Exploring the mechanisms behind cigarette smoke-induced internalization of CFTR

• Main Author: Marklew, Abigail Jane
• Published: University of Newcastle upon Tyne 2016
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.740497

Chronic obstructive pulmonary disease (COPD) the third leading cause of death, with an estimated 65 million cases worldwide. Despite this, most research to date has focused on treating the symptoms of COPD rather than the underlying mechanisms. Recently, we have shown that exposure to cigarette smoke (CS), the leading cause of COPD, results in an increase in cytosolic calcium and the rapid internalization and insolublization of the cystic fibrosis transmembrane conductance regulator (CFTR). Normal ion transport is imperative for mucus hydration and clearance, and its dysfunction after CS exposure may be responsible for the mucus dehydration and accumulation seen in COPD patients. Thus, the primary aim of this thesis was to establish the mechanism(s) behind the CS-induced internalization of CFTR. Confocal imaging and Förster resonance energy transfer demonstrated that CFTR-CFTR interactions were reduced upon internalization of CFTR, and that CFTR was internalized with a T1/2 of 27.7 min. U0126, an inhibitor of MEK1 and MEK2, abolished the internalization of CFTR by CS. Furthermore, U0126 had no effect on CS-induced Ca2+ release. These data implicate the necessity of MAPK/ERK kinases in CS-induced internalization, and suggest that this kinase activity is downstream of Ca2+ release. Furthermore, CS caused dephosphorylation of plasma membrane CFTR, and CS-induced internalization of CFTR was prevented by forskolin, suggesting that dephosphorylation of CFTR by CS may lead to its internalization. CS-induced CFTR internalization was ablated by inhibitors of endocytosis, hypertonic sucrose and dynasore. Consistent with results demonstrating that CS-internalized colocalization CFTR with clathrin light chain, these data suggest that CS-induced internalization of CFTR is both clathrin- and dynamin-dependent. CS-internalized CFTR colocalized substantially with markers of the endoplasmic reticulum. Partial colocalization of CS-internalized CFTR with markers of the early endosomes, late endosomes, and the Golgi apparatus but not recycling endosomes, suggest that CFTR is trafficked in a retrograde pathway from the plasma membrane to the endoplasmic reticulum. This thesis provides new insights into the mechanism of CS-induced CFTR internalization, and may help in the development of new therapies for CFTR correction and airway surface liquid rehydration in patients with COPD.