Effects of glial-derived neurotrophic factor on remodeling and mitochondrial function in human airway smooth muscle cells

Abstract

Airway smooth muscle (ASM) cells play important roles in airway remodeling of asthma. Our previous studies show that in vivo administration of glial-derived neurotrophic factor (GDNF) in mice induces thickening and collagen deposition in bronchial airways, whereas chelation of GDNF by GFRα1-Fc attenuates airway remodeling in the context of allergen exposure. To determine whether GDNF has direct effects on ASM, in this study, we examined GDNF in ASM cells from normal versus asthmatic humans. We found that GDNF treatment of human ASM cells had only minor effects on cell proliferation and migration, intracellular expression or extracellular deposition of collagen I (COL1), collagen III (COL3), and fibronectin. Endoplasmic reticulum (ER) stress response and mitochondrial function have been implicated in asthma. We investigated whether GDNF regulates these aspects in human ASM. We found that GDNF treatment did not affect ER stress protein expression in normal or asthmatic cells. However, GDNF treatment impaired mitochondrial morphology in ASM but without significant effects on mitochondrial respiration. Thus, it is likely that in vivo effects of GDNF on airway remodeling per se involve cell types other than those on ASM, and thus ASM may serve more as a source of GDNF rather than a target.NEW & NOTEWORTHY Our previous study suggests that glial-derived neurotrophic factor (GDNF) is involved in allergen-induced airway hyperreactivity and remodeling in vivo. Here, we show that GDNF has no direct effects in remodeling of human airway smooth muscle (ASM) but GDNF dysregulates mitochondrial morphology in human ASM in the context of asthma.

Keywords: airway smooth muscle; asthma; mitochondria; neurotrophin; remodeling.

Graphical abstract

Figure 1.

Effects of glial-derived neurotrophic factor (GDNF) and transforming growth factor (TGF)β on human airway smooth muscle (ASM) cell proliferation. A: normal or asthmatic ASM cells were treated with GDNF (1, 20, or 50 ng/mL) or TGFβ (2 ng/mL) in medium with 1% FBS for 2 days. ASM cells grown in medium with 10% FBS served as positive control. Cell proliferation was measured by CyQuant assay. B: asthmatic ASM cells were treated with or without GDNF (1 ng/mL) in medium with 1% or 10% FBS for 2 days. Cell proliferation was normalized to medium with 1% FBS. C: ASM cells in ibidi Culture-Insert 2 Well system were treated with or without GDNF (1 ng/mL) in serum-free medium for 24 h and then imaged. Open areas without migrated cells were quantified using Nikon software (D). All data are shown as means ± SD from n = 5 patient samples. Each symbol represents one patient sample. Black colored symbols: normal ASM. Red colored symbols: asthmatic ASM. Open symbols: females; Closed symbols: Males. Data were analyzed using two-way ANOVA, Turkey’s multiple comparisons test (A and D) or RM-one-way ANOVA (B).

Figure 2.

Effects of glial-derived neurotrophic factor (GDNF) and transforming growth factor (TGF)β on human airway smooth muscle (ASM) extracellular matrix (ECM) proteins. Normal or asthmatic ASM cells were treated with indicated reagents for 2 days. Control groups were treated with vehicle only. A–C: intracellular collagen type I (COL1), collagen type III (COL3), and fibronectin protein expression was measured by WES assays and normalized to total protein. Representative WES images are shown below each figure. The sample orders in the representative images are the same as the figures. D–F: ECM protein deposition was quantified using a modified Li-Cor In-Cell Western technique and normalized to cell numbers. Data are shown as means ± SD from n = 5–6 patient samples. Each symbol represents one patient sample. Black colored symbols: normal ASM. Red colored symbols: asthmatic ASM. Open symbols: females; closed symbols: Males. Data were analyzed using mixed-effects analysis, Sidak’s multiple comparisons test (A–C) or two-way ANOVA, Sidak’s multiple comparisons test (D–F).

Figure 3.

Effects of glial-derived neurotrophic factor (GDNF) and transforming growth factor (TGF)β on endoplasmic reticulum (ER) stress protein expression in human airway smooth muscle (ASM). Normal or asthmatic ASM cells were treated with indicated reagents for 12 h. ER stress protein expression in these cells were analyzed by WES assays and normalized to total protein (A–E). Data are presented as means ± SD (n = 5–6 patients/group). Each symbol represents one patient sample. Black colored symbols: normal ASM. Red colored symbols: asthmatic ASM. Open symbols: females; closed symbols: males. Data were analyzed using mixed-effects analysis, Sidak’s multiple comparisons test. The sample orders in the representative images are the same as the figures.

Figure 4.

Mitochondrial morphology of human airway smooth muscle (ASM) cells treated with glial-derived neurotrophic factor (GDNF) and transforming growth factor (TGF)β. Normal or asthmatic ASM cells were treated with GDNF and TGFβ for 48 h and then stained with MitoTracker Green. Labeled cells were imaged and analyzed to determine form factor (mitochondrial branching, A) and aspect ratio (mitochondrial branch length, B). Quantitative data are presented as means ± SD (n = 4–5 patients/group). Each symbol represents one patient sample. Black colored symbols: normal ASM. Red colored symbols: asthmatic ASM. Open symbols: females; closed symbols: males. Data were analyzed using mixed-effects analysis, Sidak’s multiple comparisons test. Representative images are shown in (C). All images for control and treatment groups were collected at the same time under the same conditions.

Figure 5.

Mitochondrial respiration in human airway smooth muscle (ASM) cells treated with glial-derived neurotrophic factor (GDNF) and transforming growth factor (TGF)β. Normal or asthmatic ASM cells were treated with GDNF and TGFβ for 24 h prior to mitochondrial stress test using a Seahorse Bioanalyzer. A: a representative time-course of mitochondrial stress test from normal ASM cells treated with vehicle. B: a representative time-course mitochondrial stress test from normal ASM cells treated with vehicle control, GDNF, or TGFβ. Basal respiration (C), maximal respiration (D), ATP-linked respiration (E), non-mitochondrial respiration (F) were calculated from Seahorse time measurements. Each symbol represents one patient sample. Black colored symbols: normal patients. Red colored symbols: asthma patients. Open symbols: females; closed symbols: males. Data were analyzed using mixed-effects analysis, Sidak’s multiple comparisons test.

Figure 6.

Mitochondrial protein expression in human airway smooth muscle (ASM) cells treated with glial-derived neurotrophic factor (GDNF) and transforming growth factor (TGF)β. Normal or asthmatic ASM cells were treated with GDNF and TGFβ for 48 h. Mitochondrial protein mitofusin (MFN)1 (A), MFN2 (B), optic atrophy protein 1 (OPA1; C), dynamin-related protein 1 (DRP1, D), and fission 1 (FIS1; E) in ASM cells were quantified with WES assays and normalized to total protein. Data are presented as means ± SD (n = 5–6 patients/group). Each symbol represents one patient sample. Black colored symbols: normal patients. Red colored symbols: asthma patients. Open symbols: females; closed symbols: males. Data were analyzed using two-way ANOVA, Sidak’s multiple comparisons test.