TGF-β1 Augments the Apical Membrane Abundance of Lemur Tyrosine Kinase 2 to Inhibit CFTR-Mediated Chloride Transport in Human Bronchial Epithelia.

Daniel F Cruz,Agnieszka Swiatecka-Urban,Carlos M Farinha,Nilay Mitash

doi:10.3389/fcell.2020.00058

Abstract

The most common disease-causing mutation in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, F508del, leads to cystic fibrosis (CF), by arresting CFTR processing and trafficking to the plasma membrane. The FDA-approved modulators partially restore CFTR function and slow down the progression of CF lung disease by increasing processing and delivery to the plasma membrane and improving activity of F508del-CFTR Cl– channels. However, the modulators do not correct compromised membrane stability of rescued F508del-CFTR. Transforming growth factor (TGF)-β1 is a well-established gene modifier of CF associated with worse lung disease in F508del-homozygous patients, by inhibiting CFTR biogenesis and blocking the functional rescue of F508del-CFTR. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein localized at the apical and basolateral membrane domain of human bronchial epithelial cells. Phosphorylation of the apical membrane CFTR by LMTK2 triggers its endocytosis and reduces the abundance of membrane-associated CFTR, impairing the CFTR-mediated Cl– transport. We have previously shown that LMTK2 knockdown improves the pharmacologically rescued F508del-CFTR abundance and function. Thus, reducing the LMTK2 recruitment to the plasma membrane may provide a useful strategy to potentiate the pharmacological rescue of F508del-CFTR. Here, we elucidate the mechanism of LMTK2 recruitment to the apical plasma membrane in polarized CFBE41o- cells. TGF-β1 increased LMTK2 abundance selectively at the apical membrane by accelerating its recycling in Rab11-positive vesicles without affecting LMTK2 mRNA levels, protein biosynthesis, or endocytosis. Our data suggest that controlling TGF-β1 signaling may attenuate recruitment of LMTK2 to the apical membrane thereby improving stability of pharmacologically rescued F508del-CFTR.

Highlights

Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cyclic adenosine monophosphate-activated anion channel
We have previously shown that lemur tyrosine kinase 2 (LMTK2) is present at the apical and basolateral membrane domain in bronchial epithelial cell models used in our studies, human bronchial epithelial (HBE) and CFBE41o- cells, and triggers CFTR endocytosis from the apical membrane by phosphorylating CFTR-Ser737 (Luz et al, 2014)
Evaluation by IHC demonstrated that LMTK2 assumes granular intracellular distribution and is enriched in the apical and basolateral membrane, consistent with the localization determined by selective cell surface biotinylation in HBE and CFBE41o- cells (Figure 1)

Summary

Introduction

Cystic fibrosis (CF), the most common autosomal recessive disease in Caucasians, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes a cyclic adenosine monophosphate (cAMP)-activated anion channel. The most common disease-causing mutation present on at least one allele in 90% of CF patients is the deletion of Phe508 (F508del), caused by an in-frame deletion of three nucleotides (Feriotto et al, 1999). This mutation causes a biosynthetic processing defect leading to intracellular retention of CFTR protein and severely impairs the CFTR channel function (Penque et al, 2000). Published data show that TGF-β1 reduces CFTR mRNA levels and prevents the corrector/potentiator mediated rescue of the CFTR channel function in primary differentiated HBE cells homozygous for the F508del (Roux et al, 2010; Snodgrass et al, 2013; Sun et al, 2014). TGF-β1 may compromise the full beneficial effect of the corrector/potentiator therapy in the CF patients who have increased TGF-β1 signaling due to the TGFβ1 gene polymorphisms, lung infection or environmental factors (Arkwright et al, 2000; Drumm et al, 2005; Collaco et al, 2008; Cutting, 2015)

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