Inhibition of human airway smooth muscle cell proliferation by β₂-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement

Abstract

Mechanisms by which beta-adrenergic receptor (beta AR) agonists inhibit proliferation of human airway smooth muscle (HASM) cells were investigated because of their potential relevance to smooth muscle hyperplasia in asthma. We hypothesized that beta AR agonists would inhibit mitogenesis in HASM cells via the beta 2AR, an increase in cAMP, and PKA activation. HASM cells were treated for 24 h with various agents and then analyzed for [3H]thymidine incorporation as a measure of cell proliferation. EGF stimulated proliferation by approximately 10-fold. The nonselective beta AR agonist isoproterenol and the beta 2AR-selective agonists albuterol and salmeterol inhibited EGF-stimulated proliferation by more than 50%, with half-maximal effects at 4.8 nM, 110 nM, and 6.7 nM, respectively. A beta 2AR-selective antagonist inhibited the isoproterenol effect with 100-fold greater potency than a beta 1AR-selective antagonist, confirming beta 2AR involvement in the inhibition of proliferation. The cAMP-elevating agents PGE2 and forskolin decreased EGF-induced proliferation, suggesting cAMP as the mediator. beta 2AR agonists and forskolin also inhibited proliferation stimulated by lysophosphatidic acid (LPA) as well as the synergistic proliferation stimulated by LPA+EGF. Importantly, PKA-selective cAMP analogs did not inhibit proliferation at concentrations that maximally activated PKA (10-100 microM), whereas a cAMP analog selective for the exchange protein directly activated by cAMP (EPAC), 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, maximally inhibited proliferation at a concentration that did not activate PKA (10 microM). These data show that beta 2AR agonists and other cAMP-elevating agents decrease proliferation in HASM cells via a PKA-independent mechanism, and they provide pharmacological evidence for involvement of EPAC or an EPAC-like cAMP effector protein instead.

Quantitative global phosphoproteomics of human umbilical vein endothelial cells after activation of the Rap signaling pathway

“…Epac itself or together with its target Rap has been implicated in the control of cell proliferation, and another major cAMP target, protein kinase A (PKA), was shown to act synergistically with these proteins in this process in several cases. [52][53][54][55][56][57] Our observation underlines the link between Epac, Rap, PKA and the cell cycle. However, further investigations are needed to determine whether their effects in HUVECs are proliferative or anti-proliferative and Rap-or PKA-dependent.…”

Prostaglandin E2 Inhibits Specific Lung Fibroblast Functions via Selective Actions of PKA and Epac-1

“…These findings were independent of the growth factor/ medium conditions used. This novel anti-proliferative role for Epac-1 in primary lung fibroblasts is supported by a recent study that suggests Epac may also be anti-proliferative in bronchial smooth muscle cells (46). The ability of Epac-1 (and subsequent activation of Rap1) to play divergent roles in mesenchymal cells versus other cell types illustrates how these cells can exhibit dichotomous responses to common signaling pathways.…”

Cyclic AMP Sensor EPAC Proteins and Their Role in Cardiovascular Function and Disease

“…66,67 Similarly, direct activators of EPAC, also inhibit mitogen-induced proliferation of cultured air way smooth muscle cells suggesting that EPAC activation may limit the remodeling observed in diseases of the airways, such as asthma and chronic obstructive pulmonary disease. 68,69 This effect of EPAC, at least in rat VSMCs, is synergistic with PKA and involves coordinated inhibition of the immediate response gene Early growth response 1 and other factors such as cyclin D1 and S-phase kinase-associated protein-2 (Skp2), that are critical for regulating cell-cycle progression ( Figure 5). 66,70 Recently, it has been reported that therapeutically relevant concentrations of the prostacyclin analog, beraprost can inhibit PDGF-induced human VSMC migration via activation of the cAMP effector EPAC.…”