Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs

Abstract

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

Fig. 1.

Expression of mutant L188Q SFTPC in vivo leads to ER stress in type II AECs. (A and B) IHC for BiP in lung sections of WT (A) and L188Q SFTPC (B) mice, both treated with Dox for 1 wk. (C and D) IHC for XBP1 in lungs from the same mice. (Magnification in A–D: ×400.) Arrows point to immunostain-positive cells. (E) Western blot analysis on whole-lung lysates for BiP from WT and L188Q SFTPC mice treated with Dox for 1 wk. β-Actin is shown as loading control. (F) Results of quantitative real-time RT-PCR for expression of BiP mRNA from lungs of mice treated with Dox for 1 wk, normalized to the housekeeping gene RPL19. (n = 4–5 per column; *P < 0.001 between columns.) (G) RT-PCR gel demonstrating splice variants for XBP1 mRNA in lungs of L188Q SFTPC and WT mice treated with Dox for 1 wk. (H) XBP1 splicing analysis by densitometry. (n = 5 per column; *P < 0.01 between columns.) Graphical data are presented as mean ± SEM.

Fig. 2.

Mutant L188Q pro-SP-C localizes to the ER in type II AECs isolated from L188Q SFTPC mice after in vivo Dox treatment for 1 wk. (A–C) Dual immunofluorescence for the myc tag (red; A) and the ER marker BiP (green; B) demonstrated strong colocalization on merged imaging (yellow; C). Blue, DAPI. (D–F) Dual immunofluorescence for the myc tag (red; D) and the Golgi marker giantin (green; E) demonstrated minimal colocalization on merged imaging (yellow; F). Blue, DAPI. (G) Western blot analysis for BiP from type II AECs isolated from WT or L188Q SFTPC mice treated with Dox in vivo for 1 wk. β-Actin is shown as loading control. (H) Real-time RT-PCR for expression of BiP mRNA, normalized to the housekeeping gene RPL19. (n = 3 per column; *P < 0.05 between columns.) (I) RT-PCR gel demonstrating a splice variant for XBP1 in type II AECs. (J) XBP1 splicing analysis by densitometry. (n = 3 per column; *P < 0.05 between columns.) Graphical data are presented as mean ± SEM.

Fig. 3.

Mice expressing mutant L188Q SFTPC had greater lung fibrosis after i.t. bleomycin (Bleo). (A and B) Trichrome-stained lung sections from Dox-treated WT mice and L188Q SFTPC mice at 3 wk after 0.04 unit of i.t. bleomycin. (Magnification: ×100.) (C) Semiquantitative fibrosis scoring of trichrome-stained lung sections from WT and L188Q SFTPC mice at 3 wk after 0.04 unit of i.t. bleomycin. (n = 5 per group; *P < 0.05 compared with other groups.) Results are representative of three separate experiments. (D) Total lung collagen content from right lower lobe (RLL) based on hydroxyproline microplate assay at 3 wk after 0.04 unit of i.t. bleomycin or saline. (n = 4–6 per group for saline and 10 per group for bleomycin; *P < 0.05 compared with other groups.) (E) Semiquantitative scoring of S100A4+ lung fibroblasts on immunostained lung sections from WT and L188Q SFTPC mice at 3 wk after i.t. bleomycin. (n = 5 per group; *P < 0.05 compared with other groups.) (F) Semiquantitative scoring of αSMA+ lung fibroblasts on immunostained lung sections from WT and L188Q SFTPC mice at 3 wk after i.t. bleomycin. (n = 5 per group; *P < 0.05 compared with other groups.) (G) Static lung compliance in WT and L188Q SFTPC-expressing mice treated with Dox at 3 wk after i.t. bleomycin. (n = 3 per group for saline and 5–8 per group for bleomycin; *P < 0.05 between bleomycin-treated groups; ^#P < 0.001 compared with respective saline-treated group.) (H) Airway resistance in WT and L188Q SFTPC-expressing mice treated with Dox at 3 wk after i.t. bleomycin. Graphical data are presented as mean ± SEM.

Fig. 4.

Mice expressing L188Q SPTPC had greater AEC death after i.t. bleomycin. (A and B) TUNEL-stained lung sections from WT (A) and L188Q SFTPC (B) mice at 1 wk after i.t. bleomycin. Arrows point to representative TUNEL+ cells. (Magnification: ×600.) (C) Semiquantitative evaluation of TUNEL+ AECs on lung sections from WT and L188Q SFTPC mice at 1 wk after i.t. bleomycin. (n = 8–11 per group; *P < 0.05 between groups.) (D and E) Western blot analysis (D) and densitometry (E) for caspase-3 using whole-lung lysates from bleomycin-treated WT and L188Q SFTPC mice. β-Actin was used as a loading control. Graphical data represent the ratio of band densities for active caspase-3 and total caspase-3 (WT normalized to value of 1). (n = 5 per group; *P < 0.05 compared with WT.) (F and G) Western blot analysis (F) and densitometry (G) for caspase-12 from lung lysates. Graphical data represent the band density of caspase-12 adjusted for β-actin (WT normalized to value of 1). (n = 5 per group; *P < 0.05 compared with WT.) Graphical data are presented as mean ± SEM.

Fig. 5.

Mutant L188Q SFTPC expression does not enhance lung inflammation after i.t. bleomycin (Bleo). (A) Total and differential cell counts in bronchoalveolar lavage (BAL) from WT and L188Q SFTPC mice at 1 wk after i.t. bleomycin. (n = 5–6 per group). (B) Bioluminescence imaging over the thorax as an indicator of NF-κB activation in lungs of WT and L188Q SFTPC mice crossed with NGL mice. Baseline photon counts represent mice with Dox treatment for 14 d. Subsequently, mice were treated with bleomycin and imaged at 3 and 7 d. Photon counting was performed after i.p. injection of luciferin (1 mg). (n= 4 per group.) Graphical data are presented as mean ± SEM.

Fig. 6.

Tunicamycin (TM) enhances lung fibrosis induced by bleomycin (Bleo). (A and B) Trichrome-stained lung sections at 2 wk after bleomycin from WT mice treated with a single i.t. injection of vehicle (DMSO; A) or tunicamycin (B) at 48 h before bleomycin. (Magnification: ×100.) (C) Semiquantitative fibrosis scoring of trichrome-stained lung sections from mice exposed to vehicle + bleomycin (vehicle + Bleo) compared with tunicamycin + bleomycin (TM + Bleo) at 2 wk after i.t. bleomycin. (n = 5–6 per group; *P < 0.05 between groups.) (D) Total collagen content of entire right lung from mice exposed to vehicle or tunicamycin followed by i.t. administration of saline or bleomycin. (n = 5 per group for saline and 7–10 per group for bleomycin; *P < 0.05 compared with other groups.) Graphical data are presented as mean ± SEM.