Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation

Abstract

Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERβ isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERβ modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17β-estradiol, ERα-agonist and/or ERβ-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERβ significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERβ can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways, and may point to a novel perception for blunting airway remodeling.

Keywords: Asthma; ERα receptor; Estrogen; Lung; PCNA; Sex steroids.

Figure 1. Effect of TNFα, IL-13 and PDGF on human ASM cell proliferation

Significant mitogenic effect in asthmatic and non-asthmatic ASM cells was observed with 24 h treatment of tumor necrosis factor alpha (20 ng/ml TNFα), Interleukin-13 (50 ng/ml IL-13) or Platelet Derived Growth Factor (2 ng/ml PDGF) by MTT (A) and CyQuant (B) assays. PDGF-induced proliferation was relatively higher compared to both the pro-inflammatory cytokine exposure. ^###p<0.001, ^##p<0.01, ^nsp>0.05 vs vehicle; ***p<0.001, **p<0.01 vs TNFα. Values are mean ± SEM from n of 4 samples each from asthmatics and non-asthmatics.

Figure 2. Mitogenic effect of PDGF on human ASM cells

Effect of different concentrations of PDGF on the rate of proliferation on human airway smooth muscle (ASM) cells was measured by MTT (A) and CyQuant (B) assays. At a dose of 2 ng/ml, maximum ASM proliferation at 24 h was observed, while further increases in concentrations of PDGF resulted in reduced proliferation. Notably, the rate of proliferation was greater in asthmatic ASM compared to non-asthmatic ASM. *Significant effect in Asthmatic cells vs. Non-asthmatic cells at the same given dose, (*p<0.05). Values are mean ± SEM from n of 4 samples each from asthmatics and non-asthmatics.

Figure 3. Effect of ERα, ERβ and GPR30 agonists on human ASM proliferation

The effect of ERα agonists (PPT and THC), ERβ agonists (WAY-200070, FERB033 and DPN) GPR30/GPER agonist (G1) on PDGF-induced cell proliferation of asthmatic and non-asthmatic human ASM cells was evaluated using (A) MTT and (B) CyQuant NF assays. ERβ agonist WAY showed the more pronounced inhibition of PDGF-induced proliferation in both asthmatic and non-asthmatic samples. ERα and GPR30 agonists did not show any consistent blunting of PDGF-induced proliferation of human ASM as compared to ERβ agonists. ^###p<0.05 vs vehicle, ***p<0.001, **p<0.01, *p<0.05, ^nsp>0.05 vs PDGF treated group. Values are means ± SEM from n of 5 samples each from asthmatics and non-asthmatics.

Figure 4. ERβ agonist blunts PDGF induced proliferation in human ASM

ERβ agonist (WAY) significantly blunted the mitogenic effect of PDGF as evaluated using (A) MTT and (B) CyQuant NF assays. The inhibitory effect of PPT on PDGF-induced ASM proliferation was substantially less compared to WAY or non-significant in non-asthmatic patients. E₂ did not show significant decrease in PDGF-induced proliferation in most settings. ^###P<0.001, ^##P<0.01, ^#P<0.05, ^nsP>0.05 vs vehicle; ***P<0.001, **P<0.01 ^nsP>0.05 vs PDGF treated group. Values are means ± SEM from n of 6 samples each from asthmatics and non-asthmatics.

Figure 5. Effect of ER siRNA on human ASM proliferation and ER Protein Expression

MTT assay was used to study the effect of ERβ agonist WAY and ERα agonist PPT on PDGF-induced proliferation in Asthmatic and Non-Asthmatic human ASM cells transfected with ERβ siRNA (Figure 5A) and ERα siRNA (Figure 5B) respectively. There was significant mitogenic effect observed in ERβ siRNA transfected cells compared to negative siRNA in both asthmatic and non-asthmatic ASM cells. ERβ siRNA transfected human ASM cells treated with WAY and WAY+PDGF produced a significant increase in proliferation as compared to negative siRNA treated group. PPT+PDGF treatment produced an increased proliferation in ERα siRNA transfected human ASM cells. Western blot analysis was performed to determine the transfection efficacy of ERβ (Figure 5C) and ERα (Figure 5D) receptor siRNA in human ASM cells. Values are means ± SEM from n of 5 samples from asthmatics and n of 6 samples from non-asthmatics for MTT assay. Values are means ± SEM from n of 3 different patient samples of asthmatics and non-asthmatics for western analysis. ***p<0.001, **p<0.01, *p<0.05, ^nsp>0.05 vs negative siRNA.

Figure 6. Effect of ER agonists on different phases of human ASM cell cycles studied using flow cytometry

Cell cycle analysis showed increased cell population in S and M phases (B and C) with PDGF treatment. Asthmatic ASM showed an increased ratio of cells in S (B) and G2/M phase (C) to G0/G1 phase (A), consistent with a proliferative tendency of asthmatic ASM. ERβ agonist WAY significantly reduced the ratio of asthmatic human ASM cells in S to G0/G1 phase (B to A) and G2/M to G0/G1 phase (C to A) suggesting arrest of cell cycle progression in G0/G1 phase, and thus inhibiting proliferation. The representative profiles of asthmatic (D) and non-asthmatic (E) human ASM cell-cycle distributions by various treatment groups is shown where M5, M6 and M7 represent percentage of cells in G0/G1, S and G2/M phase respectively. ^###p<0.001, ^##p<0.01 vs vehicle; ***p<0.001, **p<0.01, *p<0.05, ^nsp>0.05 vs PDGF treated group. Values are means ± SEM from n of 3 samples each from asthmatics and non-asthmatics.

Figure 7. PCNA, Ki67 and C/EBP gene expression in human ASM

Cells were treated with vehicle, ERα agonist (PPT) or ERβ agonist (WAY) for 2 h, followed by PDGF for 24 h. (A) PCNA, (B) Ki67, and (C) C/EBP mRNA expression were measured by real time qPCR. PDGF treated group showed a significant increase in PCNA, Ki67, and C/EBP expression; whereas pre-treatment with PPT and WAY significantly decreased the expression of these genes. ^###p<0.001 vs vehicle and ***p<0.001, **p<0.01 vs PDGF treated group. Data are presented as mean ± SEM, from n of 6 samples each from asthmatics and non-asthmatics.

Figure 8. Effect of ER agonists on PDGF induced proliferative marker proteins in human ASM

Western analysis of proliferative protein markers. (A) Asthmatic human ASM showed increased protein expression of PCNA and C/EBP-α following PDGF exposure; an effect substantially blunted by WAY but not by PPT. (B) Similarly, non-asthmatic human ASM shows decreased PDGF induced PCNA and C/EBP expression in cells pre-treated with WAY but PPT had no effect on the same. β-Actin served as a loading control ^###p<0.001, ^##p<0.01 vs vehicle and ***p<0.001, **p<0.01, ^nsp>0.05 vs PDGF treated group. Data are presented as mean ± SEM, from n of 5 samples each from asthmatics and non-asthmatics.

Figure 9. Effect of ER agonists on PDGF induced cell cycle progression proteins in human ASM

(A) Asthmatic and (B) Non-asthmatic human ASM showed increased expression of Cyclin-D1 and Cyclin E with PDGF exposure. WAY significantly decreased PDGF induced Cyclin-D1 and Cyclin-E protein expression in both asthmatic and non-asthmatic human ASM. Pre-treatment with PPT did not show any significant effect on the expression of these proteins. β-Actin served as a loading control. ^###P<0.001, ^##P<0.01 vs vehicle; ***P<0.001, **P<0.01, ^nsP>0.05 vs PDGF treated group. Data are presented as mean ± SEM, from n of 6 samples each from asthmatics and non-asthmatics.

Figure 10. Effect of ER agonists on MAPK expression in PDGF induced human ASM cells

Western analysis shows PDGF induced activation of p38, ERK and Akt in (A) Asthmatic and (B) Non-asthmatic human ASM. Pre-treatment with WAY significantly reduced this PDGF-induced phosphorylation of p38, ERK and Akt in both asthmatic and non-asthmatic human ASM. Pre-treatment with PPT significantly decreased only p38 activation in both asthmatic and non-asthmatic human ASM and the effect is significantly lesser when compared to WAY. ^###p<0.001, ^##p<0.01, ^#p<0.05 vs vehicle; ***p<0.001, **p<0.01, ^nsp>0.05 vs PDGF treated group. Data are presented as mean ± SEM, from n of 6 samples each from asthmatics and non-asthmatics.