ST6GAL1 and α2-6 Sialylation Regulates IL-6 Expression and Secretion in Chronic Obstructive Pulmonary Disease

Abstract

Chronic obstructive pulmonary disease (COPD) is a systemic disease strongly associated with cigarette smoking, airway inflammation, and acute disease exacerbations. Changes in terminal sialylation and fucosylation of asparagine (N)-linked glycans have been documented in COPD, but the role that glycosyltransferases may play in the regulation of N-linked glycans in COPD has not been fully elucidated. Recent studies suggest that modulation of ST6GAL1 (ST6 beta-galactoside alpha-2,6-sialyltransferase-1), which catalyzes terminal α2-6 sialylation of cellular proteins, may regulate inflammation and contribute to COPD phenotype(s). Interestingly, it has been previously demonstrated that ST6GAL1, a Golgi resident protein, can be proteolytically processed by BACE1 (beta-site amyloid precursor protein cleaving enzyme-1) to a circulating form that retains activity. In this study, we showed that loss of ST6GAL1 expression increased interleukin (IL)-6 expression and secretion in human bronchial epithelial cells (HBECs). Furthermore, exposure to cigarette smoke medium/extract (CSE) or BACE1 inhibition resulted in decreased ST6GAL1 secretion, reduced α2-6 sialylation, and increased IL-6 production in HBECs. Analysis of plasma ST6GAL1 levels in a small COPD patient cohort demonstrated an inverse association with prospective acute exacerbations of COPD (AECOPD), while IL-6 was positively associated. Altogether, these results suggest that reduced ST6GAL1 and α2-6 sialylation augments IL-6 expression/secretion in HBECs and is associated with poor clinical outcomes in COPD.

Keywords: COPD; ST6GAL1; bronchial epithelium; cigarette smoke; inflammation.

Figure 1

Reduced extracellular sialylation in HBEs following 3, 6 and 15-day CSE exposure. (A) Relative mRNA expression of ST6GAL1 at times indicated without (CTRL) and with cigarette smoke extract (CSE). (B–D) Flow cytometric histogram showing levels of α2-6 sialylation for HBECs cultured for 3, 6, and 15 days without (CTRL) and with (CSE). Ten thousand events were collected for each group, analyzed by SNA-FITC staining, and shown as the geometric mean of the values. Experiments were performed in triplicate and three separate experiments. SNA, Sambucus Nigra Lectin; and CSE, cigarette smoke extract. All bar graphs are means ± SEM with *p < 0.05.

Figure 2

ST6GAL1 overexpression and knockdown leads to alterations in sialylation in HBECs. (A) Bar graphs showing ST6GAL1 mRNA levels in stably transfected ST6GAL1 KD and OE cultures compared to control (CRTL) infected cells in the presence or absence of CSE. (B) Flow cytometric analysis of α2-6 sialylation using SNA-FITC labeling of HBECs (CTRL, ST6GAL1 KD, and ST6GAL1 OE) following treatment with and without CSE for 3 days. (C) Graphical data of the 10,000 events that were collected from (B) for each group and analyzed. Abbrv. SNA, Sambucus Nigra Lectin; CTRL, pLKO vector control; KD, ST6GAL1 knockdown; and OE, ST6GAL1 overexpression; NT, no treatment; CSE, cigarette smoke extract; and HBECs, human bronchial epithelial cells. Experiments were performed in triplicate using three separate experiments. All bar graphs are means ± SEM with *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3

ST6GAL1 knockdown increases IL-6 expression and secretion. (A) Relative mRNA transcript levels of IL-1β, IL-6, and IL-8 in CTRL, ST6GAL1 KD, and ST6GAL1 OE HBECs. Analysis of IL-6 (B) and IL-8 (C) protein levels from supernatants of CTRL, ST6GAL1 KD, and ST6GAL1 OE HBECs using ELISA. CTRL, pLKO vector control; KD, ST6GAL1 knockdown; OE, ST6GAL1overexpression; rel, relative; IL-6, interleukin 6; and IL-8, interleukin 8. All experiments were reproduced 3 times and done in triplicates with bar graphs indicating means ± SEM with *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 4

IL-6 expression and secretion is increased following cigarette smoke exposure and attenuated by ST6GAL1 overexpression in HBECs. (A) Relative mRNA transcript levels of IL-6 in control (CTRL) and CSE treated HBECs. (B) IL-6 protein levels from supernatants of control, and ST6GAL1 OE HBECs exposed to CSE for 24 hours. CTRL, control; CSE, cigarette smoke extract/medium; and OE, ST6GAL1 overexpression. All experiments were reproduced 3 times and done in triplicates with bar graphs indicating means ± SEM with *p < 0.05 and **p < 0.01.

Figure 5

Inhibition of ST6GAL1 secretion reduces α2-6 sialylation and increases IL-6 secretion similar to CSE. (A) Flow cytometric analysis of α2-6 sialylation using SNA-FITC labeling of HBECs (CTRL, ST6GAL1 KD, and ST6GAL1 OE) following treatment with and without iBACE and CSE for 72 hours. (B) 10,000 events were collected for each group, analyzed, and shown as the geometric mean. (C) Densitometry and a representative slot blot of secreted ST6GAL1 collected from conditioned medium following incubation with iBACE, CSE, or both. As a loading control, the supernatants were normalized to total cellular protein. (D) IL-6 secretion was determined from conditioned medium using an ELISA kit to the IL-6 ligand following same treatments as (C). Experiments were performed in triplicate using three separate experiments. SNA, Sambucus Nigra Lectin; CTRL, control; iBACE, beta-site amyloid precursor protein cleaving enzyme 1 inhibitor; and CSE, cigarette smoke extract. All bar graphs are means ± SEM with *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 6

Circulating ST6GAL1 levels are lower in COPD patients and correlate with poor outcomes. (A) GOLD group status 1-4 and associated levels of ST6GAL1 in a COPD patient cohort. Circulating levels of ST6GAL1 (B) and IL-6 (C) and their respective association with acute exacerbations in COPD. Patients were classified based on severity of the COPD using the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (30846476), which are as follows: GOLD 1: Mild (FEV1 >= 80% predicted); GOLD 2: Mod (FEV1 between 50 and 79% predicted); GOLD 3: Severe (FEV1 between 30 and 49% predicted); and GOLD 4: Very severe (FEV1 <30% predicted). Shown are the mean ± SEM, where n = subjects per group. *p < 0.05 and **p < 0.01.