BET bromodomains regulate transforming growth factor-β-induced proliferation and cytokine release in asthmatic airway smooth muscle

Abstract

Airway smooth muscle (ASM) mass is increased in asthma, and ASM cells from patients with asthma are hyperproliferative and release more IL-6 and CXCL8. The BET (bromo- and extra-terminal) family of proteins (Brd2, Brd3, and Brd4) govern the assembly of histone acetylation-dependent chromatin complexes. We have examined whether they modulate proliferation and cytokine expression in asthmatic ASM cells by studying the effect of BET bromodomain mimics JQ1/SGCBD01 and I-BET762. ASM cells from healthy individuals and nonsevere and severe asthmatics were pretreated with JQ1/SGCBD01 and I-BET762 prior to stimulation with FCS and TGF-β. Proliferation was measured by BrdU incorporation. IL-6 and CXCL8 release was measured by ELISA, and mRNA expression was measured by quantitative RT-PCR. ChIP using a specific anti-Brd4 antibody and PCR primers directed against the transcriptional start site of IL-6 and CXCL8 gene promoters was performed. Neither JQ1/SGCBD01 nor I-BET762 had any effect on ASM cell viability. JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced ASM cell proliferation and IL-6 and CXCL8 release in healthy individuals (≥ 30 nM) and in nonsevere and severe asthma patients (≥100 nM), with the latter requiring higher concentrations of these mimics. JQ1/SGCBD01 reduced Brd4 binding to IL8 and IL6 promoters induced by FCS+TGF-β. Mimics of BET bromodomains inhibit aberrant ASM cell proliferation and inflammation with lesser efficiency in those from asthmatic patients. They may be effective in reducing airway remodeling in asthma.

Keywords: Airway Smooth Muscle; Asthma; Bromodomain-containing Protein 4 (BRD4); CXCL8; Cell Proliferation; I-BET762; Interleukin 6 (IL-6); JQ1/SGCBD01; Myc (c-Myc).

FIGURE 1.

JQ1/SGCBD01, but not its negative enantiomer (JQ1⁻), suppresses FCS and TGF-β-induced cell proliferation and cytokine release. A–F, concentration-dependent effect of JQ1⁺/JQ1⁻ on FCS (2.5%) and TGF-β (1 ng/ml)-induced cellular proliferation (A and B), IL-6 release (C and D), and CXCL8 release (E and F) after 8 days. Points represent means ± S.E. from ASM cells from nine healthy (□*), nonsevere asthma (♦^#), and severe asthma (▵⁺) subjects. *, p < 0.05; ***/###/+++, p < 0.001 compared with FCS+TGF-β-stimulated cells.

FIGURE 2.

I-BET762 suppresses FCS and TGF-β-induced cell proliferation and cytokine release. A–C, the concentration-dependent effect of I-BET762 on FCS (2.5%) and TGF-β (1 ng/ml)-induced cellular proliferation (A), IL-6 release (B), and CXCL8 release (C) after 8 days. Points represent means ± S.E. from ASM cells from nine healthy (□*), nonsevere asthma (♦^#), and severe asthma (▵⁺) subjects. *, p < 0.05; **, p < 0.01; ***/###/+++, p < 0.001 compared with FCS+TGF-β-stimulated cells.

FIGURE 3.

Inhibition of c-Myc attenuates FCS and TGF-β-induced cell proliferation but not IL-6 and CXCL8 release. A–C, concentration-dependent effect of the c-Myc inhibitor (10058-F4, 0–150 μm) on FCS (2.5%) and TGF-β (1 ng/ml)-induced cell viability (A), cellular proliferation (B), and IL-6 and CXCL8 release (C) from ASM cells from healthy (□), nonsevere asthma (♦), and severe asthma (▵) subjects at 8 days. Bars/points represent means ± S.E. from nine ASM donors in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with untreated cells.

FIGURE 4.

Effect of targeting of Brd4 and c-MYC with siRNAs on FCS and TGF-β-induced cell proliferation and IL-6 release. ASM cells from healthy subjects were transfected with siRNAs designed to target Brd4 and c-MYC, or a negative (Neg) control (100 nm), before being stimulated with FCS (2.5%) and TGF-β (1 ng/ml). Cellular proliferation (A) and IL-6 release (B) were measured at 8 days. Bars represent means ± S.E. from nine ASM donors. ***, p < 0.001 compared with stimulated cells.

FIGURE 5.

Effect of JQ1/SGCBD01 on FCS and TGF-β-induced c-MYC, p21WAF1 and p27kip1 mRNA expression. A–C, effect of JQ1⁺ and JQ1⁻ pretreatment (both at 1 μm) on FCS (2.5%) and TGF-β (1 ng/ml)-induced c-MYC, (A), p21^WAF1 (B), and p27^kip1 (C) mRNA expression in ASM cells from healthy, nonsevere asthma and severe asthma subjects at 24 h. Bars represent means ± S.E. from nine ASM donors in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with unstimulated cells. +, p < 0.05; +++, p < 0.001 compared with FCS+TGF-β-stimulated cells.

FIGURE 6.

JQ1/SGCBD01 attenuates FCS and TGF-β-induced IL6 mRNA expression and decreases association with IL6 promoters. A, C, and E, effect of JQ1⁺ and JQ1⁻ pretreatment (both at 1 μm) on FCS (2.5%) and TGF-β (1 ng/ml)-induced IL6 mRNA expression in ASM cells from healthy (A), nonsevere asthma (C), and severe asthma (E) subjects at 24 h. Bars represent means ± S.E. from nine ASM donors in each group. B, D, and F, effect of JQ1⁺ and JQ1⁻ on FCS (2.5%) and TGF-β (1 ng/ml)-induced Brd4 IL6 promoter binding measured by ChIP in ASM cells from healthy (B), nonsevere asthma (D), and severe asthma (F) subjects at 24 h. Bars represent means ± S.E. from three ASM donors in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with unstimulated cells. +++, p < 0.001 compared with FCS+TGF-β-stimulated cells.

FIGURE 7.

JQ1/SGCBD01 attenuates FCS and TGF-β-induced IL8 mRNA expression and decreases association with IL8 promoters. A, C, and E, effect of JQ1⁺ and JQ1⁻ pretreatment (both at 1 μm) on FCS (2.5%) and TGF-β (1 ng/ml)-induced IL8 mRNA expression in ASM cells from healthy (A), nonsevere asthma (C), and severe asthma (E) subjects at 24 h. Bars represent means ± S.E. from nine ASM donors in each group. B, D, and F, effect of JQ1+ and JQ1- on FCS (2.5%) and TGF-β (1 ng/ml)-induced Brd4 IL8 promoter binding measured by ChIP in ASM cells from healthy (B), nonsevere asthma (D), and severe asthma (F) subjects at 24 h. Bars represent means ± S.E. from three ASM donors in each group. *, p < 0.05; **, p < 0.01 compared with unstimulated cells. +++, p < 0.001 compared with FCS+TGF-β-stimulated cells.