Apicobasal Polarity Protein PARD3 Plays an Essential Role in Human Corneal Epithelial Cells

Previously, we reported that the apicobasal polarity-determinant Pard3 regulates mouse corneal epithelial (CE) stratification and barrier function. Herein, we describe a complementary cell culture model to study the functions of PARD3 in greater detail.

PARD3-knockout (PARD3KO) cells were generated by CRISPR/Cas9-mutagenesis in hTCEpi, a telomerase-immortalized human CE cell line. We compared hTCEpi and PARD3KO cellular proliferation by CellTiter-Blue, crystal violet, xCELLigence Real Time Cell Analyzer, Ki67 staining and flow cytometry, extracellular matrix (ECM) affinity by adhesion assays and trypsinization time, actin polymerization by phalloidin staining, barrier function by Real-Time Cell Analyzer and FITC-dextran permeability assays, and relevant markers' expression by quantitative RT-PCR, immunoblots, and immunofluorescence.

Deconvolution of sequencing data, immunoblots, and immunofluorescence confirmed PARD3 ablation in PARD3KO cells, which displayed 1.44- to 1.66-fold faster proliferation with a shorter doubling time of 19.06 hours compared with 21.84 hours for hTCEpi cells. The G1-S phase regulators CCND2, CCNE, and CDK2 were upregulated, while the mitosis-promoting factor components CCNB1 and CDK1 were downregulated in PARD3KO cells, consistent with their increased stalling at S phase revealed by flow cytometry. PARD3KO cells also displayed 0.5- to 0.8-fold weaker adhesion to different components of ECM, 1.37-fold faster trypsin-mediated detachment, 1.4-fold faster gap filling, disrupted actin cytoskeleton, nuclear β-catenin, upregulated epithelial-mesenchymal transition markers ZEB1, TWIST1, SLUG, and α-smooth muscle actin, and defective barrier function coupled with decreased expression of cell junctional components.

PARD3 regulates human CE cell proliferation, ECM-binding, migration, F-actin polymerization, and barrier function. Loss of PARD3 activity pushes CE cells toward epithelial-mesenchymal transition.