β2-adrenergic receptor agonists modulate human airway smooth muscle cell migration via vasodilator-stimulated phosphoprotein

Abstract

Severe asthma manifests as airway remodeling and irreversible airway obstruction, in part because of the proliferation and migration of human airway smooth muscle (HASM) cells. We previously reported that cyclic adenosine monophosphate-mobilizing agents, including β(2)-adrenergic receptor (β(2)AR) agonists, which are mainstay of asthma therapy, and prostaglandin E2 (PGE2), inhibit the migration of HASM cells, although the mechanism for this migration remains unknown. Vasodilator-stimulated phosphoprotein (VASP), an anticapping protein, modulates the formation of actin stress fibers during cell motility, and is negatively regulated by protein kinase A (PKA)-specific inhibitory phosphorylation at serine 157 (Ser157). Here, we show that treatment with β(2)AR agonists and PGE2 induces the PKA-dependent phosphorylation of VASP and inhibits the migration of HASM cells. The stable expression of PKA inhibitory peptide and the small interfering (si) RNA-induced depletion of VASP abolish the inhibitory effects of albuterol and PGE2 on the migration of HASM cells. Importantly, prolonged treatment with albuterol prevents the agonist-induced phosphorylation of VASP at Ser157, and reverses the inhibitory effects of albuterol and formoterol, but not PGE2, on the basal and PDGF-induced migration of HASM cells. Collectively, our data demonstrate that β(2)AR agonists selectively inhibit the migration of HASM cells via a β(2)AR/PKA/VASP signaling pathway, and that prolonged treatment with albuterol abolishes the inhibitory effect of β-agonists on the phosphorylation of VASP and migration of HASM cells because of β(2)AR desensitization.

Figure 1.

Short-term incubation with β₂-adrenergic receptor (β₂AR) agonists and prostaglandin E2 (PGE2) promotes the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in human airway smooth muscle (HASM) cells. HASM cells were growth-arrested for 48 hours and incubated with different concentrations of albuterol (A), formoterol (B), PGE2, or diluent (0) for 30 minutes, followed by immunoblot analysis with anti-phospho VASP serine 157 (Ser157) or anti-total VASP antibodies. Images are representative of three independent experiments. P, phosphorylated.

Figure 2.

Propranolol inhibits the albuterol-dependent phosphorylation of VASP at Ser157. HASM cells were growth-arrested for 48 hours and incubated with 0.1–10 μM albuterol, 10 μM PGE2, or diluent in the presence or absence of 1 pM propranolol, 1 nM propranolol, or vehicle for 30 minutes, followed by immunoblot analysis with anti-phospho VASP Ser157 or anti-total VASP antibodies. (A) Representative images of three independent experiments. (B) Statistical analysis of three separate experiments. Data represent mean values ± SE. p, phosphorylated. *P < 0.001 for albuterol versus diluent. **P < 0.05 for 1 μM albuterol + 1 pM propranolol versus 1 pM propranolol. ***P < 0.01 for 10 μM albuterol + 1 pM propranolol versus 1 pM propranolol. ****P < 0.001 for PGE2 versus diluent by ANOVA (Bonferroni-Dunn test).

Figure 3.

(A and B) (R)-albuterol (where “R” stands for rectus), but not (S)-albuterol (where “S” stands for sinistre), promotes the phosphorylation of VASP in HASM cells. Growth-arrested HASM cells were incubated with 0.05, 0.5, 5, 10, or 50 μM (S)-albuterol or (R)-albuterol, 10 μM PGE2, or diluent for 10 minutes, followed by immunoblot analysis with anti-phospho VASP Ser157 and anti-total VASP antibodies. (A) Representative images from two independent experiments. (B) Statistical analysis of Ser157 VASP phosphorylation levels. Phosphorylation levels of VASP were calculated using Gel-Pro analyzer software and normalized to total VASP levels. P-VASP Ser157 in diluent-treated cells was taken as 1-fold. *P < 0.001 for (R)-albuterol versus (S)-albuterol. **P < 0.001 for PGE2 versus diluent. (C) (R)-albuterol, but not (S)-albuterol, inhibits the migration of HASM cells. HASM cells were growth-arrested for 48 hours, and then we examined their basal migration and the migration induced by 10 ng/ml PDGF in the presence of 10 μM (R)-albuterol, 10 μM (S)-albuterol, or diluent. The data represent mean values ± SE according to ANOVA (Bonferroni Dunn test) from three independent experiments. Migration in the presence of diluent was taken as 1-fold.

Figure 4.

(A) The activation of protein kinase A (PKA) is required for the albuterol-dependent inhibition of HASM cell migration. HASM cells were stably transfected with green fluorescent protein (GFP)-tagged protein kinase inhibitor (PKI) peptide (PKI-GFP), the dominant negative mutant of regulatory PKA subunit (RevAB) (RevAB-GFP), or control GFP, and were serum-deprived for 48 hours, and then migration in the presence or absence of 10 μM albuterol and 10 ng/ml PDGF was examined. The migration of diluent-treated cells stably expressing GFP was taken as 1-fold. Data represent the mean values ± SE of two independent experiments and three repetitions of each experiment, according to ANOVA (Bonferroni Dunn test). (B and C) Small interfering (si) RNA VASP suppresses the PGE2-dependent phosphorylation of VASP and attenuates the PGE2-induced inhibition of HASM cell migration. (B) HASM cells, transfected with 100 or 250 nM siRNA VASP, or control siGLO RISC-Free (GLO) siRNA, were growth-arrested and treated with 10 μM PGE2 or diluent for 30 minutes, and then immunoblot analysis with anti-phospho VASP Ser157 and anti-total VASP antibodies was performed. (C) Cells, transfected with 100 or 250 nM siRNA VASP or control siRNA, were subjected to a migration assay in Boyden chamber in the presence or absence of 10 μM PGE2 and 10 ng/ml PDGF. The data represent mean values ± SE from three independent experiments. *P < 0.001 for control siRNA + PGE2 versus control siRNA. **P < 0.01 for siRNA VASP + PGE2 versus control siRNA + PGE2. All P values were determined according to ANOVA (Bonferroni-Dunn test).

Figure 5.

β₂AR receptor agonists induce the transient phosphorylation of VASP in HASM cells. HASM cells were growth-arrested for 48 hours and incubated with different concentrations of albuterol (A), formoterol (B), PGE2, or diluent (0) for indicated times, followed by immunoblot analysis with anti-phospho VASP Ser157 or anti-total VASP antibodies. Images are representative of three independent experiments.

Figure 6.

(A) Prolonged treatment with albuterol desensitizes HASM cells to the albuterol-dependent phosphorylation of VASP. Cells were growth-arrested for 48 hours, incubated with 3 μM albuterol or diluent for 24 hours, washed twice with PBS, and maintained for 2 hours in albuterol-free media. Cells were then stimulated with 0.1, 0.3, and 1 μM albuterol, 10 μM PGE2, or diluent for 30 minutes, followed by immunoblot analysis with anti-phospho VASP Ser157 and anti-total VASP antibodies (a schematic representation of the experiment is provided in Figure E4 of the online supplement). Representative images (top) and statistical analysis (bottom) of three independent experiments are shown. The data represent mean values ± SE according to ANOVA (Bonferroni-Dunn test). *P < 0.01 for albuterol-treated cells versus diluent-treated cells, and for PGE2-treated cells versus diluent-treated cells. **P < 0.01 for cells preincubated with albuterol versus cells preincubated with diluent. (B) The albuterol-induced desensitization of HASM cells to the albuterol-dependent of VASP is time-dependent. Growth-arrested HASM cells were incubated with 1 μM albuterol for 0, 30, 60, 90, and 120 minutes, followed by washing with PBS and incubation in media with 0.1% BSA for 2 hours. Afterward, cells were treated with 1 μM albuterol, 1 μM PGE2, or diluent for 30 minutes, followed by immunoblot analysis with anti-total VASP and anti-phospho VASP Ser157 antibodies (see schematic representation in Figure E5 of the online supplement). Top: Images are representative of three independent experiments. Bottom: Data represent mean values ± SE from three separate experiments. *P < 0.01 for cells treated with albuterol versus diluent-treated cells. **P < 0.05 for cells pretreated with albuterol for 30 minutes and then treated with albuterol, versus cells pretreated with diluent for 30 minutes and then treated with albuterol. ***P < 0.01 for cells pretreated with albuterol for 60, 90, and 120 minutes and then treated with albuterol versus diluent-pretreated cells, incubated with albuterol. All P values were determined according to ANOVA (Bonferroni-Dunn test).

Figure 7.

Prolonged exposure to albuterol prevents the albuterol-dependent and formoterol-dependent, but not PGE2-dependent, inhibition of HASM cell migration. Cells were serum-deprived for 24 hours and preincubated with 1 μM albuterol or vehicle for 24 hours, and then basal (A) or PDGF-induced (B) migration in the Boyden chamber was performed for 4 hours in the presence of 1 μM albuterol, 1 μM formoterol, 1 μM PGE2, or diluent. The data represent mean values ± SE from three separate experiments, with three repetitions of each experiment. *P < 0.05 for albuterol and formoterol vs. diluent; **P < 0.001 for PGE2 vs. diluent by ANOVA (Bonferroni-Dunn test). The migration of vehicle-pretreated cells in the presence of diluent was taken as 1-fold.