Abstract
Purpose:
We previously demonstrated preferential expression of the transcription factor PPARγ (peroxisome proliferator-activated receptor γ) in limbal epithelial progenitor cell clusters ex vivo (Menzel-Severing et al., ARVO 2013). Here, we examine the potential involvement of PPARγ in corneal epithelial differentiation and regeneration.
Methods:
PPARγ expression was analyzed by real time PCR in basal limbal and corneal epithelial cells isolated by laser capture microdissection (LCM) and in cultured corneal epithelial cells. Immunofluorescence was used to localize PPARγ on cryosections of corneolimbal tissue both under resting and wound healing conditions. Cultured corneal epithelial cells (passage 1) or limbal epithelial clones were exposed to PPARγ agonists rosiglitazone (5µM) and troglitazone (1µM), PPARγ antagonist GW9662 (1,10µM), and EGF receptor inhibitor PD153035 (2µM) for 3 days. Changes in mRNA expression of corneal differentiation marker genes after activation and after siRNA-mediated downregulation of PPARγ were analyzed by real time PCR.
Results:
PPARγ expression was significantly increased (about 400-fold) in LCM-isolated limbal compared to corneal epithelial cells. Immunolabeling showed a predominantly cytoplasmic staining pattern in limbal progenitor cell clusters and a nuclear localization in basal/suprabasal cells of ocular surface epithelia. PPARγ mRNA and protein expression was increased during wound healing in an organ culture model and upon serum-induced differentiation of cultured corneal epithelial cells. The expression of keratin K3 (350-fold), K15 (25-fold), K12 (3.5-fold), Pax6 (10-fold), and connexin Cx43 (2-fold) increased following treatment of corneal epithelial or clonal cells with PPARγ agonists in combination with EGFR inhibitor PD153035 compared to untreated control cells, which could be inhibited by PPARγ antagonist GW9662. Following siRNA-mediated knockdown of PPARγ K3 expression was downregulated in cultured corneal epithelial cells by 50%. LeCre-PPARgamma transgenic mice revealed a disorganized structure of the corneal epithelium and corneal lymphangiogenesis.
Conclusions:
PPARγ has been identified as a master regulator of differentiation in various tissues. The present findings indicate a key role of PPARγ in the corneal epithelial differentiation program and a cross-talk between PPARγ and EGFR signaling pathways in the regulation of corneal epithelial proliferation and differentiation.
Keywords: 482 cornea: epithelium •
500 differentiation •
739 transcription factors