In vitro evidence of nerve growth factor effects on human conjunctival epithelial cell differentiation and mucin gene expression.
OBJECTIVE
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.
Lambiase
Alessandro
A
CIR Laboratory of Ophthalmology, University Campus Bio-Medico, Rome, Italy.
Micera
Alessandra
A
Pellegrini
Graziella
G
Merlo
Daniela
D
Rama
Paolo
P
De Luca
Michele
M
Bonini
Sergio
S
Bonini
Stefano
S
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor / Q000378:metabolism; D015153:Blotting, Western; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003228:Conjunctiva / Q000166:cytology* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005092:Exonucleases / Q000378:metabolism; D005095:Exoribonucleases; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000187:drug effects*; D020397:Goblet Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D055271:Mucin 5AC / Q000235:genetics*; D009363:Neoplasm Proteins / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020800:Receptor, Nerve Growth Factor / Q000378:metabolism; D020917:Receptor, trkA / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; C081876:CKAP4 protein, human; C524678:MUC5AC protein, human; D008565:Membrane Proteins; D055271:Mucin 5AC; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D020800:Receptor, Nerve Growth Factor; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human