Mucin-4 (MUC4), a key membrane-associated mucin of the corneal apical glycocalyx, plays an important role in maintaining corneal epithelial homeostasis and wettability. Dysregulation of MUC4 has been associated with barrier dysfunction and ocular surface inflammation. This study aimed to investigate the physiological regulatory role of MUC4 in human corneal epithelial cells (HCEs) and elucidate its underlying intracellular mechanisms. MUC4 in HCEs was silenced through lentivirus-based shMUC4 pseudovirion transduction. Cell viability was analyzed via CCK-8 reagent. Cell motility was monitored via real-time culture monitoring system. Mitochondrial function was analyzed by Seahorse analyzer. Expressions of associated proteins were determined by immunoblotting. Changes in phosphorylation levels in major signaling pathways were determined by protein arrays. MUC4 increased time-dependently in HCEs. The expression was efficiently silenced by shMUC4 transduction. MUC4 loss suppressed cell viability through increasing caspase-8 and PARP1 as well as decreasing Bcl-2 and Bcl-xL. MUC4 loss promoted cell motility by increasing the phosphorylation of FAK-Y397 and paxillin-Y118. Maximal respiration and spare respiratory capacity were lower in MUC4-silenced HCEs by decreasing expression of key mitochondrial proteins (ATP8, MT-ND1, MT-ND3, MT-ND5, MT-CO2, and MT-CO3). MUC4 loss activated FAK and triggered the activation of MAPK-associated kinases in HCEs. These findings indicated that persistent MUC4 production maintained cell viability and hindered migration in corneal epithelial cells via FAK/MAPK/P38 signaling pathway.
Chen et al. (Wed,) studied this question.