Purinergic Signaling Involvement in Thymosin β4-mediated Corneal Epithelial Cell Migration

Purpose: This study aimed to determine the effect of thymosin beta 4 (Tβ4) on human corneal epithelial cell migration and the downstream signaling pathways. Tβ4 has a role in tissue development, cell migration, inflammation, and wound healing. A previous study showed that Tβ4 directly binds to F0-F1 ATP synthase. Other studies reported the role of extracellular ATP and purinergic receptors in cell migration with several cell types. Despite advancing to the clinical stage for treatment of eye disorders, the effect of Tβ4 on human corneal epithelial cell (HCEC) migration and proliferation and the precise downstream signaling pathway(s) have not been identified. Methods: Various concentrations of Tβ4 were tested in vitro on human corneal epithelial cell proliferation using the CCK-8 Kit and on cell migration using the gap closure migration assay. Additionally, ATP levels at various time points were determined using the ATP Lite One-Step Kit. The Fluo 8 NO Wash Calcium Assay Kit was used to measure the intracellular Ca²⁺ concentration after treatment with various concentrations of Tβ4. P2X7 inhibitors were tested on ATP signaling and migration. Total- and phospho-ERK1/2 levels were determined in western blot. Results: Tβ4 enhanced HCEC proliferation and migration in a dose- and time-dependent manner. Moreover, these functions were related to increased extracellular ATP levels, intracellular Ca²⁺ influx, and ERK1/2 phosphorylation. Tβ4-mediated HCEC migration was inhibited by specific P2X7 purinergic receptor antagonists suggesting the role of this receptor in Tβ4-mediated human corneal epithelial cell migration. Conclusions: These results suggest that Tβ4-mediated HCEC proliferation and migration are associated with increased ATP levels, P2X7 R-mediated Ca²⁺ influx, and the ERK1/2 signaling pathway. This study begins to describe the mechanisms for Tβ4-mediated corneal healing and regeneration.