Rapid FAK Phosphorylation and Progressive Tight-Junction Protein Alterations Under Hyperosmotic Stress in Corneal Epithelial Cells

Dry eye disease (DED) is characterized by tear hyperosmolality and ocular surface injury, but the contribution of focal adhesion kinase (FAK)-related signaling to tight junction (TJ) protein alterations remains unclear. We combined a scopolamine hydrobromide (SCOP)-induced desiccating stress mouse model with human corneal epithelial (HCE-T) cells exposed to NaCl-induced hyperosmolarity, together with transcriptomics, immunofluorescence, immunoblotting, pharmacological inhibition, and siRNA-mediated FAK knockdown. RNA sequencing identified 823 significant large-effect differentially expressed genes; analysis of the 3741 FDR-corrected significant genes revealed significant enrichment of focal adhesion, extracellular matrix–receptor, cell adhesion, and tight junction pathways. In HCE-T cells, hyperosmotic stress increased FAK Tyr397 phosphorylation as early as 1 h without changing total FAK. At 24 h, p-FAK declined while total FAK protein levels remained stable, consistent with dephosphorylation at Tyr397; by 48 h, total FAK protein was lost, accompanied by cell injury. TJ protein abundance and junctional continuity decreased progressively with increasing osmolarity and exposure duration. PF-562271, a dual FAK/Pyk2 inhibitor, produced a concentration-dependent reduction in FAK Tyr397 phosphorylation and, at 2.5 μM, also reduced Pyk2 Tyr402 phosphorylation while partially preserving ZO-1, claudin-1, and occludin abundance. FAK knockdown independently attenuated TJ protein loss and partially preserved ZO-1 junctional continuity under 500 mOsm/L hyperosmotic stress. These findings support a time-dependent contribution of FAK-related signaling to hyperosmotic stress-induced TJ protein remodeling.