cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A

Abstract

Background and purpose: Changes in airway smooth muscle (ASM) phenotype may contribute to the pathogenesis of airway disease. Platelet-derived growth factor (PDGF) switches ASM from a contractile to a proliferative, hypo-contractile phenotype, a process requiring activation of extracellular signal-regulated kinase (ERK) and p70(S6) Kinase (p70(S6K) ). The effects of cAMP-elevating agents on these processes is unknown. Here, we investigated the effects of cAMP elevation by prostaglandin E(2) (PGE(2) ) and the activation of the cAMP effectors, protein kinase A (PKA) and exchange protein activated by cAMP (Epac) on PDGF-induced phenotype switching in bovine tracheal smooth muscle (BTSM).

Experimental approach: Effects of long-term treatment with the PGE(2) analogue 16,16-dimethyl-PGE(2) , the selective Epac activator, 8-pCPT-2'-O-Me-cAMP and the selective PKA activator, 6-Bnz-cAMP were assessed on the induction of a hypo-contractile, proliferative BTSM phenotype and on activation of ERK and p70(S6K) , both induced by PDGF.

Key results: Treatment with 16,16-dimethyl-PGE(2) inhibited PDGF-induced proliferation of BTSM cells and maintained BTSM strip contractility and contractile protein expression in the presence of PDGF. Activation of both Epac and PKA similarly prevented PDGF-induced phenotype switching and PDGF-induced activation of ERK. Interestingly, only PKA activation resulted in inhibition of PDGF-induced phosphorylation of p70(S6K) .

Conclusions and implications: Our data indicate for the first time that both Epac and PKA regulated switching of ASM phenotype via differential inhibition of ERK and p70(S6K) pathways. These findings suggest that cAMP elevation may be beneficial in the treatment of long-term changes in airway disease.

Figure 1

Activation of G_s-protein coupled EP₂ prostaglandin receptors inhibits platelet-derived growth factor (PDGF)-induced phenotypic modulation. Concentration–response curves of methacholine-induced contractions (A) and Western blot analysis of smooth muscle α-actin (α-SMA) expression (B) in bovine tracheal smooth muscle (BTSM) strips pretreated with 16,16-dimethyl (dm) prostaglandin E₂ (PGE₂) (1.5 µM and/or 15 µM) in the absence or presence of PDGF (10 ng·mL⁻¹) for 4 days. α-SMA expression obtained from BTSM strips homogenates was normalized to GAPDH. Representative immunoblots are shown. Data represent mean ± SEM of 3–10 independent experiments, performed in duplicate. Effects of 16,16-dimethyl-PGE₂ on basal and PDGF-induced increase in BTSM cell number in the absence or presence of the EP₂ receptor selective antagonist AH6809 (1 µM) (C), H89 (300 nM) (D) or the combination of Rp-cAMPS and Rp-8-Br-cAMPS (500 µM, each) (E). Data represent mean ± SEM of 4–10 independent experiments. Measurement of vasodilator-activated phosphoprotein (VASP) phosphorylation (F) in BTSM cells after 15 min treatment with 16,16-dm PGE₂ in the absence or presence of H89, AH6809 or the combination of Rp-cAMPS and Rp-8-Br-cAMPS. Representative immunoblots of 4–8 experiments are shown. VASP expression obtained from BTSM cell lysates was normalized to GAPDH. *P < 0.05; **P < 0.01; ***P < 0.001 compared with basal control; ^#P < 0.05; ^###P < 0.001 compared with PDGF; ^§§P < 0.01; ^†P = 0.02.

Figure 2

Activation of Epac and protein kinase A decreases platelet-derived growth factor (PDGF)-induced bovine tracheal smooth muscle (BTSM) cell proliferation. Expression of Epac1 and Epac2 from BTSM strip homogenates obtained from two different animals (A). Measurement of vasodilator-activated phosphoprotein (VASP) phosphorylation (B) in BTSM cells after 15 min treatment with 8-pCPT-2′-O-Me-cAMP (8-pCPT, 30 µM) or 6-Bnz-cAMP (500 µM) in the absence or presence of H89 (300 nM) or the combination of Rp-cAMPS and Rp-8-Br-cAMPS (500 µM, each). VASP expression obtained from BTSM cell lysates was normalized to GAPDH. Data represent means ± SEM of 3–9 independent experiments. Effects of the indicated concentrations of 8-pCPT and 6-Bnz-cAMP on basal and PDGF (10 ng·mL⁻¹)-induced increases in BTSM cell DNA synthesis (C) and cell number (D). Effects of 8-pCPT (30 µM) or 6-Bnz-cAMP (500 µM) on basal and PDGF-induced increase in BTSM cell number in the absence or presence of H89 (300 nM) (E) or the combination of Rp-cAMPS and Rp-8-Br-cAMPS (500 µM, each) (F). Data represent means ± SEM of 4–9 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with basal control; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 compared with PDGF; ^§P < 0.05; ^§§P < 0.01.

Figure 3

Activation of Epac and protein kinase A normalizes platelet-derived growth factor (PDGF)-induced hypo-contractility of bovine tracheal smooth muscle (BTSM) strips. Concentration–response curves of KCl- (left panels) and methacholine- (right panels) induced contractions in BTSM strips pretreated with 8-pCPT (30 µM) (A) or 6-Bnz-cAMP (500 µM) (B) in the absence or presence of PDGF (10 ng·mL⁻¹) for 4 days. smooth muscle α-actin (α-SMA) (C) and smooth muscle myosin heavy chain (sm-MHC) (D) expression from BTSM strip homogenates obtained after the same treatments. Contractile protein levels were normalized to GAPDH. Representative immunoblots are shown. Graphs represent means ± SEM of 3–10 experiments. ***P < 0.001 compared with (basal) control; ^#P < 0.05 compared with PDGF.

Figure 4

Combined activation of Epac and protein kinase A induces bovine tracheal smooth muscle (BTSM) phenotypic modulation. Effects of the combinations of the indicated concentrations of 8-pCPT (3 µM and 30 µM) and 6-Bnz-cAMP (100 µM and 500 µM) on basal and platelet-derived growth factor (PDGF) (10 ng·mL⁻¹)-induced BTSM DNA synthesis (A) and cell number (B). Data represent mean ± SEM of 4–7 independent experiments. Concentration–response curves of methacholine-induced contractions (C) in BTSM strips pretreated with the combinations of 8-pCPT and 6-Bnz-cAMP in the absence or presence of PDGF for 4 days. Data represent mean ± SEM of 3–4 independent experiments, performed in duplicate. **P < 0.01; ***P < 0.001 compared with basal control; ^###P < 0.001 compared with PDGF; ^§§P < 0.01; ^§§§P < 0.001.

Figure 5

Differential regulation of ERK and p70^S6K upon activation of Epac, protein kinase A and the G_s-protein coupled EP₂ receptor. Western blot analysis of phospho-extracellular signal-regulated kinase (ERK) (A) and phospho-p70^S6K (B) expression in bovine tracheal smooth muscle cells treated for 30 min and 120 min with 8-pCPT (30 µM), 6-Bnz-cAMP (500 µM) or 16,16-dm PGE₂ (15 µM) in the absence (control) or presence of platelet-derived growth factor (PDGF) (10 ng·mL⁻¹). Results were normalized to GAPDH. Representative immunoblots are shown. Graphs represent means ± SEM of 4–12 experiments. *P < 0.05; **P < 0.01; ***P < 0.001 compared with basal controls at 30 min and 120 min; ^#P < 0.05; ^##P < 0.01; ^###P < 0.001 compared with PDGF-treated condition.

Figure 6

Mechanisms of Epac- and protein kinase A (PKA)-mediated inhibition of platelet-derived growth factor (PDGF)-induced phenotypic modulation. PDGF induces phenotypic modulation of airway smooth muscle from a contractile phenotype to a proliferative phenotype via a mechanism involving extracellular signal-regulated kinase (ERK) and p70^S6 kinase (p70^S6K). Stimulation of Epac and PKA, respectively, via the cAMP analogues 8-pCPT-2′-O-Me-cAMP (8-pCPT) or 6-Bnz-cAMP or via endogenous cAMP following activation of the G_s-coupled EP₂ receptor (G_s-PCR) for PGE₂ inhibits the PDGF-induced phenotypic modulation. See text for detailed description.