Intrinsic regeneration mechanisms of murine lacrimal gland epithelial cells for therapeutic applications in aqueous-deficient dry eye disease.

PURPOSE: Aqueous-deficient dry eye disease (ADDE) results from lacrimal gland dysfunction leading to tear film instability and chronic ocular surface inflammation. Despite the gland's known regenerative potential, the intrinsic molecular mechanisms enabling epithelial recovery remain poorly understood. This study aimed to characterize intrinsic regeneration processes of murine lacrimal gland epithelial cells (mLGECs) and identify pathways with therapeutic potential.

METHODS

Primary mLGECs were isolated from neonatal C57BL/6J mice and characterized. Acute injury was induced by brief exposure to 20% ethanol. Metabolic activity, apoptosis, and transcriptional responses were analyzed during early recovery phases. Pathway enrichment was conducted using Gene Ontology and KEGG annotations. Regeneration-associated growth factors were validated by qPCR, ELISA, and metabolic activity and wound closure assays in the presence or absence of the MAPK inhibitor SCH772984. MAPK activation was assessed by ERK1/2 phosphorylation.

RESULTS

mLGECs retained expression of epithelial and lacrimal-specific markers and exhibited regulated secretory activity. RNA sequencing and ERK1/2 phosphorylation revealed activation of MAPK signaling, accompanied by strong upregulation of regenerative ligands including Hbegf, Areg, Ngf, and Gdnf. Transient protein induction of HBEGF and AREG was confirmed. Functionally, HBEGF, AREG, NGF, and GDNF markedly enhanced metabolic recovery and wound closure, whereas pharmacological MAPK inhibition markedly impaired recovery.

CONCLUSION

These results identify MAPK signaling as a major component of the intrinsic epithelial injury response in lacrimal gland cells. HBEGF and AREG emerged as prominent mediators associated with enhanced recovery. The findings provide a mechanistic basis for developing defined growth factor-based approaches to support lacrimal gland epithelial regeneration.