IL-6 trans-signaling increases expression of airways disease genes in airway smooth muscle

Abstract

Genetic data suggest that IL-6 trans-signaling may have a pathogenic role in the lung; however, the effects of IL-6 trans-signaling on lung effector cells have not been investigated. In this study, human airway smooth muscle (HASM) cells were treated with IL-6 (classical) or IL-6+sIL6R (trans-signaling) for 24 h and gene expression was measured by RNAseq. Intracellular signaling and transcription factor activation were assessed by Western blotting and luciferase assay, respectively. The functional effect of IL-6 trans-signaling was determined by proliferation assay. IL-6 trans-signaling had no effect on phosphoinositide-3 kinase and Erk MAP kinase pathways in HASM cells. Both classical and IL-6 trans-signaling in HASM involves activation of Stat3. However, the kinetics of Stat3 phosphorylation by IL-6 trans-signaling was different than classical IL-6 signaling. This was further reflected in the differential gene expression profile by IL-6 trans-signaling in HASM cells. Under IL-6 trans-signaling conditions 36 genes were upregulated, including PLA2G2A, IL13RA1, MUC1, and SOD2. Four genes, including CCL11, were downregulated at least twofold. The expression of 112 genes was divergent between IL-6 classical and trans-signaling, including the genes HILPDA, NNMT, DAB2, MUC1, WWC1, and VEGFA. Pathway analysis revealed that IL-6 trans-signaling induced expression of genes involved in regulation of airway remodeling, immune response, hypoxia, and glucose metabolism. Treatment of HASM cells with IL-6+sIL6R induced proliferation in a dose-dependent fashion, suggesting a role for IL-6 trans-signaling in asthma pathogenesis. These novel findings demonstrate differential effect of IL-6 trans-signaling on airway cells and identify IL-6 trans-signaling as a potential modifier of airway inflammation and remodeling.

Keywords: IL-6; IL-6 trans-signaling; airway disease; gene expression; human airway smooth muscle.

Fig. 1.

Classical and trans-IL-6 signaling in human airway smooth muscle (HASM) cells. HASM cells (n = 3) were treated with IL-6 (A) or IL-6 + soluble IL-6 receptor (sIL6R) (B), and activation of Akt, Erk, and Stat3 was assessed at 60 min and 24 h by Western blotting using pan- and phospho-antibodies. Shown are representative Western blot images. Both IL-6 and IL-6+sIL6R stimulation of HASM cells did not induce activation of phosphoinositide (PI)-3 kinase and mitogen-activated protein (MAP) kinase at both time points tested. Stat3 phosphorylation was evident under both classical and trans-IL-6 stimulation of HASM cells. Most notably, phosphorylation of Stat3 under IL-6+sIL6R, but not IL-6-only, treatment (C) was sustained for an extended period of time (24 h). These findings suggest differential kinetics of transcriptional activation by trans-IL-6 signaling in HASM cells. C, control.

Fig. 2.

Stat3-induced promoter activity in HASM cells. Transcription factor Stat3-induced promoter activity in HASM cells after stimulation of cells with IL-6 or IL-6+sIL6R was determined by luciferase assay using HASM cells stably expressing Stat3-luc. Stimulation of HASM cells with IL-6 or IL-6+sIL6R resulted in significant increase in luciferase activity (n = 3). Pretreating cells with WP1066, a Stat3 inhibitor, abrogated luciferase activity induced by IL-6 or IL-6+sIL6R, further confirming the involvement of Stat3 transcription factor in mediating classical and IL-6 trans-signaling in HASM cells (**P < 0.001). AU, arbitrary units.

Fig. 3.

Effect of IL-6 and IL-6+sIL6R on HASM cell proliferation. HASM were treated with serum-free media containing vehicle, 20, 40, and 80 ng/ml of IL-6; 20, 40, and 80 ng/ml each of IL-6+sIL6R; or 20, 40, and 80 ng/ml of sIL6R for 24 h. Bromodeoxyuridine (BrdU) incorporation was assessed as described in materials and methods. IL-6+sIL6R treatment resulted in a significant increase (**P < 0.05, Friedman test) in HASM proliferation compared with controls in a dose-dependent fashion. Data (means ± SE) are pooled from experiments using 5 individual cell lines.

Fig. 4.

Validation of gene expression in HASM cells by quantitative (q)PCR. Expression of PLA2G2A (A), MUC1 (B), and CCL11 (C) in HASM cells after treating cells with IL-6 and IL-6+sIL6R for 24 h was determined by qPCR. Note a significant increase in the expression of PLA2G2A and MUC1 upon classical and IL-6 trans-signaling treatment. CCL11 expression was significantly decreased in cells treated with IL-6+sIL6R. These findings validated our RNAseq data.