KLEIP co-localizes transiently with E-cadherin in cultured MDCK cells, suggesting that it regulates formation of cell-cell contacts.
18 Therefore, we hypothesized that E-cadherin localization could be altered in the epithelium of KLEIP-deficient corneas, and expression of E-cadherin in KLEIP
+/+ and KLEIP
−/− mice was examined. In KLEIP
+/+ corneas, both epithelial cell layers, namely the squamous cell layer and the basal cell layer, were positive for E-cadherin, and both layers displayed the physiologic architecture (
Fig. 5A, left). In contrast, squamous and basal cell layers in KLEIP
−/− mice largely were disorganized and characterized by acellular areas in the squamous cell layer, suggesting loosened cell-cell contacts and an incomplete regeneration of the corneal epithelium in KLEIP
−/− corneas (
Fig. 5A, right). Furthermore, corneas of KLEIP
−/− mice displayed several apoptotic cells, which were absent in KLEIP
+/+ corneas (
Fig. 5B). This observation suggested that high proliferative activity and massive corneal epithelium formation in KLEIP
−/− mice (
Figs. 2,
3) is a compensatory mechanism due to corneal epithelial fragility, apoptosis, and damage. Next, we hypothesized that the corneal epithelium in KLEIP
−/− mice makes corneas more prone to mechanical injury, and an experimental mechanical injury would induce rapidly a strong corneal dystrophy in KLEIP
−/− mice. To test this hypothesis, abrasions were performed by removing mechanically 40% of the cornea in 18-day-old KLEIP
+/+ and KLEIP
−/− mice, and corneal wound closure was analyzed (
Fig. 6). In KLEIP
+/+ mice a rapid corneal regeneration occurred, and after three days injured corneas were indistinguishable from untreated eyes (
Fig. 6A top, B). In contrast, corneal abrasions in KLEIP
−/− mice led rapidly to corneal opacity within seven days in all treated animals (
Fig. 6A bottom, B). Injured KLEIP
−/− corneas showed similar histologic alterations, such as epithelial hyperplasia, stromal infiltrations, and superficial keratinized cells (
Fig. 6C right) as in 15-week-old non-injured KLEIP
−/− mice (
Fig. 2). Likewise, analysis for E-cadherin expression in KLEIP
−/− corneas 12 hours after abrasion showed disorganized and acellular areas in the squamous epithelial cell layer (
Fig. 6D), which were similar to the non-injured 15-week-old KLEIP
−/− mice (
Fig. 5). Together, the data indicate that KLEIP regulates corneal epithelial integrity, and loss of KLEIP expression makes corneas more fragile and sensitive to mechanical injury.