G Protein bc-Subunit Signaling in Asthma Figure 8. IL-13-induced Gi-bc signaling mediates ERK1/2-dependent upregulation of PDE4D mRNA transcripts in HASM cells. (A) IL- 13 elicits temporal increases in PDE4D mRNA expression in HASM cells, with peak induction of transcripts detected at 12 hr. A representative experiment (B), and corresponding densitometric analysis of PDE4D mRNA expression expressed as a ratio of b-actin (C), demonstrate that IL-13 induced PDE4D transcripts at 12 hr is abrogated in HASM cells that are pretreated either with PTX, anti-Gbc blocking peptide, or inhibitors of either MEK-ERK1/2 (U0126), c-Src tyrosine kinase (SU6656) or gallein, whereas pretreatment with inhibitors of p38 MAPK (SB202190) or JNK (SP600125) has no effect. Data are mean 6 SE values from 4 experiments (**p,0.01). doi:10.1371/journal.pone.0032078.g008 in vivo and in vitro experimental conditions used herein to simulate the allergic asthmatic state. Of significance, we previously demonstrated that the same Gi-bc-driven mechanism coupled to stimulation of the c-Src/Ras/Raf/MEK-ERK1/2 pathway is also responsible for mediating the PDE4-induced proasthmatic chang- es in ASM contractility observed following prolonged heterologous or homologous b2AR-desensitization [10,11]. Given this concur- ring evidence, along with a collection of earlier findings that the proasthmatic changes in ASM responsiveness evoked by either passive sensitization with human atopic asthmatic serum or exposure to proinflammatory cytokines or rhinovirus are also abrogated by pretreatment with PTX [7–9], the compelling consideration is raised that signaling via the Gbc subunits of Gi protein plays a pivotal role in regulating the induction of altered ASM responsiveness under a variety of proasthmatic sensitizing conditions. This provocative consideration is worthy of future systematic investigation. Cell-permeable peptides coupled to various cargo molecules have been used in animals models to successfully target specific intracellular signaling pathways implicated in disease, including in murine models of allergic airway disease [49]. Our rationale for using a cell permeable anti-Gbc blocking peptide, comprised of an inert carrier MPS conjugated to the C-terminal thioredoxin-like domain of PhLP [17], is based on previous evidence demonstrat- ing that: 1) PhLP is endogenously expressed in a wide variety of tissues, including lung [50], and acts as an ubiquitous inhibitor of Gbc signaling by sequestering [50–53] or producing a conforma- tional change in Gbc protein [53]; 2) the MPS-conjugated anti- Gbc blocking peptide is a highly effective inhibitor of a2- adrenergic receptor-mediated activation of MAPK [17]; and 3) PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e32078