Anticapping VASP proteins play a critical role in actin cytoskeleton rearrangements during cell motility by directly modulating the formation of actin stress fibers at the leading edge of migrating cells. Our study shows that β2AR agonists inhibit the migration of HASM cells through the PKA-dependent phosphorylation of VASP. Importantly, prolonged exposure of HASM cells to the β2AR agonist albuterol prevented the β2AR-dependent phosphorylation of VASP as well as the inhibition of HASM cell migration, suggesting that albuterol-induced β2AR receptor tolerance abolishes the β2AR agonist-dependent inhibition of HASM cell migration through VASP.
Our data and those of others show that cAMP-mobilizing agents and glucocorticoids modulate the basal and mitogen-induced migration of HASM cells (5
). We demonstrated that cAMP-inducing agents promote the depolymerization of actin in HASM cells (8
). PKA is activated by increases in cAMP concentrations, and inhibits the lysophosphatidic acid–induced migration of bovine ASM cells (7
). The molecular mechanisms regulating these effects, however, are not well understood. Evidence suggests that cAMP-inducing agents may modulate the actin cytoskeleton during cell motility through an activation of PKA, which regulates the activity of VASP by specific phosphorylation at Ser157. The PKA-dependent of VASP was demonstrated in rat aortic vascular smooth muscle cells (20
). The stimulation of human neutrophils by a chemoattractant induces the PKA-dependent phosphorylation of VASP at Ser157 (21
). VASP also plays a critical role in neuronal function by modulating the initiation of neurites and the outgrowth and guidance of axons (22
). The negative regulatory function of VASP phosphorylation in cell migration was also suggested (23
Here, we provide evidence linking the β2AR agonist–dependent phosphorylation of VASP to the inhibition of HASM cell migration induced by cAMP-mobilizing agents. Our data show that short-term treatment with β2AR agonists induces the β2AR-dependent and PKA-dependent phosphorylation of VASP required for the β2AR agonist–induced inhibition of HASM cell migration.
Importantly, the stimulation of HASM cells with albuterol or formoterol induced a transient phosphorylation of VASP that was completely abolished after 3 hours of treatment with albuterol and 18 hours of treatment with formoterol. Because β2
AR agonists are currently used as asthma therapeutics, we investigated whether HASM cells chronically treated with β2
AR agonists will maintain their responsiveness to the albuterol-dependent and formoterol-dependent phosphorylation of VASP and their inhibition of cell migration. Interestingly, a previous report by Hastie and colleagues demonstrated a decreased phosphorylation of VASP in epithelial cells derived from patients with asthma who had been chronically treated with β2
AR agonists (24
). We report that prolonged treatment with albuterol decreases the responsiveness of HASM cells to β2
AR agonists. Thus, albuterol failed to induce the phosphorylation of VASP in HASM cells treated with albuterol for 24 hours. Further, preincubation with albuterol for 24 hours reversed the ability of albuterol and formoterol to inhibit the migration of HASM cells. Our data demonstrate that prolonged treatment with β2
AR agonists desensitizes HASM to β2
AR agonists, and suggest that the phosphorylation level of VASP may serve as a potential biomarker of airway responsiveness to β2
AR agonists and β2
AR HASM desensitization.
Interestingly, pretreatment with albuterol did not affect the inhibitory effects of PGE2 on the phosphorylation of VASP and of HASM cells, suggesting a differential mechanism of the level of receptor or downstream signaling. Our previously published studies demonstrated that PGE2 promotes the cAMP-dependent activation of PKA and phosphorylation of VASP in HASM cells via the prostaglandin E2 (EP2) receptor (25
), which, in contrast to β2
AR, is relatively resistant to agonist-specific desensitization. Indeed, the accumulation of cAMP and of PKA during both acute and chronic with agonists were markedly greater with PGE2 compared with β2
AR agonists (14
) with regard to greater efficiency in the inhibition of HASM cell growth (26
) and migration (6
). Indeed, our published data show that, in contrast to β2
AR agonists that transiently increase the phosphorylation of VASP but decrease in effectiveness by 3 hours, PGE2 promotes a PKA-dependent phosphorylation of VASP that is sustained for up to 18 hours, suggesting that prolonged exposure to PGE2 does not desensitize EP2 receptors (25
). Such differences between β-agonists and PGE2 may be explained by differential mechanisms of β2
AR and EP2 receptor desensitization in HASM cells. Our previous data demonstrate that, in HASM cells, the desensitization of the β2
AR receptor is selectively mediated by G protein-coupled receptor kinases 2/3 (GRK2/3) that exert little effect on EP2 receptor functions (26
Our data demonstrate that the desensitization of VASP to β2AR agonists impairs the inhibitory effects of β2-agonists and promotes the migration of HASM cells. In terms of clinical significance, our findings suggest that the chronic use of β2-agonists could enhance the migration of ASM cells or prime them for an enhanced mitogenic response to growth factors. Ultimately, the enhanced migration of HASM cells and mitogenic responses may promote an asthma phenotype manifested by irreversible airflow obstruction. Although our data suggest that such mechanisms may be operative in chronic severe asthma, further clinical studies are needed to characterize the role of VASP definitively in the migration and proliferation of HASM cells in mediating the irreversible airflow obstruction of chronic asthma.
Understanding the cellular and molecular mechanisms of HASM remodeling is critically important, not only for advancing our knowledge of disease pathobiology, but also because such an understanding may lead to indentifying novel molecular targets for therapeutic intervention. This study identifies a mechanism of the inhibitory effects of β2AR agonists on the migration of HASM cells. We found that the β2AR agonist–induced activation of PKA modulates the phosphorylation of VASP that inhibits the migration of HASM cells. Furthermore, we found that the prolonged treatment of HASM with β2AR agonists desensitizes airways to further treatment with β2AR agonists. Our data suggest that a prolonged use of β2AR agonists in asthma as single therapy may promote the development of tolerance to β2AR agonists and the migration of ASM cells.