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CF airway smooth muscle transcriptome reveals a role for PYK2
Daniel P. Cook, … , Kin Fai Au, David A. Stoltz
Daniel P. Cook, … , Kin Fai Au, David A. Stoltz
Published September 7, 2017
Citation Information: JCI Insight. 2017;2(17):e95332. https://doi.org/10.1172/jci.insight.95332.
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Research Article Pulmonology

CF airway smooth muscle transcriptome reveals a role for PYK2

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Abstract

Abnormal airway smooth muscle function can contribute to cystic fibrosis (CF) airway disease. We previously found that airway smooth muscle from newborn CF pigs had increased basal tone, an increased bronchodilator response, and abnormal calcium handling. Since CF pigs lack airway infection and inflammation at birth, these findings suggest intrinsic airway smooth muscle dysfunction in CF. In this study, we tested the hypothesis that CFTR loss in airway smooth muscle would produce a distinct set of changes in the airway smooth muscle transcriptome that we could use to develop novel therapeutic targets. Total RNA sequencing of newborn wild-type and CF airway smooth muscle revealed changes in muscle contraction–related genes, ontologies, and pathways. Using connectivity mapping, we identified several small molecules that elicit transcriptional signatures opposite of CF airway smooth muscle, including NVP-TAE684, an inhibitor of proline-rich tyrosine kinase 2 (PYK2). In CF airway smooth muscle tissue, PYK2 phosphorylation was increased and PYK2 inhibition decreased smooth muscle contraction. In vivo NVP-TAE684 treatment of wild-type mice reduced methacholine-induced airway smooth muscle contraction. These findings suggest that studies in the newborn CF pig may provide an important approach to enhance our understanding of airway smooth muscle biology and for discovery of novel airway smooth muscle therapeutics for CF and other diseases of airway hyperreactivity.

Authors

Daniel P. Cook, Ryan J. Adam, Keyan Zarei, Benjamin Deonovic, Mallory R. Stroik, Nicholas D. Gansemer, David K. Meyerholz, Kin Fai Au, David A. Stoltz

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Figure 7

CFTR−/− airway smooth muscle has altered PYK2 and downstream target phosphorylation.

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CFTR−/− airway smooth muscle has altered PYK2 and downstream target pho...
Airway smooth muscle cell lysates from WT, CFTR−/−, and NVP-TAE684–pretreated CFTR−/− cells were compared on an ELISA containing immobilized antibodies to unphosphorylated and phosphorylated (A) PYK2; (B) PI3K regulatory subunit p85; and (C) MAPK regulatory subunit p38. Percentage phosphorylation for each protein was calculated from the phosphorylated and the total protein fluorescent intensity. Data are shown as the fold change (Δ) versus WT mean with SEM (a total of 4 donors/genotype were pooled onto one antibody microarray slide and a minimum of 3 slides/genotype were run, thus n = 3 slides, representing 12 animal donors/genotype). One-way ANOVA followed by Dunnett’s test was used for statistical analysis in A–C. *P < 0.05. (D) Isoproterenol prerelaxed WT and CFTR−/− airway smooth muscle strip isometric force generation following acetylcholine (ACh) treatment in tracheal muscle strips. CFTR−/− airway smooth muscle strips were pretreated with control vehicle (DMSO, orange symbols) or NVP-TAE684 (gray circles), and control WT strips were pretreated with DMSO (blue symbols) (n = 5 animals per group). Data are shown as mean values with SEM and are fitted with a 4-parameter logistic regression algorithm (solid line). P < 0.05 for curve comparisons between all groups in D. n = 7–11 donors per group.

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