absent in the presence of TGF-β1.

Conclusions: Collectively, these results suggest that E2 influences unexplored pathways that may be relevant to pulmonary disease and highlights potential roles for E2 in the lung that may contribute to sex-specific differences.

Keywords: Estrogen; Estrogen receptor; Fibrosis; Lung; Transforming growth factor beta1.

Fig. 1

TGF-β1 induces EMT-like changes in mRNA expression in BEAS-2B cells. (a-f) BEAS-2B cells were exposed to TGF-β1 (0.1, 1, and 5 ng/mL) for 48 h and mRNA expression of the epithelial cell type marker, E-cadherin (CDH1, a), the mesenchymal cell type markers Vimentin (VIM, b), Snail family transcriptional repressor 1 (SNAI1, d), Cadherin 2 (CDH2, e), and Fibronectin (FN1, f), and the myofibroblast cell type marker, Alpha smooth muscle actin (ACTA2) was measured by qPCR. Target gene expression was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Data are mean ± SEM of three or four independent experiments. Different letters indicate statistically significant (p < 0.05) differences between groups as determined by one-way ANOVA and Newman-Keuls multiple comparison test

Fig. 2

E2 does not significantly affect TGF-β1-induced EMT. BEAS-2B cells were exposed to 5 ng/mL TGF-β1 in the presence or absence of 10 nM E2 for 48 h. (a-f) Expression of CDH1 (a), VIM (b), ACTA2 (c), SNAI1 (d), CDH2 (e), and FN1 (f) mRNA was measured by qPCR. Target gene expression was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Data are mean ± SEM of three or four (a-d) or two (e-f) independent experiments. Different letters indicate statistically significant (p < 0.05) differences between groups as determined by one-way ANOVA and Newman-Keuls multiple comparison test

Fig. 3

TGF-β1 down-regulates ESR1, ESR2, and GPER1 mRNA expression in BEAS-2Bs. (a) Relative expression of estrogen receptor subtypes in control cells was GPER1 > ESR1 > ESR2. ESR gene expression was normalized to GAPDH mRNA expression and calculated as a ratio to ESR1 mRNA expression. (b-d) BEAS-2B cells were exposed to TGF-β1 (0.1, 1, and 5 ng/mL) for 48 h and expression of ESR1 (n = 3; b), ESR2 (n = 2; c), and GPER1 (n = 3; d) mRNA was measured by qPCR. Target gene expression was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Data are mean ± SEM and different letters indicate statistically significant (p < 0.05) differences between groups as determined by one-way ANOVA and Newman-Keuls multiple comparison test

Fig. 4

TGF-β1 down-regulates ESR1 protein expression. BEAS-2B cells were exposed to TGF-β1 (0.1, 1, and 5 ng/mL) for 48 h and ESR1 protein expression was measured by western blot followed by densitometric analysis in ImageJ. a Representative western blot. b Fold change ESR1 protein expression was normalized to Beta-actin (ACTB) and calculated as fold change to vehicle control (0 ng/mL TGF-β1). Data are mean ± SEM normalized arbitrary density units of duplicate measurements per blot of three independent experiments. Different letters indicate statistically significant (p < 0.05) differences between groups as determined by one-way ANOVA and Newman-Keuls multiple comparison test

Fig. 5

Estrogen receptor mRNA expression is reduced in lungs of patients with severe IPF compared to healthy control subjects. a-c Gene expression of ESR1 (a), ESR2 (b), and GPER1 (c) was measured in lung tissue from patients with IPF and healthy controls by qPCR. Target gene expression was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Box plots represent 5–95% confidence intervals and asterisks (*) indicate statistically significant (p < 0.05) differences compared to controls as determined by two-tailed Mann-Whitney U test

Fig. 6

TGF-β1 and E2 exhibit distinct transcriptional profiles. a BEAS-2Bs were exposed to 5 ng/mL TGF-β1 and 10 nM E2 individually and in combination. Cells were acclimated for 24 h, then groups 2 and 3 were exposed to TGF-β1. After 24 h, groups 3 and 4 were exposed to E2, and all samples were collected 24 h thereafter. b Venn diagram highlighting distribution of differentially expressed genes [Log2(Fold Change) ≥ |0.6| and FDR-corrected p-value < 0.05] among the treatment groups. c Heat map showing the clustering and relative expression levels [Log2(Fold Change) compared to controls] of genes that were differentially expressed in at least one treatment group. Red coloring indicates up-regulation compared to controls and green coloring indicates down-regulation compared to controls, (T, TGF-β1; T + E, TGF-β1 + E2; E, E2)

Fig. 7

Orthogonal validation of RNA-Seq data. a-d Expression of select genes was validated by qPCR; ESR1 (a), Connective tissue growth factor (CTGF, b), VIM (c), and Matrix metalloproteinase 2 (MMP2, d), in an identical and independent experiment. Bars represent expression [Log2(Fold Change)] of each gene in the RNA-Seq analysis, and black dots represent expression [Log2(Fold Change)] in each sample (n = 6) in the orthogonal experiment as determined by qPCR relative to vehicle control (DMSO). Target gene expression as measured by qPCR was normalized to GAPDH mRNA expression and quantified as fold change to control using the relative ΔΔCq method. Asterisks (*) indicate differential expression compared to controls [Log2(Fold Change) ≥ |0.6| and FDR-corrected p-value < 0.05] in the RNA-Seq analysis, and pound signs (#) indicate statistically significant (p < 0.05) differences compared to vehicle controls in the qPCR data as determined by one-way ANOVA and Newman-Keuls multiple comparison test

Fig. 8

TGF-β1 and E2 cause differential regulation of genes involved in extracellular matrix turnover. A gene set enrichment analysis using Pathway Studio of genes identified by RNA-Seq revealed that exposure to 5 ng/mL TGF-β1 (top) and 10 nM E2 (bottom) caused statistically significant (p < 0.05) enrichment of the extracellular matrix turnover pathway. Gray boxes denote cellular processes involved in the extracellular matrix turnover pathway. Red proteins indicate up-regulation and blue proteins indicate down-regulation as determined by RNA-Seq