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 6

Inhibition of PIM1 signaling pathway reduces prosurvival gene expression and sensitizes IPF-derived lung fibroblasts to apoptotic cues.

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Inhibition of PIM1 signaling pathway reduces prosurvival gene expression...
(A) qPCR analysis of aging-associated prosurvival genes and those with NFATc1 binding sites in IPF-derived fibroblasts transfected with scrambled siRNA or siRNA for NFATc1 for 48 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 2-tailed, paired Student’s t test. (B) Western blot analysis of FOXM1 and BIRC5 in IPF-derived fibroblasts transfected with scrambled siRNA or siNFATc1 for 48 hours and then stimulated with 50 ng/mL of PDGF-BB or 10% FBS for an additional 24 hours. Shown is a representative blot of 2 independent experiments. (C) qPCR analysis of prosurvival genes in IPF-derived fibroblasts transfected with scrambled siRNA or siRNA for PIM1 for 48 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) qPCR analysis of prosurvival gene expression in IPF-derived lung fibroblasts cotreated with 10 μM of AZD1208 and 50 ng/mL of PDGF-BB for 24 hours. Data are shown as mean ± SEM of n = 3 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (E) IPF-derived lung fibroblasts were cotreated with 10 μM of AZD1208 and 50 ng/mL of PDGF-BB for 24 hours and analyzed by Western blotting using antibodies against phospho-BAD, total-BAD, BIRC5, and GAPDH. (F) IPF-derived lung fibroblasts were pretreated for 24 hours with 10 μM of AZD1208 and with 300 nM of staurosporine for an additional 2 hours, followed by Western blotting analysis using antibodies against procaspase-3, cleaved caspase-3, and GAPDH. (G) IPF-derived lung fibroblasts were preincubated for 24 hours with 10 μM of AZD1208, followed by treatment with 0.5 μg/mL of FAS-activating antibody for an additional 24 hours. Procaspase-3 and cleaved caspase-3 were detected by Western blotting. Shown is a representative blot of 3 independent experiments.