CCL20/CCR6 Feedback Exaggerates Epidermal Growth Factor Receptor-Dependent MUC5AC Mucin Production in Human Airway Epithelial (NCI-H292) Cells

Abstract

Mucous hypersecretion is an important feature of obstructive airway diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Multiple stimuli induce mucin production via activation of an epidermal growth factor receptor (EGFR) cascade, but the mechanisms that exaggerate mucin production in obstructive airway diseases remain unknown. In this study, we show that binding of CCL20, a G protein-coupled receptor (GPCR) ligand that is upregulated in the airways of subjects with obstructive airway diseases, to its unique GPCR CCR6 induces MUC5AC mucin production in human airway epithelial (NCI-H292) cells via metalloprotease TNF-α–converting enzyme (TACE)-dependent EGFR activation. We also show that EGFR activation by its potent ligand TGF-α induces reactivation of EGFR via binding of endogenously produced CCL20 to its receptor CCR6 in NCI-H292 cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating mucin production in the NCI-H292 cells. In NCI-H292 cells, TGF-α stimulation induced two phases of EGFR phosphorylation (EGFR-P). The second EGFR-P was TACE-dependent and was responsible for most of the total mucin induced by TGF-α. Binding of endogenously produced CCL20 to CCR6 increased the second EGFR-P and subsequent mucin production induced by TGF-α. In NHBE cells, TGF-α–induced EGFR activation did not lead to significant CCL20 production or to EGFR rephosphorylation, and less mucin was produced. We conclude that NCI-H292 cells but not NHBE cells produce CCL20 in response to EGFR activation, which leads to a second phase of EGFR-P and subsequent exaggerated mucin production. These findings have potentially important therapeutic implications in obstructive airway diseases.

ORMDL3 is an inducible lung epithelial gene regulating metalloproteases, chemokines, OAS, and ATF6

“…In addition, we demonstrated that ORMDL3 selectively regulates activation of the ER localized transcription factor ATF6α signaling branch, a pathway we have demonstrated in human lung epithelial cells is linked to SERCA-2b, which is implicated in airway remodeling in asthma (24). Finally, our studies demonstrate that ORMDL3 activates several metalloprotease, chemokine, and OAS-regulated genes, several of whom have also been implicated in the pathogenesis of asthma (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Overall, these studies provide evidence for a mechanism to link an ER localized protein such as ORMDL3 to the pathogenesis of asthma.…”

“…Transfection of ORMDL3 into primary human lung bronchial epithelial cells induced increased expression of genes with potential importance to asthma including metalloproteases (MMP-9, ADAM-8) (7-11), CC chemokines (CCL-20) (12)(13)(14)(15), and CXC chemokines (IL-8, CXC-10) (16-21), suggesting potential downstream pathways from ORMDL3 pertinent to the pathogenesis of asthma. The importance of these ORMDL3-regulated genes to asthma is suggested from studies demonstrating increased expression of many of these mediators in the airways of human asthmatics (MMP-9, ADAM-8, CCL-20, CXCL-10, IL-8) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), induction of these mediators by allergen inhalation in asthmatics (MMP-9, CCL-20, CXCL-10) (7,12,19), and inhibition of asthma outcomes in mice deficient in these genes (MMP-9, ADAM-8, CXCL-10) (8,9,11,20,21).…”

“…The importance of these ORMDL3-regulated genes to asthma is suggested from studies demonstrating increased expression of many of these mediators in the airways of human asthmatics (MMP-9, ADAM-8, CCL-20, CXCL-10, IL-8) (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17), induction of these mediators by allergen inhalation in asthmatics (MMP-9, CCL-20, CXCL-10) (7,12,19), and inhibition of asthma outcomes in mice deficient in these genes (MMP-9, ADAM-8, CXCL-10) (8,9,11,20,21). In addition to the important role of CCL-20 on T-cell recruitment (12), and epithelial mucus production (14), in mouse models of asthma, there is a critical link between CCL-20 and TRAIL for Th2 cell activation and allergic airway inflammation (15). Although asthma has predominantly been associated with expression of CC chemokines, both CC and CXC chemokines have been linked to asthma in studies in humans with asthma (12,13,(16)(17)(18)(19), and in studies in animal models (13, 20, 21) (i.e., CXCL-10 KO have significant reduction in Th2-type allergic airway inflammation and AHR) (20).…”

“…We have made the unique observation that ORMDL3 is an allergen (and Th2 cytokine) inducible STAT-6-dependent gene expressed in the lung, in particular in airway epithelial cells. In addition, we demonstrate that transfection of ORMDL3 into normal human lung bronchial epithelial cells induces expression of genes with potential importance to the pathogenesis of asthma including metalloproteases (MMP-9 and ADAM-8) (7-11), CC chemokines (CCL-20 also known as MIP-3α) (12)(13)(14)(15), CXC chemokines (IL-8 and CXCL-10) (16)(17)(18)(19)(20)(21), and oligoadenylate synthetases (OAS) anti-viral regulated genes not previously implicated in asthma. Finally, we demonstrate that ORMDL3 transfection in lung epithelial cells activates the ATF6 pathway, one of three branches of the ER localized unfolded protein response (22,23), whereas siRNA knockdown of ATF-6 in lung epithelial cells inhibited expression of SERCA2b (sarco/ endoplasmic reticulum Ca 2+ ATPase), which has been implicated in remodeling in asthma (24).…”

The Role of Tyrosine Kinases in the Pathogenesis and Treatment of Lung Disease

“…Signaling through EGFR involves dual oxidase 1 and the production of reactive oxygen species to activate TNF-alpha converting enzyme (TACE, ADAM17), which induces mucin production via a pathway involving increased TGF- shedding and EGFR phosphorylation (Shao & Nadel, 2005). Binding of CCL20 to CCR6 has been shown to exaggerate EGFR-dependent MUC5AC production by human airway epithelial cells (Kim, Lewis, & Nadel, 2011). EGFR activation also participates in CXCL8 (IL-8) production by bronchial epithelial cells (Richter et al, 2002).…”