G Protein βγ-subunit signaling mediates airway hyperresponsiveness and inflammation in allergic asthma

Abstract

Since the Gβγ 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 Gβγ 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-Gβγ 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 Gβγ-dependent and mediated by MAPK-upregulated PDE4 activity; and 2) the latter was attributed to Gβγ-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 Gβγ-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.

Figure 1. Inhibition of ERK1/2, PDE4, or Gβγ signaling prevents induced changes in agonist responsiveness in OVA-serum-sensitized ASM tissues.

Relative to vehicle- or control serum-exposed rabbit ASM tissues, tissues passively sensitized for ∼18 hr with OVA serum exhibit significantly increased contractility to ACh (A) and impaired relaxation to isoproterenol (B). Pre-treatment with either U0126, rolipram, or anti-Gβγ peptide prevents OVA serum-induced changes in ASM responsiveness. Data are mean ± SD values from 5–7 experiments. ANOVA used for multiple comparisons of mean Tmax values. *p<0.05; **p<0.01.

Figure 2. Anti-Gβγ blocking peptide prevents in vivo antigen-induced airway hyperresponsiveness in OVA-sensitized rabbits.

Relative to OVA-challenged control (non-sensitized; n = 4) rabbits, Rrs responses to MCh are significantly increased at 24 hr following antigen challenge in OVA-sensitized rabbits (n = 4). This heightened bronchoconstrictor responsiveness to MCh is suppressed in OVA-sensitized rabbits that are treated either with inhaled anti-Gβγ peptide (1 mg/Kg; n- = 4) or budesonide (0.5 mg/Kg; n = 3) prior to antigen challenge. Data are mean ± SE values. ANOVA used for multiple comparisons of mean Rrs values. *p<0.05; **p<0.01. Note: a significant difference is also detected when using the nonparametric Kruskal-Wallis test with Dunn's post-test to compare the medians of the Rrs-max responses in the control vs. non-pretreated OVA sensitized rabbits (p<0.05), whereas no significant difference is detected between the control vs. OVA-sensitized animals that are pretreated with the anti-Gβγ blocking peptide.

Figure 3. Anti-Gβγ blocking peptide suppresses pulmonary inflammation in OVA-sensitized rabbits.

Relative to controls (A), lungs isolated from antigen-challenged OVA-sensitized rabbits exhibit diffusely scattered patchy foci of inflammatory cell infiltration, including in peribronchial, perivascular, and parenchymal regions (B). Contrasting the lack of effect of pretreatment with MPS alone (C), inflammation is suppressed to a similar extent in antigen-challenged OVA-sensitized rabbits that were pretreated either with inhaled budesonide (D) or anti-Gβγ peptide (E), whereas pretreatment with gallein has relatively little anti-inflammatory effect (F). Representative photomicrographs (mag. ×100) are from 4 µM sections of H&E stained lung sections.

Figure 4. Pulmonary inflammatory response to antigen challenge in OVA-sensitized rabbits.

Representative high magnification photomicrographs (mag. ×1000) demonstrating that inflammatory infiltrate in lung tissues (A) and BALF (B) isolated from OVA-sensitized+challenged rabbits is mostly composed of neutrophils (N) with lesser amounts of mononuclear macrophages (M) and rare eosinophils (E).

Figure 5. Proinflammatory cellular and cytokine responses in antigen-challenged OVA-sensitized rabbits are suppressed by anti-Gβγ blocking peptide.

Relative to controls, total inflammatory cell count (A) and neutrophil/macrophage cellular ratio (B), are significantly increased in BALF samples from antigen-challenged OVA-sensitized rabbits. Correspondingly, BALF levels of INF-γ are significantly reduced whereas IL-13 levels are increased in OVA-sensitized+challenged animals. These proinflammatory indices are suppressed in BALF from sensitized rabbits that are treated with inhaled anti-Gβγ peptide prior to OVA challenge. Data are represented by bar plots depicting median and range values. Statistical comparisons are made using the Kruskal-Wallis test. *p<0.05 based on Dunn's post-test.

Figure 6. Inhibition of Gβγ-signaling prevents upregulation of PDE activity in lungs from OVA-sensitized/challenged rabbits and passively sensitized ASM tissues.

Relative to controls, PDE activity is significantly increased in lung tissues isolated from OVA-sensitized+challenged rabbits (A) and in OVA serum-sensitized (B) and IL-13-treated (C) isolated ASM tissues. The upregulated PDE activity is abrogated in lungs isolated from OVA-sensitized rabbits that are treated with inhaled anti-Gβγ peptide prior to antigen challenge, as well as in OVA serum- or IL-13-exposed ASM tissues that were pretreated with anti-Gβγ peptide or gallein. Data are mean ± SE values from 3–5 determinations. Comparisons are made using two-tailed Student t-test. **p<0.01.

Figure 7. Gβγ signaling regulates IL-13-induced c-Src and ERK1/2 activation in HASM cells.

(A) Immunoblots depicting IL-13-induced transient phosphorylation of c-Src^Tyr416 and ERK1/2 in HASM cells, with peak phosphorylation detected at 10 and 30 min, respectively. (B) Immunoblots depicting that, contrasting the lack of effect of pretreatment with MPS alone, IL-13-induced phosphorylation of c-Src^Tyr416 and ERK1/2 is suppressed in HASM cells pretreated with either anti-Gβγ peptide (20 µM) or gallein (10 µM). Additionally, in contrast to HASM cells transfected with adeno-LacZ (i.e., negative control), IL-13-induced phosphorylation of c-Src^Tyr416 and ERK1/2 is also suppressed in HASM cells wherein Gβγ signaling is inhibited by transfection with adeno-βARK-ct. (C) Western blot depicting interaction of Gβ and c-Src^Tyr416 in HASM cells stimulated with IL-13 in the absence and presence of inhibition of Gβγ activation. Following preparation of lysates from untreated and IL-13-treated (50 ng/ml×10 min) HASM cells, Gβ was immunoprecipitated (IP) with anti-Gβ monoclonal antibody, and subsequently immunoblotted (IB) with anti-phospho-c-Src^Tyr416 antibody (see Methods). Note, relative to untreated cells, association of Gβ and c-Src^Tyr416 proteins was significantly increased in IL-13-treated HASM cells; and formation of this protein complex was suppressed in IL-13-exposed cells that were pretreated with either anti-Gβγ peptide (20 µM) or gallein (10 µM), and also suppressed in IL-13-exposed HASM cells that were transfected with adeno-βARK-ct. The immunoblots shown in A–C are representative from 3–4 experiments.

Figure 8. IL-13-induced Gi-βγ 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 β-actin (C), demonstrate that IL-13 induced PDE4D transcripts at 12 hr is abrogated in HASM cells that are pretreated either with PTX, anti-Gβγ 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 ± SE values from 4 experiments (**p<0.01).

Figure 9. Inhibition Gβγ signaling prevents rolipram-sensitive changes in agonist responsiveness in IL-13-exposed rabbit ASM tissues.

Relative to controls, ASM tissues sensitized with IL-13 (50 ng/ml×24 hr) exhibit significantly increased contractility to ACh (A) and impaired relaxation to isoproterenol (B). Inhibition of Gβγ signaling by pretreatment with anti-Gβγ blocking peptide or gallein, or inhibition of PDE4 activity with rolipram, prevents IL-13-induced changes in ASM responsiveness. Data are mean ± SD values from 5–6 experiments. ANOVA used for multiple comparisons of mean Tmax and Rmax values. *p<0.05; **p<0.01.