Heat Shock-Related Protein 20 Peptide Decreases Human Airway Constriction Downstream of β2-Adrenergic Receptor

Abstract

Severe bronchospasm refractory to β-agonists is a challenging aspect of asthma therapy, and novel therapeutics are needed. β-agonist-induced airway smooth muscle (ASM) relaxation is associated with increases in the phosphorylation of the small heat shock-related protein (HSP) 20. We hypothesized that a transducible phosphopeptide mimetic of HSP20 (P20 peptide) causes relaxation of human ASM (HASM) by interacting with target(s) downstream of the β2-adrenergic receptor (β2AR) pathway. The effect of the P20 peptide on ASM contractility was determined in human and porcine ASM using a muscle bath. The effect of the P20 peptide on filamentous actin dynamics and migration was examined in intact porcine ASM and cultured primary HASM cells. The efficacy of the P20 peptide in vivo on airway hyperresponsiveness (AHR) was determined in an ovalbumin (OVA) sensitization and challenge murine model of allergic airway inflammation. P20 peptide caused dose-dependent relaxation of carbachol-precontracted ASM and blocked carbachol-induced contraction. The β2AR inhibitor, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551), abrogated isoproterenol but not P20 peptide-mediated relaxation. The P20 peptide decreased filamentous actin levels in intact ASM, disrupted stress fibers, and inhibited platelet-derived growth factor-induced migration of HASM cells. The P20 peptide treatment reduced methacholine-induced AHR in OVA mice without affecting the inflammatory response. These results suggest that the P20 peptide decreased airway constriction and disrupted stress fibers through regulation of the actin cytoskeleton downstream of β2AR. Thus, the P20 peptide may be a potential therapeutic for asthma refractory to β-agonists.

Keywords: airway smooth muscle relaxation; asthma; filamentous actin; ovalbumin mouse model; peptide therapeutic.

Figure 1.

Isoproterenol (ISO) and P20 peptide dose–response curves and phosphorylation of heat shock–related protein (HSP) 20 in human airway smooth muscle (ASM). (A) Representative trace of the dose–response curve of contraction with carbachol (CCH; 0.15 and 0.2 μM) and relaxation with ISO (0.001, 0.01, 0.1 μM) and (B) cumulative data of percent relaxation induced by ISO (0.001–1 μM; *P < 0.01, n = 6–9, **P < 0.001, n = 8). (C) Representative trace of the dose–response curve of contraction with CCH and relaxation with P20 peptide (1 and 2 mM) and (D) cumulative data of percent relaxation induced by P20 peptide (1 and 2 mM) and scrambled (Scr) peptide (2 mM; *P < 0.05, n = 5–8, **P < 0.001, n = 4–7). (E) Cumulative data of percent inhibition of CCH (0.15 μM) contraction induced by pretreatment with ISO (0.5 μM) and P20 peptide (2 mM) in human ASM (HASM; ***P < 0.0001, n = 4–9). (F) ISO induces phosphorylation of HSP20. Representative Western blot of the phosphorylation of HSP20 in response to ISO (0.1 μM) in untreated tissue, basal (B), CCH (0.15 μM), or 0.1 μM ISO plus 0.15 μM CCH–treated ASM. (G) The β₂-adrenergic receptor antagonist (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551, ICI) inhibits ISO-induced relaxation but does not block P20 peptide (P20)–induced relaxation of HASM. Cumulative data of percent relaxation induced by ISO (0.1 μM) and P20 peptide (1 mM) with and without the presence of 0.2 μM ICI. **Significant compared with ISO (n = 4, P < 0.001); ns, not significantly different from ICI plus P20 (n = 4, P = 0.283).

Figure 2.

P20 peptide inhibits CCH-induced contraction and alters actin cytoskeletal dynamics in porcine ASM (PASM). (A) Cumulative data showing the inhibition of CCH (0.15 μM)-induced contraction by P20 peptide (1 mM, P20; n = 3–4, ***P < 0.0001). (B) P20 peptide decreases filamentous (F)-actin in intact PASM. Top panel, representative Western blot showing actin in the supernatant (globular [G]-actin) and pellet (F-actin) of PASM treated with P20 peptide (1 mM). Bottom panel, cumulative data of percent F-actin levels show significant reduction of F-actin in P20 peptide–treated ASM compared with basal, indicating depolymerization of F-actin, *P < 0.05. (C) P20 peptide prevents CCH-induced stress fiber formation in HASM cells. Representative epifluorescence micrographs at 40× magnification of HASM cells showing stress fiber formation in cells left untreated, or treated with 0.15 μM CCH (CCH), P20 peptide (200 μM for 20 min, P20), or with P20 peptide (200 μM) for 20 minutes before CCH (0.15 μM, P20 + CCH). (D) Relative fluorescence intensity of the stress fiber formation quantitated from the ratio of the fluorescence intensity of the stress fibers over the total stained cell area using Adobe Photoshop CS5 software (**P < 0.001, ***P < 0.0001, n = 6–7). (E) P20 peptide significantly reduced platelet-derived growth factor (PDGF)-induced migration of HASM cells. HASM cells were treated with P20 peptide (200 μM, 20 min), PDGF-BB (10 ng/ml), or pretreated with P20 (200 μM, 20 min) before PDGF-BB (10 ng/ml). Cells were allowed to grow for 24 hours, and images and migratory cell counts were obtained at 0 and 24 hours (**P < 0.001, n = 6–7).

Figure 3.

P20 peptide inhibits allergen-induced airway hyperresponsiveness to methacholine in ovalbumin (OVA) mice. (A) Timeline of experimental protocol for OVA sensitization/challenge protocol and P20 peptide treatment in mice. (B) Lung resistance with increasing concentrations of intravenous methacholine normalized to baseline. **Mock/PBS versus OVA/PBS, P < 0.004; **OVA/PBS versus OVA/P20, P = 0.001; ns, not significant, mock/PBS versus OVA/P20, P = 0.333; n = 8–9 mice per group, ANOVA. (C) Ex vivo effect of CCH-induced contraction in tracheas of mock/PBS, OVA/PBS, and OVA/P20 mice. CCH (0.15 μM)-induced contraction was significantly increased from mock/PBS compared with OVA/PBS (*P = 0.04) and decreased for OVA/P20 peptide (*P < 0.05, n = 3–4) –treated mice compared with OVA/PBS-treated mice. (D) Smooth muscle relaxation in tracheas from mock/PBS and OVA/PBS mice treated ex vivo with P20 peptide (2 mM; P = 0.09, n = 3–4). (E) P20 peptide (1.5 μg/g body weight) does not significantly change the infiltration of inflammatory cells in bronchoalveolar lavage (BAL) fluid. Eos, eosinophils; Lymphs, lymphocytes; Macs, macrophages; Neuts, neutrophils; ns, not significant, P = 0.25, *P < 0.05, n = 6–8 mice.

Figure 4.

P20 peptide does not reduce allergic airway inflammation in mice. Histological sections of mice after OVA-induced airway inflammation in the presence of PBS or P20 peptide. (A) Representative sections of lung hematoxylin and eosin stain (magnification, ×200). (B) Representative sections of lung immunohistochemistry for major basic protein (magnification, ×200; n = 5 mice per group). (C) Histology score of the hematoxylin and eosin slides showing the perivascular and peribronchiolar infiltrates and interstitial pneumonia. Scoring method: 0 = no inflammation; 1 = minimal; 2 = mild; 3 = moderate; and 4 = severe inflammatory infiltration. ns, not significant.

Figure 5.

P20 peptide does not reduce mucus production or affect smooth muscle mass in the OVA mice. (A) Representative sections of lung periodic acid–Schiff stain (magnification, ×200) on Day 5 of OVA protocol. (B) Representative sections of lung immunohistochemistry for smooth muscle actin (magnification, ×200; n = 5).