Targeting the Epigenetic Regulator CBX5 Promotes Fibroblast Metabolic Reprogramming and Inhibits Lung Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is characterized by the sustained activation of interstitial fibroblasts leading to excessive collagen deposition and progressive organ failure. Epigenetic and metabolic abnormalities have been shown to contribute to the persistent activated state of scar-forming fibroblasts. However, how epigenetic changes regulate fibroblast metabolic responses to promote fibroblast activation and progressive fibrosis remains largely unknown. Here, we show that the epigenetic regulator CBX5 (chromobox protein homolog 5) is critical to the transition of quiescent fibroblasts to activated collagen-producing fibroblasts in response to bleomycin-induced lung injury. Loss of mesenchymal CBX5 attenuated fibrosis development, and this effect was accompanied by the downregulation of pathogenic fibroblast genes, including Cthrc1, Col1a1, and Spp1, and by the upregulation of metabolic genes with antifibrotic activity such as Ppara and Pparg. Single-cell RNA sequencing and immunohistochemistry analyses revealed that CBX5 expression was enriched in pathogenic fibroblasts and fibroblastic foci of IPF lungs. Bulk RNA-sequencing analysis combined with metabolic assessments demonstrated that CBX5 silencing in IPF fibroblasts potently inhibited transforming growth factor-stimulated glycolysis while enhancing AMPK signaling and mitochondrial metabolism. Finally, interruption of the CBX5 pathway in IPF fibroblasts in vitro and in IPF lung explants ex vivo synergistically potentiated the activation of metformin-induced AMP-activated protein kinase activation and inhibited collagen secretion. Collectively, our findings identify CBX5 as an epigenetic regulator linking metabolic maladaptation to the persistent activated state of lung fibroblasts during IPF progression.

Keywords: bleomycin; epigenetics; fibroblasts; metabolic reprogramming; pulmonary fibrosis.

Figure 1.

Mesenchymal CBX5 (chromobox protein homolog 5) inhibition attenuates bleomycin-induced lung fibrosis. (A) Schematic showing the experimental workflow to evaluate the effect of CBX5 deletion on bleomycin (Bleo)-induced lung fibrosis. (B) A hydroxyproline assay was performed to measure collagen content in the lungs. Mice were sham control (n = 7), wild-type (WT) mice that were administered Bleo and tamoxifen (Tam) (WT Bleo + Tam; n = 12), and CBX5 CKO mice that were administered Bleo and Tam (CBX5 CKO Bleo + Tam; n = 9). Data are shown as mean ± SEM, and P values were calculated using one-way ANOVA. (C) Ashcroft score indicates the severity of fibrosis. Mice were sham control (n = 10), WT Bleo + Tam (n = 12), and CBX5 CKO Bleo + Tam (n = 10). Data are shown as mean ± SEM, and P values were calculated using one-way ANOVA. (D) Representative images of Masson’s trichrome staining in lung sections after 21 days post–bleomycin injury. Scale bars, 50 μm. (E) Transcriptional analysis of whole-lung tissues obtained from sham control, WT Bleo + Tam, and CBX5 CKO Bleo + Tam mice. Data are shown as mean ± SEM of n = 6 replicates, and P values were calculated using one-way ANOVA.

Figure 2.

Loss of CBX5 inhibits the expression of profibrotic genes and promotes the expression of metabolic genes in lung fibroblasts. (A) Schematic showing the experimental workflow to assess the effect of CBX5 deletion on freshly isolated lung fibroblasts from Bleo-treated WT and CBX5 CKO mice. Lung fibroblasts were isolated at 14 days post–Bleo challenge by negative selection using magnetic beads conjugated with antibodies against CD31, CD45, and EpCAM. (B and C) Violin plots showing the relative gene expression by qPCR on isolated lung fibroblasts indicate that CBX5 deletion attenuated pro-fibrotic gene expression including (B) Col1a1, Cthrc1, and Spp1 and increased metabolic gene expression including (C) Ppara, Pparg, and Ppargc1a. n = 4–6, and P values were calculated using one-way ANOVA. Figure 2(A) created using BioRender.com.

Figure 3.

CBX5 expression is enhanced in idiopathic pulmonary fibrosis (IPF) lung fibroblasts. (A) Uniform Manifold Approximation and Projection (UMAP) plots derived from a publicly available single-cell RNA-sequencing (scRNA-seq) data set (Gene Expression Omnibus ID: GSE136831) of normal and IPF lung fibroblasts. UMAP plots show the expression of COL1A1 in whole-lung fibroblasts (FBs), and cell distribution in normal (blue) and IPF (red) lungs. Dotted lines indicate high COL1A1–expressing FBs. (B) Violin plots show normalized expression of COL1A1, CTHRC1, and CBX5 genes in whole-lung FBs, comparing normal and IPF lungs. (C) Representative immunohistochemistry images of human healthy and IPF lung sections showing nuclear expression of CBX5 (red). Scale bars: 50 μm (top and middle row); 25 μm (bottom row). Hematoxylin was used for counterstain. Fibroblastic foci are indicated in the images. The highest magnification image of the normal lung is an enlargement of the area indicated by the dotted line square. Arrows indicate CBX5-positive nuclei.

Figure 4.

RNA-sequencing analysis of IPF-derived lung fibroblasts identifies key pathways and gene signatures implicated in CBX5-mediated profibrotic function. (A) Principal-component analysis (PCA) displaying clusters of samples from experimental groups and the similarity of their transcriptomes (red represents scramble and purple represents siCBX5). (B) Pie chart illustrating upregulated (red) and downregulated (blue) genes in CBX5 knockdown IPF fibroblasts. (C) Volcano plot depicting differentially expressed genes after silencing CBX5 in human IPF fibroblasts. Red dots represent upregulated genes, and blue dots represent downregulated genes in CBX5-silenced fibroblasts. The x-axis denotes log₂ fold change values, and the y-axis denotes −log₁₀ (false discovery rate) values. (D) Ingenuity pathway analysis shows canonical pathways with z-score values in CBX5 knockdown cells relative to scramble. The x-axis denotes −log₁₀ (P value), and the y-axis denotes canonical pathways. A positive z-score indicates pathway activation, whereas a negative z-score indicates inhibition of a canonical pathway. (E) Heatmaps showing differentially expressed genes in the absence of CBX5. Data are displayed as z-scores of reads per kilobase per million reads (RPKM). (F and G) RNA expression (RPKM values) of representative genes from (F) the profibrotic gene signature and (G) the metabolic gene signature in CBX5-silenced IPF fibroblasts. Data are shown as mean ± SD of three independent human IPF fibroblast cells. P values were calculated using two-tailed Student’s t test.

Figure 5.

CBX5 silencing alleviates transforming growth factor β (TGFβ)–promoted profibrotic gene expression and metabolic reprogramming in IPF fibroblasts. (A) PCA displaying clusters of samples from experimental groups and the similarity of their transcriptomes (red represents control, blue represents TGFβ-treated, and orange represents TGFβ-treated siCBX5). (B) Venn diagrams illustrating upregulated or downregulated genes in CBX5 knockdown treated with TGFβ for 24 hours (false discovery rate, <0.01; log₂ fold change, ⩾1.5 or ⩽−1.5). (C) Ingenuity pathway analysis shows significant canonical pathways with z-score values in CBX5 knockdown cells relative to scramble in response to TGFβ stimulation (P ⩽ 0.05). The x-axis denotes −log₁₀(P value), and the y-axis denotes canonical pathways. The z-score represents the activation or inhibition state of a canonical pathway. (D) Heatmaps showing differentially expressed gene signatures (profibrotic genes, glycolysis, AMPK signaling, and PPAR signaling) in the absence of CBX5 with TGFβ treatment relative to control group. Data are displayed as z-scores of RPKM.

Figure 6.

AMPK activation and metabolic reprogramming in IPF fibroblasts lacking CBX5. (A) Representative images of western blotting showing reduced expression of collagen-I and attenuated H3K9 dimethylation (histone H3 lysine 9 dimethylation) together with elevated AMPK phosphorylation (Thr172) in CBX5-silenced IPF fibroblasts compared with control cells. Shown is a representative blot of three independent experiments. (B) Normal human lung fibroblasts were treated with TGFβ for 24 hours (left), or with scramble and CBX5 siRNAs for 48 hours and then treated with TGFβ for additional 24 hours (right). Shown is a representative blot of three independent experiments. (C and D) RNA expression (RPKM) of genes encoding for (C) AMPK-associated kinases and (D) and AMPK-associated phosphatases in three independent IPF fibroblasts. P values were calculated using one-way ANOVA. (E) Extracellular L-lactate level was measured in the conditioned medium of lung fibroblasts. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using one-way ANOVA. (F) Glycolysis and oxidative phosphorylation (OXPHOS) were assessed by quantifying the ATP levels in IPF fibroblasts. The level of ATP was determined by measuring the luminescence. The graph shows the average amount of ATP from three independent experiments (orange bars represent glycolytic ATP, and green bars represent mitochondrial ATP; n = 3 independent experiments).

Figure 7.

Inhibition of the CBX5 pathway in IPF fibroblasts in vitro and IPF lung explants ex vivo potentiates the anti-fibrotic effect of metformin. (A) Representative images of western blotting showing that CBX5 silencing synergizes with metformin (250 μM) to induced AMPK phosphorylation (Thr172) and decreased collagen type I (collagen-I) expression. (B) Representative images of western blotting showing G9a inhibition using selective UNC0631 inhibitor (50 nM) in combination with metformin (250 μM) synergistically induced AMPK phosphorylation and decreased expression of collagen-I. (C) Schematic showing the generation of IPF organotypic cultures ex vivo. (D) Western blotting was used to assess secreted collagen-I from IPF lung cultures. Each lane contained an equal volume of conditioned medium. Ponceau-s staining was used as a loading control. (E) Densitometry analysis for western blot quantification of secreted collagen-I was performed using ImageJ software. Data are shown as mean ± SEM of n = 12 independent cultures obtained from n = 1 IPF lung explant. P values were calculated using one-way ANOVA. Figure 7(C) created using BioRender.com.