To clarify the potential utility of targeting GRK2/3-mediated desensitization as a means of manipulating airway smooth muscle (ASM) contractile state, we assessed the specificity of GRK2/3 regulation of procontractile and relaxant G-protein-coupled receptors in ASM. Functional domains of GRK2/3 were stably expressed, or siRNA-mediated GRK2/3 knockdown was performed, in human ASM cultures, and agonist-induced signaling was assessed. Regulation of contraction of murine tracheal rings expressing GRK2 C terminus was also assessed. GRK2/3 knockdown or expression of the GRK2 C terminus caused a significant (~30–90%) increase in maximal β-agonist and histamine [phosphoinositide (PI) hydrolysis] signaling, without affecting the calculated EC50. GRK2 C-terminal expression did not affect signaling by methacholine, thrombin, or LTD4. Expression of the GRK2 N terminus or kinase-dead holo-GRK2 diminished (~30–70%) both PI hydrolysis and Ca2+ mobilization by every Gq-coupled receptor examined. Under conditions of GRK2 C-terminal expression, β-agonist inhibition of methacholine-stimulated PI hydrolysis was greater. Finally, transgenic expression of the GRK2 C terminus in murine ASM enabled ~30–50% greater β-agonist-mediated relaxation of methacholine-induced contraction. Collectively these data demonstrate the relative selectivity of GRKs for the β2AR in ASM and the ability to exploit GRK2/3 functional domains to render ASM hyporesponsive to contractile agents while increasing responsiveness to bronchodilating β-agonist.—Deshpande, D. A., Yan, H., Kong, K.-C., Tiegs, B. C., Morgan, S. J., Pera, T., Panettieri, R. A., Eckhart, A. D., Penn, R. B. Exploiting functional domains of GRK2/3 to alter the competitive balance of pro- and anticontractile signaling in airway smooth muscle. FASEB J. 28, 956–965 (2014). www.fasebj.org
Inhaled β-agonists are effective at reversing bronchoconstriction in asthma, but the mechanism by which they exert this effect is unclear and controversial. PKA is the historically accepted effector, although this assumption is made on the basis of associative and not direct evidence. Recent studies have asserted that exchange protein activated by cAMP (Epac), not PKA, mediates the relaxation of airway smooth muscle (ASM) observed with β-agonist treatment. This study aims to clarify the role of PKA in the prorelaxant effects of β-agonists on ASM. Inhibition of PKA activity via expression of the PKI and RevAB peptides results in increased β-agonist-mediated cAMP release, abolishes the inhibitory effect of isoproterenol on histamine-induced intracellular calcium flux, and significantly attenuates histamine-stimulated MLC-20 phosphorylation. Analyses of ASM cell and tissue contraction demonstrate that PKA inhibition eliminates most, if not all, β-agonist-mediated relaxation of contracted smooth muscle. Conversely, Epac knockdown had no effect on the regulation of contraction or procontractile signaling by isoproterenol. These findings suggest that PKA, not Epac, is the predominant and physiologically relevant effector through which β-agonists exert their relaxant effects.
Although G protein-coupled receptor (GPCR) kinases (GRKs) have been shown to mediate desensitization of numerous GPCRs in studies using cellular expression systems, their function under physiological conditions is less well understood. In the current study, we employed various strategies to assess the effect of inhibiting endogenous GRK2/3 on signaling and function of endogenously expressed G s-coupled receptors in human airway smooth muscle (ASM) cells. GRK2/3 inhibition by expression of a Gbetagamma sequestrant, a GRK2/3 dominant-negative mutant, or siRNA-mediated knockdown increased intracellular cAMP accumulation mediated via beta-agonist stimulation of the beta-2-adrenergic receptor (beta 2AR). Conversely, neither 5'-( N-ethylcarboxamido)-adenosine (NECA; activating the A2b adenosine receptor) nor prostaglandin E2 (PGE 2; activating EP2 or EP4 receptors)-stimulated cAMP was significantly increased by GRK2/3 inhibition. Selective knockdown using siRNA suggested the majority of PGE 2-stimulated cAMP in ASM was mediated by the EP2 receptor. Although a minor role for EP3 receptors in influencing PGE 2-mediated cAMP was determined, the GRK2/3-resistant nature of EP2 receptor signaling in ASM was confirmed using the EP2-selective agonist butaprost. Somewhat surprisingly, GRK2/3 inhibition did not augment the inhibitory effect of the beta-agonist on mitogen-stimulated increases in ASM growth. These findings demonstrate that with respect to G s-coupled receptors in ASM, GRK2/3 selectively attenuates beta 2AR signaling, yet relief of GRK2/3-dependent beta 2AR desensitization does not influence at least one important physiological function of the receptor.
Excessive smooth muscle growth occurs within the context of inflammation associated with certain vascular and airway diseases. The inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor-α (TNF-α) have been shown previously to inhibit mitogen-stimulated smooth muscle growth through a mechanism presumed to be dependent on the induction of cyclooxygenase-2, prostaglandins, and activation of the cAMP-dependent protein kinase (PKA). Using both molecular and pharmacological strategies, we demonstrate that the mitogenic effects of IL-1β and TNF-α on cultured human airway smooth muscle (ASM) cells are tightly regulated by PKA activity. Suppression of induced PKA activity by either corticosteroids or cyclooxygenase inhibitors converts the cytokines from inhibitors to enhancers of mitogen-stimulated ASM growth, and biological variability in the capacity to activate PKA influences the modulatory effect of cytokines. Promitogenic effects of IL-1β are associated with delayed increases in p42/p44 and phosphoinositide-3 kinase activities, suggesting a role for induced autocrine factors. These findings suggest a mechanism by which mainstream therapies such as corticosteroids or cyclooxygenase inhibitors could fail to address or exacerbate the pathogenic smooth muscle growth that occurs in obstructive airway and cardiovascular diseases.
Inhaled β-agonists are effective airway smooth muscle (ASM)-relaxing agents that help reverse bronchoconstriction in asthma, but their ability to affect the aberrant ASM growth that also occurs with asthma is poorly understood. β-Agonists exhibit PKA-dependent antimitogenic effects in several cell types. However, recent studies suggest that Epac, and not PKA, mediates the antimitogenic effect of cAMP in both ASM and fibroblasts. This study aims to clarify the roleof PKA in mediating the effect of GS-coupled receptors on human ASM growth. Pretreatmentof ASM cultures with β-agonists albuterol, isoproterenol, or salmeterol (100 nM to 10 µM) caused a significant (-25–30%) inhibition of EGF-stimulated ASM thymi-dine incorporation and cell proliferation, whereas a much greater inhibition was observed from pretreatment with PGE2 (75–80%). However, all agents were ineffective in cells expressing GFP chimeras of either PKI (a PKA inhibitor) or a mutant PKA regulatory subunit relative to the control cells expressing GFP. The antimitogenic efficacy of PGE2 in inhibiting control cultures was associated with greater ability to stimulate sustained PKA activation and greater inhibition of late-phase promitogenic p42/p44 and PI3K activities. These findings suggest that therapeutic approaches enabling superior PKA activation in ASM will be most efficacious in deterring ASM growth.—Yan, H., Deshpande, D. A., Misior, A. M., Miles, M. C., Saxena, H., Riemer, E. C., Pascual, R. M., Panettieri, R. A., Penn. R. B. Anti-mitogenic effects of β-agonists and PGE2 on airway smooth muscle are PKA dependent. FASEB J. 25, 389–397 (2011). www.fasebj.org
