Glial-derived neurotrophic factor in human airway smooth muscle

Abstract

Airway smooth muscle (ASM) cells modulate the local airway milieu via production of inflammatory mediators and growth factors including classical neurotrophins, such as brain-derived neurotrophic factor (BDNF). The glial cell-derived neurotrophic factor (GDNF) family of ligands (GFLs) are nonclassical neurotrophins and their role in the airway is barely understood. The major GFLs, GDNF and Neurturin (NRTN) bind to GDNF family receptor (GFR) α1 and α2 respectively that pair with Ret receptor to accomplish signaling. In this study, we found GDNF is expressed in human lung and increased in adult asthma, while human ASM expresses GDNF and its receptors. Accordingly, we used human ASM cells to test the hypothesis that ASM expression and autocrine signaling by GFLs regulate [Ca²⁺ ]_i . Serum-deprived ASM cells from non-asthmatics were exposed to 10 ng/ml GDNF or NRTN for 15 min (acute) or 24 h (chronic). In fura-2 loaded cells, acute GDNF or NRTN alone induced [Ca²⁺ ]_i responses, and further enhanced responses to 1 µM ACh or 10 µM histamine. Ret inhibitor (SPP86; 10 µM) or specific GDNF chelator GFRα1-Fc (1 µg/ml) showed roles of these receptors in GDNF effects. In contrast, NRTN did not enhance [Ca²⁺ ]_i response to histamine. Furthermore, conditioned media of nonasthmatic and asthmatic ASM cells showed GDNF secretion. SPP86, Ret inhibitor and GFRα1-Fc chelator markedly decreased [Ca²⁺ ]_i response compared with vehicle, highlighting autocrine effects of secreted GDNF. Chronic GDNF treatment increased histamine-induced myosin light chain phosphorylation. These novel data demonstrate GFLs particularly GDNF/GFRα1 influence ASM [Ca²⁺ ]_i and raise the possibility that GFLs are potential targets of airway hyperresponsiveness.

Keywords: Ret receptor; airway; asthma; calcium; neurotrophin.

Figure 1:

Glial-derived neurotrophic factor family of ligands (GFLs) and their receptors in airways. Glial-derived neurotrophic factor (GDNF) is expressed in whole lung, with greater expression in lungs of mild-moderate asthmatics (A). In non-asthmatic airway smooth muscle (ASM) cells, the GFLs -GDNF (B) and neurturin (NRTN; C) are expressed, with pro-inflammatory cytokines such as TNFα or IL-13 increasing them, albeit to different extents. ASM cells also express the GFL receptors Ret (D), GFRα1 (E) and GFRα2 (F), with cytokines increasing only GFRα1. *p<0.05, vs. normal or control. Data represented as mean ± SEM; N=9–11 for B-F, and N=19 for non-asthmatics and N=5 asthmatics in A. * indicates significant effect (p<0.05)

Figure 2:

Acute effect of GDNF and NRTN on [Ca²⁺]_i responses in human ASM: Non-asthmatic ASM cells were treated with GDNF (A) or NRTN (B) in concentrations ranging from 1 ng/ml to 100 ng/ml. Acute treatment (15 min) with GDNF or NRTN induced varying degrees of transient [Ca²⁺]_i responses. Amplitude of [Ca²⁺]_i response was increased by mid-concentrations of either ligand. Data represented as mean ± SEM from N of 4 patients each. * indicates significant effect (p<0.05)

Figure 3:

Enhancing effect of GDNF on agonist-induced [Ca²⁺]_i responses in ASM cells: In non-asthmatic human ASM cells, short-term pre-treatment with GDNF (15 min) enhanced subsequent [Ca²⁺]_i responses to the bronchoconstrictor agonists ACh (A) or histamine (B) compared to the agonist alone. In contrast, pre-treatment with NRTN did not have an enhancing effect (C, D). Data represented as mean ± SEM from N of 4–5 patients each. * indicates significant effect (p<0.05)

Figure 4:

Role of Ret and GFRαs in GDNF and NRTN effects on [Ca²⁺]_i in ASM: Non-asthmatic human ASM cells were subjected to GDNF (A) or NRTN (B) treatment in the presence or absence of Ret inhibitor (SPP86;10 µM; 24 h) or GFR chelators (GFRα1-Fc vs. GFRα2-Fc; 1µg/ml; 24 h) and [Ca²⁺]_i response were measured. Ret inhibition and GFRα1-Fc both significantly blunted GDNF effects while Ret inhibition or GFRα2-Fc were less effective in blunting NRTN effects, albeit significant. Data represented as mean ± SEM from N of 3–4 patients each. * indicates significant effect (p<0.05)

Figure 5:

Chronic effect of GDNF and NRTN on [Ca²⁺]_i response to histamine: (A) Representative traces showing [Ca²⁺]_i response to histamine in non-asthmatic ASM cells following 24h exposure to GDNF, NRTN or vehicle. (B) The amplitude of [Ca²⁺]_i responses to histamine were increased by chronic pre-exposure to GDNF but not NRTN. Data represented as mean ± SEM from N of 5 patients each. * indicates significant effect (p<0.05).

Figure 6:

Effect of ASM-derived GDNF on [Ca²⁺]_i response to histamine: GDNF in non-asthmatic and asthmatic human ASM conditioned media were quantified by ELISA (A). Amplitude of histamine-induced [Ca²⁺]_i response was reduced with 24 h pre-exposure to Ret inhibitor SPP86 (B) or GFR$\mathrm{\alpha }1$-Fc (C) highlighting an autocrine effect of secreted GDNF on ASM [Ca²⁺]_i. Data represented as mean ± SEM from N of 5–7 patients each. *indicates significant effect (p<0.05).

Figure 7:

Effect of GFLs on histamine induced myosin light chain (MLC) phosphorylation: Human ASM cells were pre-treated with GDNF or NRTN for 24 h, followed by stimulation with 1 µM histamine (Hist) for 5 min. Cells were lysed and immunoblotted for pMLC and β-actin for loading control. Chronic GDNF treatment increased the histamine induced phosphorylation of MLC compared to vehicle. Data represented as mean ± SEM from N of 6 patients each. *p<0.05, ***p<0.001 vs. vehicle (-Histamine). ^#p<0.05 vs. vehicle (+Histamine).

Figure 8:

Schematic of GDNF and NRTN effects in ASM. Human ASM can produce GDNF family ligands (GFLs) such as GDNF and NRTN that can have autocrine/paracrine effects by inducing [Ca²⁺]_i responses by themselves, and promoting responses to agonist-induced [Ca²⁺]_I, thus promoting contractility.