The TCF4 Gene Regulates Apoptosis of Corneal Endothelial Cells in Fuchs Endothelial Corneal Dystrophy

Abstract

Purpose: Fuchs endothelial corneal dystrophy (FECD) is a progressive corneal disorder characterized by excessive extracellular matrix (ECM) accumulation and corneal endothelial cell death. CTG trinucleotide repeat expansion in the transcription factor 4 (TCF4) gene represents the most significant genetic risk factor. This study aimed to elucidate the role of TCF4 in FECD pathogenesis through comprehensive proteomic analysis.

Methods: Corneal endothelial cells isolated from patients with FECD harboring TCF4 trinucleotide repeat expansion were immortalized to establish an FECD cell model (iFECD). CRISPR/Cas9-mediated genome editing was employed to generate TCF4-knockout iFECD cells. Whole-cell proteome analysis was performed using liquid chromatography-mass spectrometry, followed by pathway enrichment analysis of differentially expressed proteins (DEPs). The effects of TCF4 deletion on TGF-β-mediated protein aggregation and cell death were evaluated using Western blot analysis, flow cytometry, and aggresome detection assays.

Results: Proteomic analysis identified 88 DEPs among 6510 detected proteins. Pathway analysis revealed significant enrichment in ECM-associated pathways, oxidative stress responses, and cellular motility. TCF4 deletion attenuated TGF-β-induced cell death in iFECD cells. Concordantly, Western blot analysis demonstrated that TCF4 deletion suppressed TGF-β2-mediated cleavage of caspase-3 and poly (ADP-ribose) polymerase. Flow cytometric analysis of Annexin V-positive cells confirmed reduced apoptosis in TCF4-deleted cells following TGF-β2 treatment. Additionally, aggresome detection assays revealed that TCF4 deletion diminished TGF-β2-induced protein aggregation.

Conclusions: This study demonstrates a crucial role for TCF4 in FECD pathogenesis, particularly in ECM regulation and protein aggregation-induced cell death.

Figure 1.

Knockout of the bHLH in TCF4 in the FECD cell model (iFECD). (A) iFECD cells were established from patient-derived corneal endothelial cells. Using CRISPR/Cas9, either the bHLH region or 20 bases in exon 9 of TCF4 were knocked out (iFECD TCF4ΔbHLH, iFECD TCF4^−/−). Phase-contrast microscopy images show that iFECD retains a polygonal, monolayer structure, similar to iFECD TCF4ΔbHLH. Scale bar: 200 µm. (B) PCR analysis showed genomic DNA product sizes of approximately 900 bp in iFECD and 700 bp in iFECD TCF4ΔbHLH. Experiments were repeated independently at least three times with consistent results; representative images are shown. (C) Western blotting confirmed the suppression of TCF4-A (54 kDa, NM_001243234.2) and TCF4-B (72 kDa, NM_001083962.2). Experiments were conducted in three independent replicates with reproducible results. (D) Densitometric analysis of TCF4 protein expression levels. Values were normalized to GAPDH and are expressed as mean ± SEM from three independent experiments. (E) Sanger sequencing verified the absence of the bHLH domain in the TCF4 region. Red and blue lines indicate bases upstream and downstream of the bHLH domain, respectively.

Figure 2.

Identification of DEPs between FECD cell model (iFECD) and iFECD TCF4ΔbHLH. (A) The volcano plot provides an overview of protein expression in iFECD compared to iFECD TCF4ΔbHLH. Mass spectrometry identified 6510 proteins, with 88 DEPs: 52 upregulated (red dots) and 36 downregulated (blue dots). The gray-shaded areas mark thresholds of |log₂ (fold change)| ≥ 0.5 and P < 0.05. (B) The heatmap shows hierarchical clustering of iFECD and iFECD TCF4ΔbHLH, displaying variations in protein abundance with row z-scores from −2 (blue) to +2 (red). The heatmap visually splits into two distinct clusters, representing the similarity within the iFECD and iFECD TCF4ΔbHLH groups.

Figure 3.

GO analysis of DEPs between FECD cell model (iFECD) and iFECD TCF4ΔbHLH. GO analysis was performed on the 88 DEPs associated with TCF4 deletion. Significantly enriched GO terms were identified with a P value threshold of <0.05. The GO terms are categorized into three groups: BP, CC, and MF. In BP, significant enrichments include response to oxidative stress, response to toxic substance, and cellular response to chemical stress. In CC, enrichments include the apical part of the cell, collagen-containing ECM, and cell–cell junction. In MF, significant enrichments include actin binding, ECM structural constituent, and cadherin binding.

Figure 4.

Enrichment analyses of DEPs between FECD cell model (iFECD) and iFECD TCF4ΔbHLH. (A) Reactome pathway analysis showed DEPs enriched in carbohydrate metabolism, ECM organization, transport of inorganic cations/anions and amino acids/oligopeptides, cell surface interactions at the vascular wall, and collagen formation. (B) KEGG pathway analysis demonstrated enrichment in proteoglycans in cancer, sphingolipid metabolism, protein digestion and absorption, ECM–receptor interaction, and ferroptosis.

Figure 5.

Confirmation of altered ECM molecules at the mRNA level using RNA-seq data. Expression levels of ECM-related mRNAs corresponding to DEPs were analyzed using three previously published RNA-seq data sets, including our own.^,, ANTXR1 (A), SULF1 (B), and COL1A2 (C) show consistent upregulation across all data sets in corneal endothelial cells from patients with FECD compared to controls. FLNB (D) was upregulated in Nakagawa et al. and Chu et al., while CCN1 (E) showed increased expression in Nikitina et al. and Chu et al. SDC1 (F) exhibited decreased expression in Nakagawa et al. but increased expression in Nikitina et al. COL8A1 (G) showed upregulation only in Nakagawa et al. LUM (H) and HAPLN1 (I) showed no significant changes in any data set.

Figure 6.

Effect of TCF4 knockout on TGF-β2–mediated ECM production and apoptosis. (A) The bHLH region or 20 bases in exon 9 of TCF4 in iFECD were knocked out using CRISPR/Cas9 (iFECD TCF4^−/−, iFECD TCF4ΔbHLH). Cells were cultured in serum-free medium for 24 hours, then treated with or without TGF-β2 (10 ng/mL) for 24 hours. Phase-contrast images show that iFECD forms a monolayer with polygonal morphology. TGF-β2–induced cell death in iFECD but not in iFECD TCF4^−/− or iFECD TCF4ΔbHLH. Scale bar: 200 µm. (B) Sanger sequencing confirmed the deletion of 20 bases in exon 9 of TCF4 in iFECD TCF4^−/−. Red lines indicate the deleted bases. (C) Western blotting showed TGF-β2–induced cleavage of caspase-3 and PARP in iFECD, which was suppressed in iFECD TCF4^−/− and iFECD TCF4ΔbHLH. (D) Flow cytometric analysis of Annexin V–positive apoptotic cells in response to TGF-β2 treatment. TGF-β2 treatment substantially increased the percentage of Annexin V–positive cells to 31.4% ± 2.0% in iFECD cells. Both iFECD TCF4^−/− and iFECD TCF4ΔbHLH cells demonstrated resistance to TGF-β2–induced apoptosis, showing lower percentages of Annexin V–positive cells (19.8% ± 1.3% and 18.0% ± 1.6%, respectively; P = 5.28 × 10⁻² and P = 3.02 × 10⁻², compared to TGF-β2–treated iFECD). Data are presented as mean ± SEM from three independent experiments. (E) Western blotting confirmed suppression of TCF4 in both iFECD TCF4^−/− and iFECD TCF4ΔbHLH. TGF-β2 upregulated Snail1 in iFECD, but this upregulation was suppressed in both mutant cell lines. ZEB1 expression was unaffected by TGF-β2 in all cell lines. Fibronectin levels increased in iFECD but not in either iFECD TCF4^−/− or iFECD TCF4ΔbHLH with TGF-β2 treatment. (F) Phosphorylation of Smad2 and Smad3 by TGF-β2 was confirmed in both iFECD and iFECD TCF4^−/−, while this phosphorylation was suppressed in iFECD TCF4ΔbHLH. All experiments were performed independently at least three times with reproducible results.

Figure 7.

Effect of TCF4 knockout on TGF-β2 mediated unfolded protein deposition. (A) iFECD and iFECD TCF4^−/− cells were cultured with or without TGF-β2 (10 ng/mL) for 24 hours. Fibronectin production and unfolded protein deposition were evaluated by immunofluorescent staining and aggresome staining, respectively. Immunofluorescent staining showed that TGF-β2 increased fibronectin expression in iFECD but showed a lesser increase in iFECD TCF4^−/−. Aggresome staining indicated that TGF-β2 induced unfolded protein partially colocalizing with fibronectin in iFECD. In contrast, TGF-β2 did not induce unfolded protein in iFECD TCF4^−/−. To ensure reproducibility, all experiments were performed in triplicate (n = 3 independent experiments), yielding similar results. Representative images are presented. Scale bar: 50 µm. (B) Colocalization between aggresome and fibronectin signals was quantified using Manders’s coefficient. Values are expressed as mean ± SEM from three independent experiments.