Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation
Tingting Weng, … , Eric J. Wagner, Michael R. Blackburn
Tingting Weng, … , Eric J. Wagner, Michael R. Blackburn
Published February 28, 2019
Citation Information: J Clin Invest. 2019;129(5):1984-1999. https://doi.org/10.1172/JCI122106.
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Research Article Pulmonology

Cleavage factor 25 deregulation contributes to pulmonary fibrosis through alternative polyadenylation

Abstract

Idiopathic pulmonary fibrosis (IPF) is a deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) is a key factor that drives the disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors through the alteration of miRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrated that CFIm25, a global regulator of APA, was downregulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in α–smooth muscle actin–positive (α-SMA–positive) fibroblasts. Following CFIm25 knockdown in healthy human lung fibroblasts, we identified 808 genes with shortened 3′-UTRs, including those involved in the TGF-β signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key profibrotic factors was suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrated that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhanced pulmonary fibrosis after bleomycin exposure. Collectively, our results identified CFIm25 downregulation as an important mechanism for elevating profibrotic gene expression in pulmonary fibrosis.

Authors

Tingting Weng, Junsuk Ko, Chioniso P. Masamha, Zheng Xia, Yu Xiang, Ning-yuan Chen, Jose G. Molina, Scott Collum, Tinne C. Mertens, Fayong Luo, Kemly Philip, Jonathan Davies, Jingjing Huang, Cory Wilson, Rajarajan A. Thandavarayan, Brian A. Bruckner, Soma S.K. Jyothula, Kelly A. Volcik, Lei Li, Leng Han, Wei Li, Shervin Assassi, Harry Karmouty-Quintana, Eric J. Wagner, Michael R. Blackburn

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Figure 1

CFIm25 is downregulated in fibrotic lungs.

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CFIm25 is downregulated in fibrotic lungs. (A) A representative Western ...
(A) A representative Western blot showing protein expression of CFIm25, FN, and GAPDH from 6 healthy and 7 IPF lung specimens. CFIm25 densitometric analysis shows significantly downregulated CFIm25 levels in IPF lungs. **P < 0.01, by unpaired t test versus healthy control. (B) Immunohistochemistry for CFIm25 (brown) and α-SMA (pink) showing cellular localization in control and IPF lung specimens. Arrow indicates CFIm25-positive cells. Arrowhead indicates α-SMA–positive but CFIm25-negative cells. Scale bars: 100 μm. Original magnification ×100. (C) Western blot shows CFIm25, FN, and β-actin protein expression levels in primary fibroblast lines derived from healthy or IPF lungs. (D–F) Mice were i.p. injected with PBS or bleomycin biweekly for 4 weeks. (D) Western blotting was performed to analyze the protein expression of CFIm25 in whole-lung lysates on day 33 after PBS, or 7, 17, 28, and 33 days after the first bleomycin exposure. The different lanes represent samples collected from distinct mice. β-Actin was used as a protein loading control. (E) Immunofluorescence was carried out to determine CFIm25 (pink) and α-SMA (green) colocalization in lungs from mice exposed to PBS or bleomycin for 33 days. Arrows indicates CFIm25-positive cells; arrowheads indicatesα-SMA–positive but CFIm25-negative cells. Scale bars: 100 μm. (F) Primary fibroblasts were isolated from day-33 PBS or bleomycin-injected mouse lungs. Western blotting was performed to determine CFIm25, FN, and β-actin protein levels.