Abstract
Purpose: :
Glial cell derived neurotrophic factor (GDNF) has been identified to be produced by corneal epithelial progenitor cells and reported to promote cell migration and proliferation. However, the underlining mechanism remains to be elucidated. This study was to investigate whether GDNF activates β-catenin/TCF4/survivin signaling pathway in a corneal epithelial wound healing model.
Methods: :
Fresh donor corneal tissues were used to make cryosections and to generate primary human corneal epithelial cells (HCECs) in 12-well plates from cultured limbal explants. When the cells grew to confluence, a 2mm wide zone of epithelial cells were scraped to create an in vitro wound model. To knockdown GDNF/Tcf4 signaling, primary HCECs were transfected with the small interfering RNA (siRNA, 10-50 nM) specific for GDNF or Tcf4 with a non-coding sequence siRNA-fluorescein (siRNA-F) used as a negative control. The mRNA expression was evaluated by reverse transcription and Real-time PCR, and their proteins were detected by immonofluorescent staining and Western blot analysis.
Results: :
GDNF and TCF4 proteins were found to be immunolocalized in the basal cells of limbal epithelium where corneal epithelial stem cells locate. The immunoreactivity of β-catenin was stronger in basal than suprabasal layers of limbal epithelium. Primary cultured HCECs could heal the 2mm wide wound within 48 hours. In 4-24 hours after wounding, the mRNA expression of GDNF, β-catenin, Tcf4 and survivin were significantly upregulated, accompanied by activation of β-catenin and Tcf4 proteins evidenced by nuclear translocation of their immunoreactivities from the cytoplasm. Interestingly, the proliferation markers nuclear transcription factor p63 and cyclin D1 were upregulated, while cyclin-dependent kinase inhibitor p57 was downregulted at both mRNA and protein levels. Furthermore, siRNA-GDNF and siRNA-Tcf4 knocked down their expressions and delayed the wound healing, with a reversed pattern of Tcf4 signaling: decreased Tcf4, survivin, p63 and cyclin D1, and increased p57.
Conclusions: :
These findings demonstrate that limbal progenitor cell derived GDNF enhances corneal epithelial wound healing via activation of the β-catenin/Tcf4/survivin signaling pathway.
Keywords: cornea: epithelium • wound healing • proliferation