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Ewa Anna Meyer, Mindy Call, Matthias Zenkel, Naresh Polisetti, Friedrich E Kruse, Winston Kao, Ursula Schlotzer-Schrehardt; Cornea epithelial homeostasis: the importance of the transcription factor PPARγ. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5647.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported on a preferential expression of the transcription factor PPARγ (peroxisome proliferator-activated receptor γ) in limbal epithelial progenitor cells (Meyer et al., ARVO 2014). The purpose of this study was to examine the potential involvement of PPARγ in corneal epithelial differentiation and proliferation.
<br /> Knockdown of PPARγ expression was achieved in cultured limbal epithelial cells using specific siRNA. Activation of the PPARγ pathway was performed by exposure of the cells to the PPARγ agonist troglitazone (5µM) with and without the PPARγ inhibitor, GW9662 (10µM). The effects of PPARγ knockdown and activation on the regulation of candidate genes related to differentiation and proliferation in vitro were monitored for 1-7 days by real time PCR and BrdU assay. Both non-inducible and inducible transgenic mouse models using the Pax6 surface ectoderm enhancer and P0 promoter to drive the expression of cre or reverse tetracycline transcriptional activator were utilized to examine the role of PPARγ in maintaining corneal epithelium homeostasis in vivo.
Following siRNA-mediated knockdown of PPARγ mRNA, limbal epithelial cells showed a significant downregulation of differentiation-related genes, such as KRT3, KRT12, KRT15, ZO-1, K19, MUC1 and PAX6 (up to 10-fold) up to 7 days post-transfection, but only a mild downregulation of genes related to an undifferentiated phenotype, such as ABCG2, CEBPD, p63DeltaN and SOX9. Markers of proliferation, including CCND1 and MKI67, were also downregulated (up to 4-fold) and cell proliferation was reduced by about 30% in PPARγ-silenced cells. By immunocytochemistry, cultured cells showed clear colocalization of nuclear PPARγ labeling with KRT3, KRT15 and ZO-1, which was markedly decreased after PPARγ silencing. Consistently, expression levels of PPARγ, KRT3, KRT12, and KRT15 increased following treatment with the PPARγ agonist troglitazone (up to 13-fold), which could be largely inhibited by the PPARγ antagonist GW9662. Mice lacking PPARγ in the limbus showed structural alterations of the corneal surface associated with a decrease in KRT12 and KRT15.
<br /> PPARγ has been identified as a master regulator of differentiation and proliferation in a number of tissues. Changes in keratin expression following knockdown or activation of the PPARγ pathway suggest a key role for PPARγ in regulating corneal epithelial homeostasis.
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