Mucin granules are degraded in the autophagosome-lysosome pathway as a means of resolving airway mucous cell metaplasia

Abstract

Exacerbations of muco-obstructive airway diseases such as COPD and asthma are associated with epithelial changes termed mucous cell metaplasia (MCM). The molecular pathways triggering MCM have been identified; however, the factors that regulate resolution are less well understood. We hypothesized that the autophagosome-lysosome pathway is required for resolution of MCM by degrading cytoplasmic mucins. We found increased intracellular levels of Muc5ac and Muc5b in autophagy-deficient mice. This difference was not due to defective mucin secretion. Instead, we found that Lamp1-labeled lysosomes surrounded mucin granules of mucous cells indicating that granules were being degraded. Using a model of resolution of mucous cell metaplasia in mice, we found increased lysosomal proteolytic activity that peaked in the days after inflammation. Autophagy-deficient mice had persistent accumulation of mucin granules that failed to decline due to reduced mucin degradation. We applied these findings in vitro to human airway epithelial cells (AECs). Activation of autophagy by mTOR inhibition led to degradation of mucin granules in AECs. Our findings indicate that during peak and resolution phases of MCM, mucin granules can be degraded by autophagy. The addition of mucin degradation to the existing paradigm of production and secretion may more fully explain how the secretory cells handle excess amounts of cytoplasmic mucin and offers a therapeutic target to speed resolution of MCM in airway disease exacerbations. Introduction:Mammalian cells utilize two primary degradation systems to recycle proteins: the proteasome and the autophagosome-lysosome pathways. While there is some overlap, these pathways are largely independently regulated and serve different functions(1). These protein degradation systems are necessary for the cell to balance metabolic demands by recycling proteins to amino acids for new synthesis. The autophagosome-lysosome pathway is highly conserved across species(2, 3). It can be utilized in bulk protein breakdown (macro-autophagy or referred to as autophagy) in response to nutrient demands for new amino acids. Cells can also utilize chaperone-mediated autophagy that utilizes a specific amino acid motif targeted by cytoplasmic chaperones that bypass the autophagosome and directly insert proteins in the lysosome for degradation. Finally, cells can utilize selective autophagy by targeting proteins, protein aggregates, and organelles including mitochondria(4, 5) and even cilia components(6) to the autophagosome for degradation in the lysosome. We (7,8), and others(9-13), have observed that autophagy markers are increased in models of human airway disease, and in asthma and COPD airways. This led us to infer that autophagy played a key role in the response to airway inflammation in the epithelium.Mucociliary clearance is a vital feature of innate immunity (14,15). There are two primary secretory mucins in the murine and human airways, MUC5B and MUC5AC, that provide the biophysical properties of mucous gel layer (16,17)...