(440) Novel mechanism of TGF-β1 signaling in human bronchial epithelia

Background: Transforming Growth Factor (TGF)-β1emerges as an airway surface liquid inhibitor and antagonist limiting residual and corrected CFTR activity in the CF airway. The effects are mediated by 4 mechanisms: (1) TGF-β gene functions as a modifier associated with worse lung disease in patients homozygous for F508del; (2) TGF-β1 inhibits CFTR biogenesis and impairs the functional rescue of F508del-CFTR by elexacaftor/tezacaftor/ivacaftor (Trikafta); (3) TGF-β1 inhibits the ancillary chloride channels and causes dysfunction of the residual mucocilliary clearance; and (4) TGF-β1 drives inflammation, goblet cell hyperplasia, and pulmonary abnormalities with effects dependent on CFTR. The TGF-β signaling is mediated via transmembrane receptors TβRII and TβRI. After stimulation with TGF-β1, the constitutively active TβRII phosphorylates TβRI that, in turn, activates Smad2 and Smad3. Activation of TGF-β1 signaling, initiates negative feedback loop mediated by Smad7 that recruits the catalytic subunit of Protein Phosphatase 1 (PP1c), preventing activation of TβRI by TβRII. Little is currently known how stimulation with TGF-β1 inactivates the Smad7-PP1c negative feedback to allow activation of the signaling pathway.

Methods: Studies were performed in parental human bronchial epithelial CFBE41o- cells homozygous for the F508del-CFTR gene mutation and validated in primary differentiated human bronchial epithelial cells. RNA sequencing (RNAseq) was performed on Illumina platform and pathway analysis was done by Ingenuity Pathway Analysis software (QIAGEN Bioinformatics). Transient transfections of siRNA and plasmid DNA, and western blotting were performed by standard methods.The translocation of Smads from the cytosol to nucleus was examined by FIVEphoton Nuclear Protein Isolation-Translocation Assay (NPI-1).

Results: First, we showed that TGF-β1 inhibits PP1c by increasing its inhibitory phosphorylation at PP1c-Thr³²⁰. Next, to identify the specific mechanism, we performed RNAseq in cells treated with TGF-β1 or vehicle control. We identified Lemur Tyrosine Kinase 2 (LMTK2), in fact a Ser/Thr kinase and transmembrane protein, as the PP1c inhibitor. As expected, siRNA mediated knockdown (KD) of LMTK2 prevented inhibition of PP1c. LMTK2 KD decreased expression of TβRII, TβRI, and Smad7 suggesting that LMTK2 regulates the proximal TGF-β1 signaling pathway at the cell membrane. These results extend our previously published data that TGF-β1 recruits LMTK2 from the sub-membranous compartments to the plasma membrane in recycling vesicles (Cruz et al. 2020 Front. Cell Dev. Biol., February 2020). LMTK2 KD had no effect on the activation or cyto-nuclear translocation of Smad2 but specifically inhibited activation of Smad3 in the cytoplasm.

Conclusions: In human airway epithelia, activation of TGF-β1 signaling requires recruitment of LMTK2 to the cell membrane where it organizes a positive feedback loop allowing activation of TβRI. The mechanism is specific for the Smad3 signaling and does not affect the Smad2 pathway. To our knowledge, the data are the first to show the differential regulation in the canonical TGF-β1 signaling by LMTK2. Elucidating the airway-specific mechanisms of TGF-β1 signaling is important to understand the role of the cytokine in CF.

Acknowledgements: This work was supported by PD/BD/114384/2016 (to D.F.C); Gilead Génese PGG/039/2014 and ERS Romain Pauwels 2012 (to C.M.F.); Center Grants UIDB/04046/2020 and UIDP/04046/2020 (to BioISI - Portugal); NIH R01 HL144539 & CFF SWIATE18G0 (to A.S-U.).

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