Understanding the Regulation of TGF‐β1 Signaling Pathway in Human Bronchial Epithelial Cells: the Role of PP1 and LMTK2
Authors: Cruz Daniel F. , Cihil Kristine M. , Köhn Maja , Farinha Carlos M. , Swiatecka‐Urban Agnieszka 
Published: June 2021

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 Information

Supported 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.