Expression of the Transcription Factors Sp1 and Sp3 Is Altered by the Cell Density in Primary Cultures of Ocular Cells

Abstract

Abstract: : Purpose: The proliferative state of any given cell is a process that require continuous alterations in the adhesive properties of these cells. Cell-cell and cell-extracellular matrix (EM) adhesions are primarily based on integrin-type receptors. Expression of the genes encoding these integrins is, in turn, regulated at the transcription level by the binding of transcription factors to a variety of cis-acting regulatory elements present in the promoter sequence of such genes. Among such nuclear proteins, members of the Sp1 family are certainly among the most interesting candidates since they are expressed in most, if not all, cells, including those from ocular tissues. The purpose of this study was to determine whether expression of Sp1/Sp3 is altered while actively proliferating cells progress toward cell quiescence in vitro. Methods: Western blot analyses were conducted to follow the expression of both Sp1 and Sp3 in subconfluent and postconfluent (quiescent) rabbit corneal epithelial cells (RCECs), human corneal epithelial cells (HCECs) and retinal pigment epithelial (RPE) cells. Recombinant plasmids bearing the Sp1-dependent PARP promote fused to the CAT reporter gene were transiently transfected in both confluent and midconfluent, primary cultured ocular cells (RCECs, HCECs, and RPE). Electrophoretic mobility shift assays (EMSAs) were conducted to assess the expression of Sp1/Sp3 in nuclear extracts from both confluent and subconfluent cells.Results: EMSAs and Western blot analyses provided evidence that substantial amounts of both Sp1 and Sp3 were expressed in all types of subconfluent cells. However, as these cells reach confluency and progress toward terminal differentiation, expression of both these transcription factors is dramatically reduced. Transfection analyses conducted with recombinant plasmids bearing the Sp1-dependent rPARP gene promoter provided evidence that the ability of Sp1/Sp3 to positively influence rPARP promoter activity dramatically diminish as primary cultured cells reach confluency.Conclusion: Extinction of Sp1/Sp3 likely through a yet uncharacterized proteolytic pathway may preclude primary cultured ocular cells to progress through terminal differentiation. None Supported by a grant from the Canadian Institutes of Health Research (CIHR)

Keywords: transcription factors • gene/expression • wound healing