Differential Expression of the Transcription Factors Sp1/Sp3 Dictates the Stratification Properties of Primary Cultured Human Corneal Epithelial Cells on Tissue-Engineered Corneal Substitutes

Abstract

Abstract: : Purpose:Primary cultured cells are widely used for the production of tissue engineered substitutes and gain popularity as a model for gene expression studies. However, as such cells are passaged in culture, they often loose their ability to proliferate by progressing toward terminal differentiation (TD), a process that might be determined by alterations in the expression of transcription factors whose functions are required for cell adhesion and differentiation. Here, we investigated whether the high unstability of primary cultured human corneal epithelial cells (HCECs) is related to varying levels of expression for the transcription factors Sp1/Sp3. Methods:HCECs were obtained from post-mortem eyes and primary cultured on irradiated Swiss-3T3. Expression of Sp1/Sp3 was monitored by Western blot and electrophoretic mobility shift assays (EMSAs). The Sp1/Sp3 regulatory influence was evaluated by transfection of HCECs with a recombinant plasmid bearing the Sp1/Sp3-dependent rPARP gene promoter fused to the CAT reporter gene. HCECs that express varying levels of Sp1/Sp3 were also used for the production of human corneal substitutes. Results:Expression of Sp1/Sp3 was found to vary considerably between HCECs isolated from different human eyes. In addition, Sp1/Sp3 expression was abrogated as HCECs progressed toward TD, which is consistent with the reduced activity directed by the Sp1/Sp3-dependent rPARP promoter as HCECs are passaged in culture. Culturing HCECs on tissue-engineered corneal substitutes indicated that cells with the best proliferative and stratifying properties are those that also weakly transcribe the rPARP promoter and have a delayed peak of expression for both Sp1/Sp3. Conclusion:Determination of the precise moment maximal expression of both Sp1/Sp3 and mature PARP protein is occurring in passaged HCECs might reveal as a good predictor for selecting HCECs that are the most appropriate for the production of tissue-engineered corneal substitutes as well as for conducting detailed gene promoter studies in vitro.

Keywords: 372 cornea: epithelium • 417 gene/expression • 605 transcription factors