In vitro evidence of nerve growth factor effects on human conjunctival epithelial cell differentiation and mucin gene expression.

PURPOSE

Mucins released into the tear film are crucial to maintaining a healthy ocular surface. Alterations in goblet cell numbers and mucin secretion are observed in chronic ocular surface inflammatory diseases. Nerve growth factor (NGF) plays a crucial role in healing and inflammation of the ocular surface. The aim of this study was to evaluate in vitro the effect of NGF on conjunctival goblet cell differentiation and mucin production and secretion.

METHODS

Human conjunctival epithelial cells were exposed to increasing NGF concentrations (1 to 250 ng/mL) and analyzed to quantify cell growth (MTT/Ki67/BrdU), goblet cell differentiation (PAS/MUC5AC confocal staining), and mucin mRNA expression (real-time PCR). Secreted and cellular MUC5AC were also analyzed by sandwich-ELISA and FACS, respectively. To confirm the biological effects of NGF, the same evaluations were performed on primary cultures, and changes in markers of stemness (p63) and commitment (14-3-3 sigma) were also investigated.

RESULTS

In cell cultures, NGF induced a dose-dependent increase of goblet cell numbers, MUC5AC production, storage, and release. Additionally, in primary cultures, NGF induced an increase of abortive colonies and 14-3-3 sigma protein, and a decrease of p63 mRNA and protein, suggesting a differentiating effect of NGF on human conjunctival epithelium.

CONCLUSIONS

These findings show that NGF might play a role in the complex mechanism leading to conjunctival epithelium differentiation and mucin secretion. In addition to the known roles of NGF in promoting ocular surface healing and sensitivity, its effects on conjunctival goblet cells support a rationale to investigate the therapeutic effectiveness of NGF in dry eye disease.