Figure 5. Cited2 deficiency results in decreased expression of Pax6 and Klf4 in mouse cornea.

Pax6 and Klf4 mRNA expression was quantitatively examined in corneas from mice at 6 weeks of age by Real-time PCR. Around 50% reduction of Pax6 mRNA expression was detected in Cited2^flox/flox;Le-Cre⁺ corneas (n=3) as compared to Cited2^flox/flox littermate controls (n=3) (a). In the meantime, Klf4 mRNA expression in Cited2^flox/flox;Le-Cre⁺ corneas (n=3) decreased to about 20% of that in Cited2^flox/flox littermate controls (n=3) (b). Effect of Cited2 on Klf4 transcription was measured in vitro by examining the transcriptional activation of luciferase reporter containing 1Kb Klf4 promoter (Klf4-Luc) in cells without or with Cited2 overexpression. Human corneal epithelial (HCE) cells were transfected with Klf4-Luc reporter (40ng) without or with Cited2 expression plasmid (from 20ng to 400ng). Cited2 overexpression in HCE cells significantly increased the activity of Klf4-Luc reporter in a dose-dependent manner (c). Similar results were obtained from two other epithelial cell lines, HEK 293 and NumMG (c). The effect of Cited2 on Klf4 transcription was further dissected by measuring the response of various truncated mouse Klf4 promoter in the absence and presence of Cited2 overexpression. HEC cells were transfected with 20ng of truncated Klf4 promoter constructs and 200ng of Cited2 expression plasmid (d). The effect of AP-2α on the Klaf promoter was also analyzed in a similar strategy and showed significantly decreased reporter activity in response to AP-2α expression (e).