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(Investigative Ophthalmology and Visual Science. 2001;42:1980-1985.)
© 2001 by The Association for Research in Vision and Ophthalmology, Inc.

Involvement of Sp1 Elements in the Promoter Activity of Genes Affected in Keratoconus

Yasuhiro Maruyama1,2, Xinping Wang1,2, Yuhong Li1,2, Joel Sugar1 and Beatrice Y. J. T. Yue1

1 From the Department of Ophthalmology and Visual Sciences, College of Medicine, University of Illinois at Chicago.

PURPOSE. Keratoconus is a progressive disease that thins and scars the corneal stroma. In keratoconus corneas, levels of degradative enzymes, including lysosomal acid phosphatase (LAP) and cathepsin B, are elevated, and those of the inhibitors {alpha}1-proteinase inhibitor ({alpha}1-PI) and {alpha}2-macroglobulin ({alpha}2-M) are reduced, especially in the epithelial layer. An increased expression of the transcription factor Sp1 was also demonstrated. The role of Sp1 in regulation of the genes affected in keratoconus was examined in this study.

METHODS. DNA segments, containing 5'-flanking promoter sequences of the {alpha}1-PI, LAP, cathepsin B, and {alpha}2-M genes were ligated into the secreted alkaline phosphatase (SEAP) reporter gene vector. These constructs, along with the pSVß-galactosidase control vector, were transfected into cultured human corneal epithelial and stromal cells and skin fibroblasts. Cotransfection with the Sp1 expression vector was performed in parallel. SEAP and ß-galactosidase enzyme activities were assayed.

RESULTS. In corneal epithelial cells, as in stromal cells, {alpha}1-PI promoter activity was suppressed by cotransfection of pPacSp1. The LAP, cathepsin B, and {alpha}2-M promoters were functional in corneal cells, whereas activities of these promoters were much lower in skin fibroblasts. Cotransfection experiments indicated that the up- or downregulation of LAP, cathepsin B, and {alpha}2-M observed in keratoconus-affected corneas was not mediated by Sp1.

CONCLUSIONS. These results support the theory that the corneal epithelium, along with the stroma, is involved in keratoconus. An upstream role of Sp1 is indicated and the Sp1-mediated downregulation of the {alpha}1-PI gene may be a key event in the disease development.




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