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 7

Inhibition of CD44 nuclear accumulation inhibits IL-8–mediated self-renewal.

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Inhibition of CD44 nuclear accumulation inhibits IL-8–mediated self-rene...
(A) CD44hi IPF MPCs transduced with CD44 mutant NLS construct (Mu), WT CD44, or empty vector (EV) were treated with recombinant IL-8 (5 ng/mL) or vehicle control (Con). Sox2 protein expression and nuclear (N), membrane (M), and cytoplasmic (C) CD44 levels were quantified by Western Blot analysis (left panel). Lamin, Cadherin, and GAPDH served as loading controls for N, M, and C fractions, respectively. Densitometry values summarizing Western blot data in right panels. (B) CD44hi IPF MPCs transduced with CD44 Mu, WT CD44, or EV were incorporated into methylcellulose gels and treated with IL-8 (5 ng/mL) or vehicle control (Con). Self-renewal was assessed in the colony-forming assay. (C and D) CD44 was knocked down in CD44hi IPF MPCs using CD44 shRNA (CD44-shRNA). Scrambled shRNA (Scr-shRNA) served as control. Cells were treated with recombinant IL-8 (5 ng/mL) or Con. Shown are Sox2 expression quantified by qPCR (left panel) and Western Blot analysis (right panels). Densitometry values summarizing Western blot data in graph below (C). Self-renewal was assessed using the colony-forming assay (D). For C and D, IPF 422, IPF424, and IPF 442 were used in these figures. Data are expressed as mean ± SEM. n ≥ 3 independent experiments for each experimental condition or group except the Western blot 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.