Lipid Synthesis Is Required to Resolve Endoplasmic Reticulum Stress and Limit Fibrotic Responses in the Lung

Abstract

Endoplasmic reticulum (ER) stress is evident in the alveolar epithelium of humans and mice with pulmonary fibrosis, but neither the mechanisms causing ER stress nor the contribution of ER stress to fibrosis is understood. A well-recognized adaptive response to ER stress is that affected cells induce lipid synthesis; however, we recently reported that lipid synthesis was downregulated in the alveolar epithelium in pulmonary fibrosis. In the present study, we sought to determine whether lipid synthesis is needed to resolve ER stress and limit fibrotic remodeling in the lung. Pharmacologic and genetic manipulations were performed to assess whether lipid production is required for resolving ER stress and limiting fibrotic responses in cultured alveolar epithelial cells and whole-lung tissues. Concentrations of ER stress markers and lipid synthesis enzymes were also measured in control and idiopathic pulmonary fibrosis lung tissues. We found that chemical agents that induce ER stress (tunicamycin or thapsigargin) enhanced lipid production in cultured alveolar epithelial cells and in the mouse lung. Moreover, lipid production was found to be dependent on the enzyme stearoyl-coenzyme A desaturase 1, and when pharmacologically inhibited, ER stress persisted and lung fibrosis ensued. Conversely, lipid production was reduced in mouse and human fibrotic lung, despite there being an increase in the magnitude of ER stress. Furthermore, augmenting lipid production effectively reduced ER stress and mitigated fibrotic remodeling in the mouse lung after exposure to silica. Augmenting lipid production reduces ER stress and attenuates fibrotic remodeling in the mouse lung, suggesting that similar approaches might be effective for treating human fibrotic lung diseases.

Keywords: alveolar epithelium; endoplasmic reticulum stress; lipid synthesis; pulmonary fibrosis; unfolded protein response.

Figure 1.

Tunicamycin (TM) induces lipid synthesis in mouse lung epithelial 12 (MLE12) cells. (A) Western blots for activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE-1α), phosphorylated protein kinase R–like endoplasmic reticulum kinase (pPERK), PERK, phosphorylated eukaryotic elongation factor 2 (pelf2), elf2, and CCAAT/enhancer binding protein homologous protein (CHOP) in MLE12 cells cultured in media containing either vehicle (DMSO) or TM (1 μg/ml) for 24 hours (with GAPDH loading control). (B) 78 kD glucose-regulated protein (anti-Grp78) antibody (green) staining in control and TM-exposed MLE12 cells (blue represents DAPI nuclear stain). (C) Spliced X-box binding protein 1 (Xbp1) concentrations in control and TM-treated cells. (D) Western blots for fatty acid synthase (FAS), stearoyl–coenzyme A desaturase (SCD1), diacylglycerol O-acyltransferase 1 (DGAT1), sterol regulatory element–binding protein 1 (SREBP1c), and liver X receptor (LXR)-α in DMSO- or TM-exposed cells. (E) Boron dipyrromethene (BODIPY) neutral lipid staining of DMSO- and TM-treated cells. (F) Triacylglyceride and phospholipid concentrations in whole-cell lysates of DMSO- and TM-treated MLE12 cells. Immunoblots are representative of at least two different blots, and densitometric analyses (bar graphs) are representative of six or more mouse specimens (*P < 0.05 and **P  < 0.01 vs. DMSO, respectively). Scale bars: 20 μm. Data are expressed as mean ± SE, and statistical significance was assessed using an unpaired Student’s t test. H3 = histone 3.

Figure 2.

Inhibition of SCD1 activity induces endoplasmic reticulum stress and promotes the production of profibrotic mediators. (A) Western blots for ATF6, IRE-1α, pPERK, PERK, and CHOP in MLE12 cells cultured in media alone or in media containing SCD1 inhibitor (1 μM), tunicamycin (TM; 1 μg/ml), or SCD1 inhibitor plus TM (with GAPDH loading control). (B) mRNA amounts for GRP78 in MLE12 cells treated with SCD1 inhibitor, TM, or SCD1 inhibitor plus TM. (C) Neutral lipid staining in MLE12 cells under various conditions. Scale bars: 20 μm. (D) Triacylglyceride concentrations in whole-cell lysates. (E) Transforming growth factor (TGF)-β1 concentrations and expression of latent TGF-β–binding proteins (LTBP) 1 and LTBP4 in MLE12 cells. Images are representative of two different blots (n = 6 per group), and results of densitometric analysis are depicted in bar graphs. Statistical significance was assessed by analysis of variance: *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group. HPRT = hypoxanthine-guanine phosphoribosyltransferase; inh = inhibitor.

Figure 3.

Treatment with palmitate attenuates endoplasmic reticulum stress in response to tunicamycin. (A) Expression of endoplasmic reticulum stress markers ATF6, IRE-1α, pPERK, PERK, pelf2, elf2, CHOP, and cleaved caspase 3 (C-casp 3) in MLE12 cells cultured with BSA or BSA plus palmitate (PA; 100 μM) in the presence or absence of TM (with GAPDH loading control). (B) ATF6 expression in MLE12 cells grown in chamber slides and treated with BSA, PA, TM, or TM + PA for 24 hours. Scale bars: 20 μm. (C) Treatment with palmitate reduced transcript amounts of GRP78 and CHOP in response to TM. (D) Treatment with palmitate reduced expression of ATF6, IRE-1α, pPERK, PERK, pelf2, elf2, CHOP, and cleaved caspase 3 in MLE12 cells, and these effects were abolished by adding SCD1 inhibitor to the culture media. Images are representative of two different blots, and results of densitometric analysis are depicted in bar graphs (n = 6, per group). Statistical significance was assessed by ANOVA: *P < 0.05 and **P < 0.01 versus control group.

Figure 4.

TM augments lipid production in the lung. (A) Western blots for ATF6, IRE-1α, pPERK, PERK, pelf2, and elf2 in control and TM-exposed (2 μg/mouse in 75 μl twice weekly) lung tissues (with GAPDH loading control). (B) Representative lung tissue sections from mice receiving TM treatment or DMSO show an increase in GRP78 staining in alveolar epithelial type II cells. Scale bars: 50 μm. (C) Western blots for FAS, DGAT1, SCD1, SREBP1c, and LXRα in whole-lung tissues after either DMSO or TM (with GAPDH or H3 loading control). (D) Triglyceride and phospholipid concentrations in the lung after twice-weekly instillation of DMSO or TM. (E) Liquid chromatography–mass spectrometry analysis of lung tissue after DMSO or TM treatment. Statistical significance was assessed for A–D with Student’s t test: *P < 0.05 and **P < 0.01 versus control group (n = 6 in each group). For liquid chromatography–mass spectrometry, statistical significance was assessed by ANOVA: *P < 0.05 versus control group (n = 4 per group).

Figure 5.

Inhibition of SCD1 induces endoplasmic reticulum stress and promotes fibrotic responses in the lung. (A) Western blots for ATF6, IRE-1α, pPERK, PERK, pelf2, and elf2 in mice treated with vehicle, SCD1 inhibitor (30 mg/kg daily), TM (20 μg/ml in 100 μl twice weekly), or SCD1 inhibitor plus TM (with GAPDH loading control). (B) Treatment with SCD1 inhibitor blocks TM-induced triglyceride accumulation in the lung. (C) TGF-β1, LTBP1, and LTBP4 concentrations in whole-lung tissues from mice treated with vehicle, TM, SCD1 inhibitor, or TM plus SCD1 inhibitor. (D) High-power view of trichrome-stained lung tissues (blue, collagen) and (E) collagen concentrations in lung tissues from mice treated with vehicle control, TM, SCD1 inhibitor, or TM plus SCD1. Scale bars: 40 μm. Statistical significance was assessed by ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group (n = 6 per group). NS = not significant.

Figure 6.

Overexpression of SREBP1c attenuates endoplasmic reticulum stress in MLE12 cells after exposure to silica dust. (A) Western blots for FAS, SCD1, and DGAT1 in MLE12 cells transduced with control or SREBP1c-expressing vectors (with GAPDH loading control). (B) Overexpression of SREBP1c increases triglyceride and phospholipid production in MLE12 cells. (C) Western blots for SREBP1c, FAS, SCD1, and DGAT in MLE12 cells transduced with control or SREBP1c-expressing vectors and cultured with or without silica dust. (D) Western blots for ATF6, IRE-1α, pPERK, PERK, pelf2, elf2, and CHOP in control and SREBP1c-expressing cells cultured with or without silica dust. (E) Transcript amounts for Tgfβ1 were significantly reduced in silica-exposed MLE12 cells overexpressing SREBP1c. Images are representative of two different blots, and results of densitometric analysis are depicted in bar graphs. Statistical significance was assessed by ANOVA: *P < 0.05 and **P < 0.01 versus control group (n = 6 or greater in all groups).

Figure 7.

Treatment of mice with LXR agonists enhances lipid synthesis, decreases endoplasmic reticulum stress, and attenuates lung fibrosis in response to silica. (A) Transcript amounts for Srebp1, Scd1, and Fas in lungs of mice exposed to vehicle, silica, or silica plus T0901317 (30 mg/kg). (B) Western blots for SREBP1c at 14 days after silica administration in mice treated with or without T0901317 (with H3 loading control). (C) Triglyceride concentrations in lungs of mice treated with vehicle, silica, or silica plus T0901317. (D) Western blots for ATF6, IRE-1α, pPERK, PERK, pelf2, elf2, and cleaved caspase 3 protein in lungs of mice treated with vehicle, silica, or silica plus T0901317 (with Gapdh loading control). (E and F) Treatment with T0901317 reduced TGF-β1 and hydroxyproline concentrations in lungs of silica-exposed mice. (G) Low-power view of hematoxylin and eosin–stained lungs of silica-exposed mice treated with or without T0901317. Scale bars: 100 μm. Statistical significance was assessed by ANOVA: *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group.