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Yasuhiro Maruyama, Xinping Wang, Yuhong Li, Joel Sugar, Beatrice Y. J. T. Yue; Involvement of Sp1 Elements in the Promoter Activity of Genes Affected in Keratoconus. Invest. Ophthalmol. Vis. Sci. 2001;42(9):1980-1985.
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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 α1-proteinase inhibitor
(α1-PI) and α2-macroglobulin (α2-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 α1-PI,
LAP, cathepsin B, and α2-M genes were ligated into the secreted
alkaline phosphatase (SEAP) reporter gene vector. These constructs,
along with the pSVβ-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 β-galactosidase enzyme activities
results. In corneal epithelial cells, as in stromal cells, α1-PI promoter
activity was suppressed by cotransfection of pPacSp1. The LAP,
cathepsin B, and α2-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 α2-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 α1-PI gene may
be a key event in the disease development.
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