Transcriptional analysis of lung fibroblasts identifies PIM1 signaling as a driver of aging-associated persistent fibrosis
Tho X. Pham, Jisu Lee, Jiazhen Guan, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Steven K. Huang, Daniel J. Tschumperlin, Giovanni Ligresti
Tho X. Pham, Jisu Lee, Jiazhen Guan, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Steven K. Huang, Daniel J. Tschumperlin, Giovanni Ligresti
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Research Article Aging Pulmonology

Transcriptional analysis of lung fibroblasts identifies PIM1 signaling as a driver of aging-associated persistent fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.

Authors

Tho X. Pham, Jisu Lee, Jiazhen Guan, Nunzia Caporarello, Jeffrey A. Meridew, Dakota L. Jones, Qi Tan, Steven K. Huang, Daniel J. Tschumperlin, Giovanni Ligresti

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Figure 7

PIM1 inhibition inhibits profibrotic gene expression and collagen secretion in organotypic IPF lung cultures ex vivo.

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PIM1 inhibition inhibits profibrotic gene expression and collagen secret...
(A) Schematic of organotypic culture of IPF lungs. (B) H&E and trichrome staining of IPF lung tissue explants after 5 days in culture showing intact tissue architecture of nonfibrotic area (top panels) and distorted architecture of fibrotic area (bottom panels). Scale bar: 50 μm. FF, fibrotic foci; V, vein; Av, alveoli. (C) qPCR analysis of ECM gene expression in IPF lung explants treated with 10 μM of AZD1208 or DMSO control in combination with or without 10 ng/mL of TGF-β for 5 days. n ≥ 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) qPCR analysis of pathogenic lung fibroblasts gene markers in IPF lung explants treated with 10 μM of AZD1208 or DMSO in the presence or absence of 10 ng/mL of TGF-β for 5 days. n ≥ 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (E) qPCR analysis of prosurvival genes in IPF lung explants treated with 10 μM of AZD1208 or DMSO in the presence or absence of 10 ng/mL of TGF-β for 5 days. n = 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (F) Soluble collagen-I secreted from IPF lung explants into the media was evaluated by Western blot analysis. Each lane contained equal volume of conditioned medium of different lung sections obtained from single IPF lung explants. Ponceau staining was used as loading control for secreted collagen-I.