Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers.

Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14 and mutations in the -YRKLLEGEE- sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the -YRKLLEGEE- sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

Established epithelial cell lines that retain their differen-tiation potential and growth regulatory characteristics can provide valuable tools for studying gene regulation, extra-cellular matrix synthesis or growth factor response. They are also useful for drug development and toxicity testing. Experiments were therefore carried out to optimize culture conditions for the long-term, serial transfer of corneal epithelial cells in the presence of 3T3 feeder layers; and to establish a permanent cell line. In such experiments, rabbit corneal epithelial cells were seeded at low inoculation densities, and transferred every 5 days. After 80 popula-tion doublings, an epithelial cell line, RCE1, emerged. The cell line is heteroploid, with an average population doubling time of 15.5 hours (vs 18 hours for primary cultures). When RCE1 cells reached confluence, they strat-ified to form a three-to five-layered epithelium and expressed the differentiation-related keratin pair K3/K12 as shown by immunoblot and immunostaining. Biosyn-thetic labeling of proliferating, confluent and stratified cultures further showed that RCE1 cells expressed keratin pairs K5/K14, K6/K16 and K3/K12, thus mimicking faith-fully the stage-dependent differentiation of primary cultures of rabbit corneal keratinocytes. The results demonstrated that RCE1 cells provide a useful model for studying corneal cell growth and differentiation.