Yap1 is required for the corneal epithelial progenitor cell proliferation and expansion

Abstract

Purpose: Limbal stem cells proved a rich cellular resource for fast renewal of corneal epithelium during normal turnover and rapid replacement of the damaged epithelial cells upon injury. This study aims to dissect the signaling transduction networks that tightly control the corneal stem cell proliferation and differentiation. It is well documented that stem cell/progenitor cell expansion and organ size are controlled by Hippo signaling cascade, causing phosphorylation of the transcriptional coactivator Yap1, which is in turn recognized and sequestered in the cytoplasm by 14-3-3. Cytoplasmic retention of Yap1 is essential for stem cell cycle arrest. In this study we investigated the functional role of Yap1 in corneal stem cell proliferation and epithelial differentiation.

Methods: Yap1 expression in the mouse cornea was studied by immunofluorescence staining. Yap1 cellular distribution was investigated in the corneas of the WT and 14-3-3s mutated mice, and correlated to the corneal plaque formation. The functional role of Yap1 in the corneal progenitor cells was studied in vitro by knocking down the expression of Yap1 using lentivirus-shRNA. The proliferation of the cornea epithelial cell was examined by BrdU incorporation. The functional role of Yap1 in cornea epithelium was further investigated in vivo by conditional knockout of Yap1 in the corneal epithelium using the doxycycline induced Yap1^fl/fl/Krt12^rtTA/rtTA/TetO-Cre^+/+ mice.

Results: Yap1 was constantly expressed in the nuclei of the limbal basal stem cells and this nuclear localization persisted in the corneal basal cells that are composed of transient amplifying cells. But it was translocated to the cytoplasm in the differentiating suprabasal cells. 14-3-3 mutation abolished the cytoplasmic sequestration of the Yap1 in the differentiating corneal epithelial cells, causing hyperproliferation and impaired differentiation. Knockdown of Yap1 expression in the cultured WT corneal epithelial cells blocked the cell cycle progress, and knockout of Yap1 in the corneal epithelial basal cells in vivo inhibited corneal epithelial cell proliferation and restricted the generation of new progenitors for epithelial replacement.

Conclusions: Yap1 is required for proliferation and expansion of the corneal stem cells and basal progenitor cells. Yap1 must be sequestered in the cytoplasm by 14-3-3 to initiate the cell cycle arrest and corneal epithelial differentiation.