Inhibition of p21 Activated Kinase (PAK) Reduces Airway Responsiveness In Vivo and In Vitro in Murine and Human Airways Wyn C. Hoover1,2, Wenwu Zhang 3 , Zhidong Xue1,2, Huanling Gao1,2, Jonathan Chernoff4, D. Wade Clapp1,2,5., Susan J. Gunst3., Robert S. Tepper1,2*. 1 Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 2 Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 3 Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America, 4 Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America, 5 Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America Abstract The p21-activated protein kinases (Paks) have been implicated in the regulation of smooth muscle contractility, but the physiologic effects of Pak activation on airway reactivity in vivo are unknown. A mouse model with a genetic deletion of Pak1 (Pak12/2) was used to determine the role of Pak in the response of the airways in vivo to challenge with inhaled or intravenous acetylcholine (ACh). Pulmonary resistance was measured in anesthetized mechanically ventilated Pak12/2 and wild type mice. Pak12/2 mice exhibited lower airway reactivity to ACh compared with wild type mice. Tracheal segments dissected from Pak12/2 mice and studied in vitro also exhibited reduced responsiveness to ACh compared with tracheas from wild type mice. Morphometric assessment and pulmonary function analysis revealed no differences in the structure of the airways or lung parenchyma, suggesting that that the reduced airway responsiveness did not result from structural abnormalities in the lungs or airways due to Pak1 deletion. Inhalation of the small molecule synthetic Pak1 inhibitor, IPA3, also significantly reduced in vivo airway responsiveness to ACh and 5-hydroxytryptamine (5-Ht) in wild type mice. IPA3 inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. The results demonstrate that Pak is a critical component of the contractile activation process in airway smooth muscle, and suggest that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness. Citation: Hoover WC, Zhang W, Xue Z, Gao H, Chernoff J, et al. (2012) Inhibition of p21 Activated Kinase (PAK) Reduces Airway Responsiveness In Vivo and In Vitro in Murine and Human Airways. PLoS ONE 7(8): e42601. doi:10.1371/journal.pone.0042601 Editor: Martin R. J. Kolb, McMaster University, Canada Received May 4, 2012; Accepted July 10, 2012; Published August 10, 2012 Copyright: 2012 Hoover et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH-HL29289, HL074099, and American Asthma Foundation 08-0049. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rtepper@iupui.edu . These authors contributed equally to this work. Introduction Asthma is characterized by repeated episodes of reversible airway obstruction and airway hyperresponsiveness to non-specific stimuli. An effective approach for the reduction of airway hyperresponveness and obstruction has been to inhibit airway smooth muscle contraction using bronchodilators. While beta- adrenergic bronchodilators have been the primary therapy for decades; increasing concerns about the long term safety and efficacy of these agents have led to a need for novel approaches to reduce airway smooth muscle responsiveness. The p21-activated protein kinases (Paks) have been implicated in the regulation of cell motility and contractility in many eukaryotic cell types. We therefore hypothesized that Pak might provide a novel target for the reduction of airway hyperrespon- siveness [1–5]. Although Pak has been previously implicated in the regulation of smooth muscle contractility, the physiologic effects of Pak activation on airway reactivity in vivo are unknown [6–8]. Pak 1, 2 an 3 isoforms are expressed in airway smooth muscle and have been implicated in the regulation of cytoskeletal dynamics in multiple cell types [9,10]. Pak1 is implicated in the regulation of smooth muscle cell motility and contraction and has been described as the dominant isoform in smooth muscle tissues [1,8,11]. In the present study, we used a mouse model with a genetic deletion of Pak1 to determine whether Pak regulates airway reactivity under physiologic conditions, and whether it could provide a target for the suppression of airway responsiveness [12]. We also tested the effects of a synthetic small molecule Pak inhibitor, IPA3, on airway reactivity in vivo [13]. IPA-3 is an allosteric Pak inhibitor that specifically inhibits the auto-activation of Pak isoforms 1, 2 and 3 [13,14]. In addition, we evaluated the effects of Pak inhibition on the responsiveness of murine and human airway smooth muscle tissues in vitro. Our findings demonstrate that the suppression of Pak activity reduces airway responsiveness, and suggest that Pak could be an important molecular target for the suppression of airway reactivity in vivo. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e42601