Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl

CFTR is an integral transmembrane glycoprotein and a cAMP-activated Cl− channel. Mutations in the CFTR gene lead to Cystic Fibrosis (CF)–an autosomal recessive disease with majority of the morbidity and mortality resulting from airway infection, inflammation, and fibrosis. The most common disease-associated mutation in the CFTR gene–deletion of Phe508 (ΔF508) leads to a biosynthetic processing defect of CFTR. Correction of the defect and delivery of ΔF508-CFTR to the cell surface has been highly anticipated as a disease modifying therapy. Compared to promising results in cultured cell this approach was much less effective in CF patients in an early clinical trial. Although the cause of failure to rescue ΔF508-CFTR in the clinical trial has not been determined, presence of factor(s) that interfere with the rescue in vivo could be considered. The cytokine TGF-β1 is frequently elevated in CF patients. TGF-β1 has pleiotropic effects in different disease models and genetic backgrounds and little is known about TGF-β1 effects on CFTR in human airway epithelial cells. Moreover, there are no published studies examining TGF-β1 effects on the functional rescue of ΔF508-CFTR. Here we found that TGF-β1 inhibits CFTR biogenesis by reducing mRNA levels and protein abundance in primary differentiated human bronchial epithelial (HBE) cells from non-CF individuals. TGF-β1 inhibits CFTR biogenesis without compromising the epithelial phenotype or integrity of HBE cells. TGF-β1 also inhibits biogenesis and impairs the functional rescue of ΔF508-CFTR in HBE cells from patients homozygous for the ΔF508 mutation. Our data indicate that activation of TGF-β1 signaling may inhibit CFTR function in non-CF individuals and may interfere with therapies directed at correcting the processing defect of ΔF508-CFTR in CF patients.

Background: The CFTR-Ser737 site has a phospho-dependent inhibitory effect on Cl− secretion.Results: LMTK2 phosphorylation of CFTR-Ser737 facilitates endocytosis, reduces cell surface density of CFTR, and inhibits Cl− secretion.Conclusion: Targeting LMTK2 regulates the cell surface density of CFTR Cl− channels.Significance: Targeting LMTK2 in CF patients may stabilize ΔF508-CFTR pharmacologically rescued to the cell surface.

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Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior ( i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

CFTR is a PKA activated Cl- channel expressed in the apical membrane of fluid transporting epithelia. We previously demonstrated that c-Cbl decreases CFTR stability in the plasma membrane by facilitating its endocytosis and lysosomal degradation in human airway epithelium. The most common mutation associated with cystic fibrosis, deletion of Phe508 (∆F508), leads to a temperature sensitive biosynthetic processing defect in the CFTR protein. Mature ∆F508-CFTR that has been rescued by low temperature or chemical chaperones is partially functional as a Cl- channel but has decreased plasma membrane stability due to altered post-maturational trafficking. Our present data demonstrate that c-Cbl controls the post-maturational trafficking of rescued ∆F508-CFTR. Partial depletion of c-Cbl increased stability of the plasma membrane associated mature ∆F508-CFTR and the ∆F508-CFTR mediated Cl- secretion. These data indicate that correcting the post-maturational trafficking of ∆F508-CFTR may represent a therapeutic approach complementary to the biosynthetic rescue. Because c-Cbl functions as an adaptor and scaffolding protein during CFTR endocytosis, we propose that interfering with the c-Cbl mediated endocytic recruitment of ∆F508-CFTR may increase stability of ∆508-CFTR in the plasma membrane after its biosynthetic rescue.

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Cystic fibrosis (CF) is a multi‐organ disease, mostly affecting the epithelial tissues in the lungs, and is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene that encodes a Cl− channel expressed at the apical membrane of epithelial cells. The most common mutation, F508del, leads to CFTR retention in the endoplasmic reticulum, a defect that can be rescued by small molecule correctors, such as VX‐809, according to in vitro studies.Most CF patients have increased levels of Transforming Growth Factor (TGF)‐β1, which blocks the functional rescue of the F508del‐CFTR by correctors in primary differentiated human bronchial epithelial (HBE) cells. TGF‐β1 initiates a signaling pathway by binding to TGF‐β receptor (TβR)‐II, which phosphorylates and activates TβR‐I; in turn, this receptor activates receptor (R)‐Smads, initiating intracellular signaling. In non‐stimulated cells, Protein Phosphatase (PP)1 dephosphorylates TβR‐I, protecting it from constitutive activation by TβR‐II; however, it remains unknown how TGF‐β1 blocks the PP1‐mediated inhibition of TβR‐I to initiate its signaling cascade. We hypothesized that after TGF‐β1 stimulus, PP1 is inhibited through phosphorylation on its residue T320, thereby allowing the activation of TβR‐I. Lemur tyrosine kinase‐2 (LMTK2), which was later found to be a serine/threonine kinase, organizes a protein network that mediates the inhibitory phosphorylation of the catalytic subunit of PP1 (PP1c), in transfected HeLa cells. PP1 is exquisitely substrate‐specific due to interactions with different proteins regulating its localization and catalytic activity and it is unknown whether LMTK2 may also inactivate PP1 in the airway.Our aim is to understand the regulation of the TGF‐β1 signaling pathway, particularly the activation of the proximal signaling cascade at the level of TβRI/TβR‐II by PP1c in HBE cells.Experiments performed using immortalized human bronchial epithelial (CFBE41o‐) cells demonstrate that TGF‐β1 increases the inhibitory phosphorylation of PP1c. In turn, PDP3, an activator of PP1, was found to prevent the inhibitory phosphorylation of PP1‐T320, leading to a subsequent attenuation of the TGF‐β1 signaling via R‐Smads. To understand how PP1 is regulated after TGF‐β1 stimulus, we examined mRNA expression of the PP1‐ and LMTK2‐interacting proteins by mRNA sequencing. Changes in the expression level of 27 genes were observed, including PPP1R15 and p35. Proteomic analysis using liquid chromatography tandem mass spectrometry was initiated to identify PP1‐interacting proteins in CFBE41o‐ cells, and to evaluate how these protein‐protein interactions are affected by TGF‐β1, thereby allowing us to identify the proteins that are involved in the inhibition of PP1c.Our studies may lead to novel therapeutic targets blocking abnormal TGF‐β1 signaling, improving the functional rescue of F508del‐CFTR by correctors. Such a strategy will also benefit many patients with other forms of lung disease co‐mediated by TGF‐β1.Support or Funding InformationSupported by FCT‐PD/BD/114384/2016 (to D. C.). Gilead Génese PGG/039/2014 and ERS Romain Pauwels 2012 (to C.M.F.); center grant UID/MULTI/04046/2013 (to BioISI ‐ Portugal); NIH Grants R01HL090767, R01HL090767‐02S1, R56HL127202, P30 DK072506, Cystic Fibrosis Foundation SWIATE14P0 and University of Pittsburgh Founding (to A.S‐U.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.