A CD44/Brg1 nuclear complex confers mesenchymal progenitor cells with enhanced fibrogenicity in idiopathic pulmonary fibrosis
Libang Yang, … , Peter B. Bitterman, Craig A. Henke
Libang Yang, … , Peter B. Bitterman, Craig A. Henke
Published April 6, 2021
Citation Information: JCI Insight. 2021;6(9):e144652. https://doi.org/10.1172/jci.insight.144652.
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Research Article Pulmonology

A CD44/Brg1 nuclear complex confers mesenchymal progenitor cells with enhanced fibrogenicity in idiopathic pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease. We previously identified fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of patients with IPF who serve as drivers of progressive fibrosis. Recent single-cell RNA sequencing work revealed that IPF MPCs with the highest transcriptomic network entropy differ the most from control MPCs and that increased CD44 was a marker of these IPF MPCs. We hypothesize that IPF MPCs with high CD44 (CD44hi) expression will display enhanced fibrogenicity. We demonstrate that CD44-expressing MPCs are present at the periphery of the IPF fibroblastic focus, placing them in regions of active fibrogenesis. In a humanized mouse xenograft model, CD44hi IPF MPCs are more fibrogenic than CD44lo IPF MPCs, and knockdown of CD44 diminishes their fibrogenicity. CD44hi IPF MPCs display increased expression of pluripotency markers and enhanced self-renewal compared with CD44lo IPF MPCs, properties potentiated by IL-8. The mechanism involves the accumulation of CD44 within the nucleus, where it associates with the chromatin modulator protein Brahma-related gene 1 (Brg1) and the zinc finger E-box binding homeobox 1 (Zeb1) transcription factor. This CD44/Brg1/Zeb1 nuclear protein complex targets the Sox2 gene, promoting its upregulation and self-renewal. Our data implicate CD44 interaction with the epigenetic modulator protein Brg1 in conveying IPF MPCs with cell-autonomous fibrogenicity.

Authors

Libang Yang, Hong Xia, Karen Smith, Adam Gilbertsen, Daniel Beisang, Jonathan Kuo, Peter B. Bitterman, Craig A. Henke

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Figure 8

IL-8 promotes the formation of a nuclear CD44/BRG1/Zeb1 protein complex that regulates CD44hi IPF MPC self-renewal.

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IL-8 promotes the formation of a nuclear CD44/BRG1/Zeb1 protein complex ...
(A) CD44hi IPF MPCs were treated with IL-8 or vehicle (Con). Nuclear CD44 was immunoprecipitated, and Western blot analysis for CD44 and BRG1 was performed. Immunoprecipitation using isotype antibody served as control. Densitometry values below Western blot. (B) BRG1 was knocked down in CD44hi IPF MPCs using BRG1 shRNA (BRG1-shRNA). Scrambled shRNA (Scr-shRNA) served as control. Cells were treated with IL-8 or vehicle (Con). Sox2 expression was quantified by Western blot analysis (left panel). Densitometry values in graph below. Self-renewal was quantified using the colony-forming assay (right panels). (C) CD44hi IPF MPCs were treated with IL-8 or vehicle (Con). Zeb1 protein levels were quantified by Western blot analysis. Densitometry values in graph below. (D) CD44hi IPF MPCs were treated with IL-8 or vehicle (Con). Nuclear CD44 was immunoprecipitated, and Western blot analysis for Zeb1 was performed. Immunoprecipitation using isotype antibody served as control. Densitometry values in graph below. (E and F) Zeb1 was knocked down in CD44hi IPF MPCs using Zeb1 shRNA. Scrambled shRNA (Scr-shRNA) served as control. The cells were treated with IL-8 or vehicle (Con). Zeb1 and Sox2 expression were quantified by Western blot analysis (densitometry values in graph below) (E), and self-renewal was quantified using the colony-forming assay (F). (G) CD44hi IPF MPCs were treated with IL-8 or vehicle (Con). Zeb1 was immunoprecipitated from nuclear fractions, and qPCR for Sox2 was performed. Immunoprecipitation using isotype antibody (IgG) served as control. IPF 422, 424, and 442 were used in these figures. Data are expressed as mean ± SEM. n ≥ 3 independent experiments for each experimental condition, except the Western blot, which is from a single experiment representative of 3 independent replicates. Data are expressed as mean ± SEM. P values were determined by 2-tailed Student’s t test.