Inhibition of PDIA3 in club cells attenuates osteopontin production and lung fibrosis

Abstract

Background: The role of club cells in the pathology of idiopathic pulmonary fibrosis (IPF) is not well understood. Protein disulfide isomerase A3 (PDIA3), an endoplasmic reticulum-based redox chaperone required for the functions of various fibrosis-related proteins; however, the mechanisms of action of PDIA3 in pulmonary fibrosis are not fully elucidated.

Objectives: To examine the role of club cells and PDIA3 in the pathology of pulmonary fibrosis and the therapeutic potential of inhibition of PDIA3 in lung fibrosis.

Methods: Role of PDIA3 and aberrant club cells in lung fibrosis was studied by analyses of human transcriptome dataset from Lung Genomics Research Consortium, other public resources, the specific deletion or inhibition of PDIA3 in club cells and blocking SPP1 downstream of PDIA3 in mice.

Results: PDIA3 and club cell secretory protein (SCGB1A1) signatures are upregulated in IPF compared with control patients. PDIA3 or SCGB1A1 increases also correlate with a decrease in lung function in patients with IPF. The bleomycin (BLM) model of lung fibrosis showed increases in PDIA3 in SCGB1A1 cells in the lung parenchyma. Ablation of Pdia3, specifically in SCGB1A1 cells, decreases parenchymal SCGB1A1 cells along with fibrosis in mice. The administration of a PDI inhibitor LOC14 reversed the BLM-induced parenchymal SCGB1A1 cells and fibrosis in mice. Evaluation of PDIA3 partners revealed that SPP1 is a major interactor in fibrosis. Blocking SPP1 attenuated the development of lung fibrosis in mice.

Conclusions: Our study reveals a new relationship with distally localised club cells, PDIA3 and SPP1 in lung fibrosis and inhibition of PDIA3 or SPP1 attenuates lung fibrosis.

Keywords: idiopathic pulmonary fibrosis; interstitial fibrosis.

Figure 1

Elevated expression of PDIA3 and SCGB1A1 in patients with IPF. (A) PDIA3 mRNA levels in control (n=132) and patients with IPF (n=160). (B) and (C) Western blot and quantitation of PDIA3 protein expression, control (n=17) and patients with IPF (n=15). Significance was based on unpaired two-tailed t-test and non-parametric Spearman correlation (rs) non-parametric Mann-Whitney t-test. Error bars represent ±SEM for the densitometry and ±SD for the mRNA analysis. GAPDH, glyceraldehyde phosphate dehydrogenase; IPF, idiopathic pulmonary fibrosis; PDIA3, protein disulfide isomerase A3.

Figure 2

PDIA3 and SCGB1A1 are increased in the parenchyma in a mouse model of fibrosis. (A) Bleomycin (BLM) or PBS challenge and harvest regimen. (B) Time-dependent alterations in hydroxyproline content *p<0.05 as compared with 24-day PBS and #p<0.05 as compared with 4-day BLM samples by ANOVA; error bars±SEM (n=5–9 mice/group). (C, D) Western blot analysis and quantitation of PDIA3 normalised to the expression of β-actin in the lung lysates; *p<0.05 compared with 24-day PBS and #p<0.05 as compared with 4-day BLM samples by ANOVA (n=3 mice/group); error bars±SEM. (E and F) Representative images from confocal microscopy stained for SCGB1A1 (green), PDIA3 (red) and nucleus (blue) and quantitation of SCGB1A1 and PDIA3 in the parenchyma. Secondary antibody (without primary) staining on fibrotic mouse lungs is used as the negative control. Unpaired t-test, (n=3 mice/group); error bars±SEM. Scale bar 50 µm. (G, H) Western blot analysis and quantitation of SCGB1A1 normalised to the expression of β-actin in the lung lysates; *p<0.05 compared with 24-day PBS and #p<0.05 as compared with 4-day BLM samples by ANOVA (n=3 mice/group); error bars±SEM. (I) SCGB1A1 mRNA levels in control (n=132) and patients with IPF (n=160). ANOVA, analysis of variance; PBS, phosphate-buffered saline; PDIA3, protein disulfide isomerase A3.

Figure 3

Pdia3 ablation in SCGB1A1 cells decreases lung fibrosis in mice. (A) BLM or PBS-instillation, doxycycline treatment and harvest regimen. (B) Collagen content in the upper right lung lobe is measured by hydroxyproline content (n=5–11 mice/group). (C and D) Measurement of collagen and fibronectin mRNAs (n=5–11 mice/group). (E and F) Representative histochemical images of Masson’s trichrome (blue) stained lung sections and histological scoring (n=4–6 mice/group). (G and H) Representative confocal immunofluorescence images stained for SCGB1A1 and DAPI and quantitation of parenchymal SCGB1A1 (arrows) immunofluorescence (n=4 mice/group). *p<0.05 as compared with PBS and #p<0.05 as compared with Ctr-BLM samples by ANOVA; error bars±SEM. Arrows indicate SCGB1A1 immunoreactivity in the parenchyma. Scale bar 50 µm. Pooled samples from two experiments. ANOVA, analysis of variance; BLM, bleomycin; PBS, phosphate-buffered saline; PDIA3, protein disulfide isomerase A3.

Figure 4

LOC14 decreases SCGB1A1 cells and lung fibrosis in mice. (A) BLM or PBS challenge, LOC14 treatment and harvest regimen (0.015, 0.15 and 1.5 mg/kg LOC14 were administered into the lung via oropharyngeal aspiration). (B) Collagen content in the upper right lung lobe, measured by hydroxyproline content (n=5–17 mice/group). (C and D) Measurement of collagen and fibronectin mRNAs (n=5–7 mice/group). (E and F) Representative histochemical images of Masson’s trichrome (blue) stained lung sections and histological scoring (n=5 mice/group). (G) Measurement of Scgb1a1 mRNA (n=5–7 mice/group). (H and I) Representative confocal immunofluorescence images stained for SCGB1A1 and DAPI and quantitation of parenchymal SCGB1A1 (arrows) immunofluorescence (n=4 mice/group). *p<0.05 as compared with PBS and #p<0.05 as compared with other-BLM samples by ANOVA; error bars±SEM. Arrows indicate SCGB1A1 immunoreactivity in the parenchyma. Scale bar 50 µm. Pooled samples from two experiments. ANOVA, analysis of variance; BLM, bleomycin; PBS, phosphate-buffered saline.

Figure 5

Analysis of PDIA3 interacting partners and identification of SPP1 as a potential mediator of lung fibrosis. (A) Heat map of interacting partners of PDIA3 in fibrotic mouse lung analysed 14-day post-BLM challenge (n=3 samples/group, ‘C’ immunoprecipitation using non-specific IgG as a control). Identified proteins with a p value <0.05 (two-tailed t-test: 14-day post-BLM challenge vs PBS) are represented. The number beside the gene symbol indicates how many members in the cluster associated with that protein. The scale of the colour intensity is arbitrary. (B) Volcano plot depicting the significance of interactions of PDIA3 and SPP1 post immunoprecipitation and mass spectrometry. Fold changes of 2 and p value at 0.05 (two-tailed t-test: 24-day post-BLM challenge vs PBS) are indicated by dotted lines on the X-axis and y-axis, respectively. (C) Western blot analysis of immunoprecipitated PDIA3 and SPP1 in lung lysates (n=4 samples/group). (D) ELISA for SPP1 in bronchoalveolar lavage fluid samples of 24-day post-PBS (n=10) or BLM (n=12) challenged mice. (E) ELISA for SPP1 in lung tissue samples of control (n=18) and patients with IPF (n=16). (F) mRNA expression of SPP1 in control (n=132) and patients with IPF (n=160) from LGRC cohort. (G) Correlation of SPP1 mRNA expression with %DLCO in control (n=92) and patients with IPF (n=145) from LGRC cohort. Significance was based on unpaired two-tailed t-test and non-parametric Pearson correlation, non-parametric Mann-Whitney t-test. Error bars represent ±SEM for mouse samples and ±SD for human samples. ANOVA, analysis of variance; BLM, bleomycin; PBS, phosphate-buffered saline; PDIA3, protein disulfide isomerase A3.

Figure 6

Club cell-specific Pdia3 ablation or inhibition decreases SPP1 and SPP1 blocking antibody attenuates lung fibrosis in mice. (A, B) Measurement of SPP1 protein in lung tissue and BALF of Ctr and ΔEpi-Pdia3 mice. *p<0.05 as compared with PBS groups, #p<0.05 as compared with the Ctr-BLM group by ANOVA, error bars±SEM (n=5–13 mice/group). Outlier removed in BALFΔEpi-Pdia3 (n=1). (C) Measurement of SPP1 by ELISA in PBS or BLM challenged in combination with dimethyl sulfoxide or LOC14 treated mice. *p<0.05 as compared with PBS groups, #p<0.05 as compared with other-BLM groups by ANOVA, error bars±SEM (n=5–9 mice/group). (D) BLM or PBS challenge, VC/IgG/SPP1-Ab treatment and harvest regimen. (E) Collagen content in the upper right lobe is measured by hydroxyproline content (n=10–16 mice/group). (F and G) Representative histochemical images of Masson’s trichrome (blue) stained lung sections and histological scoring (n=5–8 mice/group). *p<0.05 as compared with PBS group and #p<0.05 as compared with other-BLM groups by ANOVA; error bars±SEM. Scale bar 400 µm. ANOVA, analysis of variance; BALF, bronchoalveolar lavage fluid; BLM, bleomycin; PBS, phosphate-buffered saline; PDIA3, protein disulfide isomerase A3.