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A. E. Griep, I. F. Yamben, R. Rachel; Loss of Scrib in the Mouse Lens Results in Epithelial to Mesenchymal Transition. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4775.
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PDZ proteins such as Scrib, play roles in cell cycle regulation, adhesion, polarity in invertebrates. Scrib also has been shown to act as a tumor suppressor in Drosophila and its deregulation has been correlated with several human cancers. It has been proposed that loss of Scrib may contribute to cancer by leading to epithelial to mesenchymal transition (EMT). EMT also is associated with forms of cataract such as ASC and PCO. In this study, we asked if the loss of Scrib in derivatives of the lens placode, such as the lens and corneal epithelium, might result in EMT.
Mice carrying a conditional null allele of Scrib were crossed to Lens-Cre mice. Paraffin sections of variously aged Scribf/f (control) and Scribf/f;cre (CKO) embryos or eyes were prepared. Sections were stained with hematoxylin and eosin to assess ocular morphology. Sections were immunostained with antibodies to E-cadherin and ZO-1 to assess lens epithelial character and with antibodies to Smad4 and Snail to test for TGFβ signaling. Frozen sections were prepared and immunostained with antibodies to α Smooth Muscle Actin (αSMA), a marker for EMT. Western blotting was used to measure the levels of E-cadherin protein. Frozen sections were stained with antibodies to Keratin 12 (K12) and αSMA to assess the integrity of the corneal epithelium.
At P10, the lens epithelial cells of the CKO mice were flattened and elongated. E-cadherin levels were reduced in the CKO epithelium and the epithelium now was positive for αSMA. Nuclear staining for Smad4 and Snail was observed in CKO lens epithelium but not in controls, suggesting that the EMT in the CKO lens arose at least in part due to TGFβ signaling. Changes in the pattern of E-cadherin staining were observed as early as day E11.5 and by E17.5 E-cadherin staining was frequently absent along cell membranes. The loss of E-cadherin was confirmed by immunoblotting extracts from P2 control and CKO lenses. From E13.5 through E17.5 there was a progressive loss of ZO-1 from the apical membrane of the epithelial cells. By E17.5, nuclear Smad4 and αSMA staining were apparent. The corneal epithelium appeared flattened, K12 staining was absent in some cells and some cells were positive for αSMA.
These data show that Scrib is required to maintain the integrity of both the lens and corneal epithelia. Furthermore, loss of Scrib is sufficient to lead to EMT, a cell fate change that occurs in certain forms of cataract, and the mechanism involves at least in part the acquisition of the capacity of the lens epithelial cells to respond to TGFβ signaling.
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