Glutaredoxin deficiency promotes activation of the transforming growth factor beta pathway in airway epithelial cells, in association with fibrotic airway remodeling

Abstract

S-glutathionylation of reactive protein cysteines is a post-translational event that plays a critical role in transducing signals from oxidants into biological responses. S-glutathionylation can be reversed by the deglutathionylating enzyme glutaredoxin (GLRX). We have previously demonstrated that ablation of Glrx sensitizes mice to the development of parenchymal lung fibrosis(1). It remains unclear whether GLRX also controls airway fibrosis, a clinical feature relevant to asthma and chronic obstructive pulmonary disease, and whether GLRX controls the biology of airway epithelial cells, which have been implicated in the pathophysiology of these diseases. In the present study we utilized a house dust mite (HDM) model of allergic airway disease in wild type (WT) and Glrx^-/- mice on a C57BL/6 background prone to develop airway fibrosis, and tracheal basal stem cells derived from WT mice, global Glrx^-/- mice, or bi-transgenic mice allowing conditional ablation of the Glrx gene. Herein we show that absence of Glrx led to enhanced HDM-induced collagen deposition, elevated levels of transforming growth factor beta 1 (TGFB1) in the bronchoalveolar lavage, and resulted in increases in airway hyperresponsiveness. Airway epithelial cells isolated from Glrx^-/- mice or following conditional ablation of Glrx showed spontaneous increases in secretion of TGFB1. Glrx^-/- basal cells also showed spontaneous TGFB pathway activation, in association with increased expression of mesenchymal genes, including collagen 1a1 and fibronectin. Overall, these findings suggest that GLRX regulates airway fibrosis via a mechanism(s) that involve the plasticity of basal cells, the stem cells of the airways.

Keywords: Asthma; Basal cells; COPD; Fibrosis; S-glutathionylation.

Graphical abstract

Fig. 1

Assessment of inflammation, mucus metaplasia and smooth muscle cells in WT or Glrx^-/- mice following repeated exposure to house dust mite. A: Western Blot analysis for GLRX in homogenized lung tissue of WT or Glrx^-/- mice exposed to saline or house dust mite (HDM). ACTB (β-actin): loading control. B: Total (left) and differential cell counts (right) in BAL fluid from WT or Glrx^-/- mice after exposure to saline or HDM. C: Periodic acid-Schiff (PAS) staining in WT or Glrx^-/- mice exposed to HDM or saline (scale bar = 50 μm). Quantification of airway mucus staining intensity was determined by positive staining areas using Metamorph. D: Alpha-smooth muscle actin (ACTA2) immune-reactivity in WT or Glrx^-/- mice exposed to saline or HDM (scale bar = 50 μm). WT PBS n = 8, WT HDM n = 10, Glrx^-/- PBS n = 8, Glrx^-/- HDM n = 10 mice. ***p < 0.001, ANOVA.

Fig. 2

Assessment of airway hyperresponsiveness (AHR) in WT or Glrx^-/- mice exposed to saline or HDM. Mice were tracheostomized and ventilated, and respiratory mechanics evaluated at baseline, or in response to ascending doses of the bronchoconstricting agent, methacholine. Shown are the Newtonian resistance (Rn, a marker of airway stiffness or central airway resistance, top), tissue resistance (G, middle), and tissue elastance (H, bottom) parameters. (*p < 0.05; **p < 0.01; ***p < 0.001). WT PBS n = 8, WT HDM n = 10, Glrx^-/- PBS n = 8, Glrx^-/- HDM n = 10 mice.

Fig. 3

Evaluation of airway fibrosis in WT or Glrx^-/- mice exposed to saline or HDM. A. Masson's Trichrome staining in WT or Glrx^-/- mice exposed to HDM or saline (scale bar = 50 μm). B: Assessment of hydroxyproline content in the right superior lobe 24 h after the final challenge. C: Evaluation of TGFB1 level in the BAL fluid by enzyme-linked immunosorbent assay (ELISA). WT PBS n = 13, WT HDM n = 9, Glrx^-/- PBS n = 10, Glrx^-/- HDM n = 13 mice.

Fig. 4

Characteristics of primary airway basal cells isolated from WT or Glrx^-/- mouse tracheas. A: Expression of the basal cell marker, cytokeratin 5 (KRT5) in WT and Glrx^-/- basal cells via immunofluorescence analysis (scale bar = 50 μm). B: Expression of the epithelial marker, E-cadherin (CDH1) in WT or Glrx^-/- basal cells evaluated via immunofluorescence (scale bar = 20 μm). C: Assessment of the epithelial marker, E-cadherin (CDH1) in WT or Glrx^-/- basal cells exposed to HDM (50 μg/ml, 7 days) evaluated via Western blot analysis. ACTB (β-actin): loading control. D: Assessment of protein S-glutathionylation in WT or Glrx^-/- basal cells evaluated via Western blot analysis. GAPDH (glyceraldehyde-3-phosphate dehydrogenase): loading control. GLRX blot confirms the absence of GLRX proteins in Glrx^-/- airway basal cells. Shown is a representative blot out of 3 independent experiments.

Fig. 5

Primary Glrx^-/- basal cells spontaneously secrete TGFB1 and express mesenchymal proteins. A: mRNA expression levels of Col1a1 and Fn1 in WT vs. Glrx^-/- MTB cells in response to TGFB1 (5 ng/ml, 48 h). B: Western Blot analysis of ACTA2 in WT vs. Glrx^-/- MTB cells stimulated with TGFB1 (5 ng/ml, 3 days). ACTB (β-actin): loading control.

Fig. 6

Constitutive activation of the TGFβ pathway in primary airway basal cells lacking Glrx. A: Assessment of phosphorylation(s) of SMAD2 in WT vs. Glrx^-/- basal cells (scale bar = 50 μm). B: mRNA expression levels of Col1a1 and Fn1 in WT vs. Glrx^-/- MTB cells in response to the ALK5 inhibitor, SB431542 (10 μM, 48 h) (*p < 0.05; **p < 0.01; ***p < 0.001). C: Assessment of TGFB1 levels in supernatants of WT vs. Glrx^-/- epithelial cells differentiated under air liquid interface conditions (ALI), in the absence of stimuli, or following exposure to HDM (50 μg/ml, 24 h).

Fig. 7

Acute ablation of Glrx from airway basal cells isolated from Krt5Cre-ERT2:Glrx^LoxP/LoxP bi-transgenic mice results in secretion of TGFB1. A: Design of mice with a LoxP-flanked Glrx allele. B: PCR screening of Glrx^LoxP/LoxP transgenic mice. C: Schematic of TMX-mediated ablation of Glrx from airway basal cells. D: Deletion of GLRX protein in airway basal cells from Krt5Cre-ERT2:Glrx^LoxP/LoxP bi-transgenic mice following administration of 500 nM 4hydroxytamoxifen (TMX) for 2 days. ACTB (β-actin): loading control. E: TMX-mediated ablation of GLRX from airway basal cells prior to their differentiation under ALI results in spontaneous TGFB1 secretion when assessed at day 25 of ALI culture. Results are obtained from 2 separate primary cell cultures, each initiated by pooling 3 tracheas from either Krt5CreERT2 single transgene or Krt5CreERT2:Glrx^loxP bitransgenic mice. **p < 0.01 ANOVA.

Fig. 7

Acute ablation of Glrx from airway basal cells isolated from Krt5Cre-ERT2:Glrx^LoxP/LoxP bi-transgenic mice results in secretion of TGFB1. A: Design of mice with a LoxP-flanked Glrx allele. B: PCR screening of Glrx^LoxP/LoxP transgenic mice. C: Schematic of TMX-mediated ablation of Glrx from airway basal cells. D: Deletion of GLRX protein in airway basal cells from Krt5Cre-ERT2:Glrx^LoxP/LoxP bi-transgenic mice following administration of 500 nM 4hydroxytamoxifen (TMX) for 2 days. ACTB (β-actin): loading control. E: TMX-mediated ablation of GLRX from airway basal cells prior to their differentiation under ALI results in spontaneous TGFB1 secretion when assessed at day 25 of ALI culture. Results are obtained from 2 separate primary cell cultures, each initiated by pooling 3 tracheas from either Krt5CreERT2 single transgene or Krt5CreERT2:Glrx^loxP bitransgenic mice. **p < 0.01 ANOVA.