Involvement of Sp1 elements in the promoter activity of genes affected in keratoconus
| Autor: | Y, Maruyama, X, Wang, Y, Li, J, Sugar, B Y, Yue |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Adult
Sp1 Transcription Factor Corneal Stroma Acid Phosphatase Genetic Vectors Epithelium Corneal Fibroblasts Alkaline Phosphatase Keratoconus Transfection beta-Galactosidase Cathepsin B Gene Expression Regulation alpha 1-Antitrypsin Humans alpha-Macroglobulins Child Promoter Regions Genetic DNA Primers Skin |
| Zdroj: | Investigative ophthalmologyvisual science. 42(9) |
| ISSN: | 0146-0404 |
| Popis: | 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 alpha1-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.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 pSV beta-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 beta-galactosidase enzyme activities were assayed.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.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. |
| Databáze: | OpenAIRE |
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