Exploring the mechanisms behind cigarette smoke-induced internalization of CFTR

PhD ThesisChronic 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

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Uses its C-Terminus to Regulate the A2B Adenosine Receptor

CFTR is an apical membrane anion channel that regulates fluid homeostasis in many organs including the airways, colon, pancreas and sweat glands. In cystic fibrosis, CFTR dysfunction causes significant morbidity/mortality. Whilst CFTR’s function as an ion channel has been well described, its ability to regulate other proteins is less understood. We have previously shown that plasma membrane CFTR increases the surface density of the adenosine 2B receptor (A2BR), but not of the β2 adrenergic receptor (β2AR), leading to an enhanced, adenosine-induced cAMP response in the presence of CFTR. In this study, we have found that the C-terminal PDZ-domain of both A2BR and CFTR were crucial for this interaction, and that replacing the C-terminus of A2BR with that of β2AR removed this CFTR-dependency. This observation extended to intact epithelia and disruption of the actin cytoskeleton prevented A2BR-induced but not β2AR-induced airway surface liquid (ASL) secretion. We also found that CFTR expression altered the organization of the actin cytoskeleton and PDZ-binding proteins in both HEK293T cells and in well-differentiated human bronchial epithelia. Furthermore, removal of CFTR’s PDZ binding motif (ΔTRL) prevented actin rearrangement, suggesting that CFTR insertion in the plasma membrane results in local reorganization of actin, PDZ binding proteins and certain GPCRs

Little Cigars are More Toxic than Cigarettes and Uniquely Change the Airway Gene and Protein Expression

Little cigars (LCs) are regulated differently than cigarettes, allowing them to be potentially targeted at youth/young adults. We exposed human bronchial epithelial cultures (HBECs) to air or whole tobacco smoke from cigarettes vs. LCs. Chronic smoke exposure increased the number of dead cells, lactate dehydrogenase release, and interleukin-8 (IL-8) secretion and decreased apical cilia, cystic fibrosis transmembrane conductance regulator (CFTR) protein levels, and transepithelial resistance. These adverse effects were significantly greater in LC-exposed HBECs than cigarette exposed cultures. LC-exposure also elicited unique gene expression changes and altered the proteomic profiles of airway apical secretions compared to cigarette-exposed HBECs. Gas chromatography-mass spectrometry (GC-MS) analysis indicated that LCs produced more chemicals than cigarettes, suggesting that the increased chemical load of LCs may be the cause of the greater toxicity. This is the first study of the biological effects of LCs on pulmonary epithelia and our observations strongly suggest that LCs pose a more severe danger to human health than cigarettes

Improving the efficiency and effectiveness of an industrial SARS-CoV-2 diagnostic facility.

On 11th March 2020, the UK government announced plans for the scaling of COVID-19 testing, and on 27th March 2020 it was announced that a new alliance of private sector and academic collaborative laboratories were being created to generate the testing capacity required. The Cambridge COVID-19 Testing Centre (CCTC) was established during April 2020 through collaboration between AstraZeneca, GlaxoSmithKline, and the University of Cambridge, with Charles River Laboratories joining the collaboration at the end of July 2020. The CCTC lab operation focussed on the optimised use of automation, introduction of novel technologies and process modelling to enable a testing capacity of 22,000 tests per day. Here we describe the optimisation of the laboratory process through the continued exploitation of internal performance metrics, while introducing new technologies including the Heat Inactivation of clinical samples upon receipt into the laboratory and a Direct to PCR protocol that removed the requirement for the RNA extraction step. We anticipate that these methods will have value in driving continued efficiency and effectiveness within all large scale viral diagnostic testing laboratories