G Protein bc-Subunit Signaling Mediates Airway Hyperresponsiveness and Inflammation in Allergic Asthma Gustavo Nino1,2., Aihua Hu1., Judith S. Grunstein1, Joseph McDonough1, Portia A. Kreiger3,4, Maureen B. Josephson1, John K. Choi3, Michael M. Grunstein1* 1 Division of Pulmonary Medicine, Children’s Hospital of Philadelphia Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 2 Division of Pediatric Pulmonary and Sleep Medicine, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America, 3 Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia Research Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America, 4 Department of Pathology, Nemours/A.I. duPont Hospital for Children, Wilmington, Delaware, United States of America Abstract Since the Gbc subunit of Gi protein has been importantly implicated in regulating immune and inflammatory responses, this study investigated the potential role and mechanism of action of Gbc signaling in regulating the induction of airway hyperresponsiveness (AHR) in a rabbit model of allergic asthma. Relative to non-sensitized animals, OVA-sensitized rabbits challenged with inhaled OVA exhibited AHR, lung inflammation, elevated BAL levels of IL-13, and increased airway phosphodiesterase-4 (PDE4) activity. These proasthmatic responses were suppressed by pretreatment with an inhaled membrane-permeable anti-Gbc blocking peptide, similar to the suppressive effect of glucocorticoid pretreatment. Extended mechanistic studies demonstrated that: 1) corresponding proasthmatic changes in contractility exhibited in isolated airway smooth muscle (ASM) sensitized with serum from OVA-sensitized+challenged rabbits or IL-13 were also Gbc-dependent and mediated by MAPK-upregulated PDE4 activity; and 2) the latter was attributed to Gbc-induced direct stimulation of the non- receptor tyrosine kinase, c-Src, resulting in downstream activation of ERK1/2 and its consequent transcriptional upregulation of PDE4. Collectively, these data are the first to identify that a mechanism involving Gbc-induced direct activation of c-Src, leading to ERK1/2-mediated upregulation of PDE4 activity, plays a decisive role in regulating the induction of AHR and inflammation in a rabbit model of allergic airway disease. Citation: Nino G, Hu A, Grunstein JS, McDonough J, Kreiger PA, et al. (2012) G Protein bc-Subunit Signaling Mediates Airway Hyperresponsiveness and Inflammation in Allergic Asthma. PLoS ONE 7(2): e32078. doi:10.1371/journal.pone.0032078 Editor: Ferenc Gallyas, University of Pecs Medical School, Hungary Received September 26, 2011; Accepted January 23, 2012; Published February 22, 2012 Copyright: 2012 Nino 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 National Institutes of Health (NIH) grants HL-061038 and HL097739. The funder 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: grunstein@email.chop.edu . These authors contributed equally to this work. Introduction G proteins play critical roles in regulating the allergic asthmatic phenotype, including the induction of airway hyperresponsiveness (AHR) and inflammation [1]. The G proteins are heterotrimers comprised of a, b and c subunits and, upon activation by G protein-coupled receptors (GPCRs) that respond to a host of bronchoactive and proinflammatory stimuli, the Ga subunit undergoes an exchange of GTP for GDP and becomes dissociated from the Gbc subunits [2]. Both the free Ga and Gbc subunits can then activate different effectors, importantly including those stimulating the MAPK signaling pathways that regulate various immune and inflammatory cell functions [3]. The MAPK pathways are also implicated in regulating different aspects of airway smooth muscle (ASM) function and inflammation under proasthmatic conditions, including activation of transcription factors and other downstream effectors that mediate the release of proinflammatory cytokines and chemokines which can alter ASM contractility and growth [4–6]. Previous studies demonstrated that the class of pertussis toxin (PTX)-sensitive G proteins that inhibit adenylate cyclase activity (i.e., Gi proteins) plays a particularly important role in mediating the heightened agonist-induced constrictor responses and impaired b2-adrenoceptor (b2AR)-induced relaxation responses exhibited in isolated ASM tissues exposed to different proasthmatic conditions, including passive sensitization with serum from atopic asthmatic patients [7], proinflammatory cytokine exposure [8], and inoculation with rhinovirus [9]. More recently, we reported that PTX-sensitive proasthmatic changes in ASM responsiveness are also exhibited in ASM tissues following their prolonged heterologous or homologous b2AR desensitization, and that this altered ASM function is attributed to upregulated phosphodies- terase 4 (PDE4) activity induced by activation of the Gbc subunit of Gi protein [10,11]. Specifically, Gbc signaling was found to activate the non-receptor tyrosine kinase, c-Src, which stimulates the Ras/c-Raf1/MEK signaling pathway leading to downstream activation of the MAPK, ERK1/2, the latter evoking transcrip- tional upregulation of PDE4 activity [10,11]. Collectively, these findings were consistent with the prevailing concept that GPCR- dependent and receptor-independent stimulation of Ras-mediated ERK1/2 activation uses proximal signals generated by the bc subunits of G protein coupled to c-Src activation [12–16]. In light PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e32078