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 5

The CXCR1/IL-8 axis regulates CD44hi IPF MPC self-renewal.

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The CXCR1/IL-8 axis regulates CD44hi IPF MPC self-renewal. (A) CXCR1/2 e...
(A) CXCR1/2 expression was quantified in CD44hi and CD44lo IPF MPCs by qPCR (left panels) and Western blot analysis (right middle panel; densitometry values right panel). n = 2 cell lines tested (IPF 422 and 424). (B) IL-8 expression was quantified by qPCR (left panel). IL-8 secretion was quantified by ELISA (right panel). n =3 cell lines tested (IPF 422, 424, and 442). (C) CD44hi IPF MPCs (IPF 422, 424, and 442) were treated with recombinant IL-8 or control (Con). CXCR1 expression was quantified by Western blot analysis (densitometry values right panel). (D) CD44hi IPF MPCs (IPF 422, 424, and 442) incorporated into methylcellulose gels were treated with IL-8. Colony number quantified. (E) CD44hi and CD44lo IPF MPCs (IPF 422 and 424) were treated with IL-8. Oct3/4, Nanog, and Sox2 expression was quantified by qPCR (left panel) and Western blot analysis (right 2 panels; densitometry values below Western blots). (F) CD44hi and CD44lo IPF MPCs (IPF 422, 424, and 442) incorporated into methylcellulose gels were treated with IL-8. Shown is colony-forming assay. (G) CD44hi IPF MPCs (IPF 422, 424, and 442) were treated with IL-8 in the presence of the IL-8 receptor inhibitor Reparixin (IL-8 + R) or vehicle (IL-8 + DMSO). Cells not treated with IL-8 served as a control (Con). Sox2, Nanog, and Oct3/4 levels were quantified by Western blot analysis (left panels, densitometry values left middle panel). Colony-forming assay (right panels). n ≥ 3 independent experiments for each experimental condition except E, where n = 2 independent experiments were performed, and Western blot, which is from a single experiment representative of 3 independently conducted replicates. Data are expressed as mean ± SEM. P values determined by 2-tailed Student’s t test, except in G, where P values were determined by 1-way ANOVA.