Cystic Fibrosis (CF), the most common recessive disease among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-activated chloride ion channel. Ninety percent of CF patients carry at least one copy of the ?F508 allele. Recent studies have shown that two small molecules, the folding corrector VX-809 and the potentiator VX-770, act in combination and partially restore ?F508-CFTR ion channel function in human bronchial epithelial (HBE) cells. However, these compounds are only marginally effective for patients with the ?F508 mutation. A key limitation is that most CF patients produce high levels of Transforming Growth Factor (TGF)-?1. Our published work showed that clinically relevant levels of TGF-?1 repress ?F508-CFTR transcription in HBE cells, acting upstream of modulators to block rescue of ?F508-CFTR. High TGF-?1 levels also prime CF patients for inflammation, epithelial-mesenchymal transformation (EMT), and fibrosis. TGF-?1 initiates signal transduction by stimulating the constitutively active TGF-?1 receptor (T?R)-II to interact with and phosphorylate T?R-I at the plasma membrane. By contrast, Protein Phosphatase 1 (PP1) protects T?R-I from constitutive activation by T?R-II in non-stimulated cells. It is unknown how TGF-?1 blocks PP1 interaction with T?R-I to activate signaling. Our preliminary work in HBE cells indicates that the scaffold organized by Lemur Tyrosine Kinase (LMTK2) at the basolateral plasma membrane favors TGF-?1 signaling by inactivating the catalytic subunit of PP1 (PP1C), thus allowing activation of T?R-I and signal transduction. Moreover, our data indicate that activating PP1C blocks TGF-?1 signaling in HBE cells. Our central hypothesis is that TGF-?1 stabilizes the LMTK2 scaffold to activate signaling leading to inflammation, fibrosis, and transcriptional repression of ?F508- CFTR in HBE cells. In so doing, LMTK2 allows TGF-?1 to antagonize ?F508-CFTR protein rescue by small molecules, and worsens outcomes. Targeting the LMTK2 scaffold thus represents a novel therapeutic strategy for CF to control TGF-?1 signaling, attenuate inflammation and fibrosis, and facilitate rescue of ?F508-CFTR in HBE cells.
In Aim 1 we will examine TGF-?1 effects on the protein-protein interactions between T?R-I, LMTK2, and PP1C in HBE cells.
In Aim 2, we will test whether TGF-?1 recruits and/or activates LMTK2 at the basolateral plasma membrane in HBE cells.
In Aim 3, we will elucidate whether TGF-?1 signaling can be attenuated by blocking LMTK2 inactivation of PP1C in HBE cells. We will use state-of-the-art research tools. Because TGF-?1 signaling is cell-type and cell-context dependent we will use HBE cells expressing ?F508- CFTR, which exhibit many of the characteristics associated with CF airway disease in vivo and are an ideal model for pre-clinical experimentation. We anticipate that our studies will lead to novel therapy targeting excessive TGF-?1 signaling triggered by high TGF-?1 levels present in most CF patients, to preserve airway integrity, and to allow small molecules to restore the ?F508-CFTR function.
A protein called CFTR helps to control the movement of salt and fluids and helps to maintain normal lung function. Another protein called TGF-beta is elevated in lungs of most patients with cystic fibrosis and destroys the ability of CFTR to control the movement of salt and fluids in the lung cells, promotes lung scarring, and prevents the new treatments for cystic fibrosis to function. Knowing how to prevent the TGF-? mediated inhibition of CFTR will help to treat cystic fibrosis.
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