Airway smooth muscle inflammation is regulated by microRNA-145 in COPD

Abstract

Chronic obstructive pulmonary disease (COPD) is a common, highly debilitating disease of the airways, primarily caused by smoking. Chronic inflammation and structural remodelling are key pathological features of this disease, in part caused by the aberrant function of airway smooth muscle (ASM) cells under the regulation of transforming growth factor (TGF)-β. miRNA are short, noncoding gene transcripts involved in the negative regulation of specific target genes, through their interactions with mRNA. Previous studies have proposed that mRNA-145 (miR-145) may interact with SMAD3, an important downstream signalling molecule of the TGF-β pathway. TGF-β was used to stimulate primary human ASM cells isolated from healthy nonsmokers, healthy smokers and COPD patients. This resulted in a TGF-β-dependent increase in CXCL8 and IL-6 release, most notably in the cells from COPD patients. TGF-β stimulation increased SMAD3 expression, only in cells from COPD patients, with a concurrent increased miR-145 expression. Regulation of miR-145 was found to be negatively controlled by pathways involving the MAP kinases, MEK-1/2 and p38 MAPK. Subsequent, overexpression of miR-145 (using synthetic mimics) in ASM cells from patients with COPD suppressed IL-6 and CXCL8 release, to levels comparable to the nonsmoker controls. Therefore, this study suggests that miR-145 negatively regulates pro-inflammatory cytokine release from ASM cells in COPD by targeting SMAD3.

Keywords: COPD; inflammation; microRNA.

Figure 1

Effect of increasing concentrations of transforming growth factor–β (TGF‐β) on airway smooth muscle (ASM) CXCL8 (A) and IL‐6 release (B), SMAD3 (C) and miR‐145 (D) expression from the ASM cells of non‐smokers, smokers and patients with COPD at 24 h. Points represent the means ± SEMs from nine ASM donors in each group. *^/$/# P < 0.05; **^/$$/## P < 0.01; ***^/###/$$$ P < 0.001. Asterisks indicate comparison with no TGF‐β control. Hash signs indicate COPD vs. nonsmoker ASM cells. Dollar signs indicate COPD vs. smoker ASM cells. Plus signs indicate smoker vs. nonsmoker ASM cells.

Figure 2

Effect of inhibitors of IKK2 and MAP kinases upon TGF‐β‐induced IL‐6 and CXCL8 release, and miR‐145 expression from the ASM cells of nonsmokers and patients with COPD at 24 h. ASM cells were pretreated for 60 min with the indicated concentrations of the inhibitors of IKK‐2 (TPCA‐1), MEK‐1/2 (PD098059), JNK‐1/2 (SP600125) and p38 MAP kinase (SB203580). Following exposure to vehicle control or TGF‐β (1 ng·mL⁻¹) for 24 h, the release of IL‐6 and CXCL8 was determined by ELISA. miR‐145 expression was measured by RT‐PCR. Points represent the means ± SEM of nine ASM donors in each group. *^/# P < 0.05; ^## P < 0.01; ^### P < 0.001. Asterisks indicate stimulated nonsmoker comparison with no TGF‐β control. Hash signs indicate stimulated COPD comparison with no TGF‐β control.

Figure 3

Effects of the overexpression of miR‐145 in the ASM cells of nonsmokers, smokers and patients with COPD at 24 h. ASM cells were electroporated in the presence of buffer, control mimic or miR‐145 mimic. Cells were then exposed to vehicle control or 1 ng·mL⁻¹ TGF‐β and the release of CXCL8 (A) and IL‐6 (B) was measured by ELISA at 24 h. Furthermore, p‐SMAD3 was measure by western blotting (C). Points represent the means ± SEMs from nine ASM donors in each group. *^/# P < 0.05; **^/##/++ P < 0.01; ***^/### P < 0.001.

Figure 4

SMAD3 dependent regulation of IL‐6 & CXCL8 release by TGF‐β‐induced miR‐145 expression. In response to TGF‐β stimulation, MEK‐1/2 and p38 activation results in increased miR‐145 expression. The concurrent increase in expression and phosphorylation of SMAD3, is regulated by miR‐145 to prevent further generation and release of IL‐6 & CXCL8.