POU5F1 bridges Hedgehog signaling and epithelial remodeling in COPD

Abstract

Airway epithelium remodeling is a hallmark of chronic obstructive pulmonary disease (COPD) pathogenesis. Hedgehog signaling is activated during airway epithelial repair to warrant proliferation and during cell differentiation to establish a fully functional epithelium with optimal mucociliary clearance. Consequently, it was found to be altered in COPD patients. Using transcriptomic analysis on air-liquid interface airway epithelial cells during differentiation upon Hedgehog pathway inhibition, we highlighted potential regulators of COPD-associated epithelial remodeling. Furthermore, the alteration of POU5F1 (OCT3/4) was validated in COPD airway epithelial cells and lung tissues. Although further investigations are required, these findings uncovered essential clues tethering respiratory epithelial cell plasticity and Hedgehog signaling.

Keywords: COPD; Hedgehog; Pou5f1; airways; epithelial cells.

FIGURE 1

Transcriptomic profiling of AB5E1-treated AECs. (A) Volcano plot showing the gene distribution with the p-value adjusted on the y-axis and log2 of the fold change between AB5E1-treated and untreated cells on the x-axis. DEGs are color-coded: pink, log2FC = −0.5 < DEG < log2FC = 0.5; blue, downregulated genes with log2FC < −1; red, upregulated genes with log2FC > 1; green, DEGs corresponding to LOC, LINC, or miRNA with log2FC > 1. (B) Heatmap of the 18 most identified DEGs in the AB5E1-treated cell transcriptomes. (C) Bubble plot representing cell populations on the y-axis and genes on the x-axis. The size of the dots represents the proportion of expressing cells, and the color intensity represents the median of expression levels. The data were extracted from the full HLCA dataset [except for #, for which data were extracted from the partial HLCA dataset (Sikkema et al., 2023)]. (D) Histogram representing a count of genes in GO terms (biological process, cellular component, annotated keywords) for the 371 DEGs (log2FC = −0.5 < DEG < log2FC = 0.5). *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

POU5F1 is altered in COPD patients. (A) Dot plot with mean ± SD showing POU5F1 expression analysis from RNAseq data (GSE137557). The proliferative basal cells (n = 8) were collected for analysis before the air switch, and the fully differentiated cells (n = 6) were collected for analysis after 28 days of differentiation. *p < 0.05; **p < 0.01; non-COPD vs. COPD. FPKM: Fragments per kilobase million. (B) Dot plot with mean ± SD showing fold change according to the 2^−ΔΔCT method for the POU5F1 expression analysis on basal cells and (C) differentiated cells. The average threshold cycle (Ct) values were respectively at ALI7 in non-COPD (n = 6) vs. COPD (n = 6) AECs: 22.5 Ct vs. 22 Ct for GAPDH, and 31.5 Ct vs. 31 Ct for POU5F1; at ALI35 in non-COPD vs. COPD AECs: 22.7 Ct vs. 23.2 Ct for GAPDH, and 31 Ct vs. 31.7 Ct for POU5F1. **p < 0.01; ns: non-significant; non-COPD vs. COPD. (D) Representative micrographs showing bronchial epithelia of non-COPD individuals and COPD patients immunostained for POU5F1 (red), epithelial cytokeratins (KP, green), and cell nuclei (DAPI, blue). Magnification corresponding to the selected area is shown. (E) Dot plot with mean ± SD representing POU5F1 pixel mean grey values of non-COPD (n = 6, black) individuals and COPD (n = 8, red) patients. *p < 0.05; non-COPD vs. COPD. (F) Linear regression representing a correlation between POU5F1 pixel mean gray levels and FEV₁/FVC in bronchial epithelia of COPD patients (n = 8). (G) Linear regression representing a correlation between POU5F1 pixel mean gray levels and FEV₁ in bronchial epithelia of COPD patients (n = 8).