HGF Stimulation of Rac1 Signaling Enhances Pharmacological Correction of the Most Prevalent Cystic Fibrosis Mutant F508del-CFTR

Abstract

Cystic fibrosis (CF), a major life-limiting genetic disease leading to severe respiratory symptoms, is caused by mutations in CF transmembrane conductance regulator (CFTR), a chloride (Cl(-)) channel expressed at the apical membrane of epithelial cells. Absence of functional CFTR from the surface of respiratory cells reduces mucociliary clearance, promoting airways obstruction, chronic infection, and ultimately lung failure. The most frequent mutation, F508del, causes the channel to misfold, triggering its premature degradation and preventing it from reaching the cell surface. Recently, novel small-molecule correctors rescuing plasma membrane localization of F508del-CFTR underwent clinical trials but with limited success. Plausibly, this may be due to the mutant intrinsic plasma membrane (PM) instability. Herein, we show that restoration of F508del-CFTR PM localization by correctors can be dramatically improved through a novel pathway involving stimulation of signaling by the endogenous small GTPase Rac1 via hepatocyte growth factor (HGF). We first show that CFTR anchors to apical actin cytoskeleton (via Ezrin) upon activation of Rac1 signaling through PIP5K and Arp2/3. We then found that such anchoring retains pharmacologically rescued F508del-CFTR at the cell surface, boosting functional restoration by correctors up to 30% of wild-type channel levels in human airway epithelial cells. Our findings reveal that surface anchoring and retention is a major target pathway for CF pharmacotherapy, namely, to achieve maximal restoration of F508del-CFTR in patients in combination with correctors. Moreover, this approach may also translate to other disorders caused by trafficking-deficient surface proteins.

Spleen Tyrosine Kinase as a Target Therapy for <b><i>Pseudomonas aeruginosa</i></b> Infection

“…This nonreceptor tyrosine kinase has been reported to phosphorylate CFTR, leading to decreased levels of CFTR in the plasma membrane [66,67]. This role of SYK in regulating protein trafficking has been reported previously for other substrates, e.g., trafficking a resident of the trans-Golgi network (TGN) 38 [68], the engaged highaffinity IgE receptor (FcεRI) [69], and the small GTPase Rac1 [70] (shown to play a role in CFTR trafficking and membrane anchoring [71]). Recent findings have shown that phosphorylation of CFTR by SYK results in reducing the abundance of CFTR in the plasma membrane [72].…”

High-Content siRNA Screen Reveals Global ENaC Regulators and Potential Cystic Fibrosis Therapy Targets

“…These genes deserve future examination to elucidate how WT-CFTR (but not F508del-CFTR) prevents the ENaC-mediated Na + hyperabsorption in non-CF individuals. Notably, 4 out of these 11 hits (AKAP14, KIFAP3, MAST3, and ARHGEF11) are cytoskeletonrelated proteins and ARHGEF11 is a regulator of Rho, which we recently showed to be critical for the plasma membrane rescue of F508del-CFTR (Moniz et al, 2013). Interestingly, these four hits were also found to affect secretory traffic in a previous screen (Simpson et al, 2012).…”

LMTK2-mediated Phosphorylation Regulates CFTR Endocytosis in Human Airway Epithelial Cells

“…Hence, understanding regulation of the post-maturational trafficking of CFTR is critical to design effective treatments for CF. Although numerous adaptors have been shown to mediate the post-maturational trafficking of CFTR little is known how this process is regulated (7, 9, 10, 22–30). …”