Keratoconus (KC) is a blinding disease that progressively thins and scars the central cornea. We have instituted a program and have accumulated a significant body of information regarding the basic biologic defects in KC. We have shown, at both the protein and mRNA levels, that the expression of degradative enzymes such as acid hydrolases is elevated in KC corneas as compared to controls, whereas that of inhibitors such as alpha1-proteinase inhibitor (alpha1-PI) is down-regulated. Such alterations have led us to theorize that degradation processes may be one of the mechanisms affected in KC. Furthermore, the coordinated regulation of multiple genes hints of involvement of transcriptional controls. A survey of several transcription factors revealed increased SP1 expression in KC corneas. We now hypothesize that SP1 may be an important factor regulation the enzyme and/or inhibitor genes affected in KC conditions. Preliminary data showed that SP1 does confer a down-regulatory effect on the promoter activity of the alpha1-PI gene in corneal stromal cells. In this renewal application, we propose to further test the SP1 hypothesis. We will determine whether, and to what extent, SP1 up- regulates or down-regulates the expression of the enzyme and inhibitor genes altered in KC. The promoter regions of these genes will be ligated to reported genes and the constructs will be used to transfect human corneal stromal and epithelial cells for promoter activities. Co- transfection with an SP1 expression vector will be conducted to assess the effects of enhanced SP1 expression on the promoter activities. The levels of enzymes and inhibitors in corneal cells overexpressing SP1 will be studied in both tissue in both tissue culture and organ culture settings to determine whether overexpression on SP1 results in enhanced expression of enzymes or suppressed expression of inhibitors. The transfected corneal cells will also be mixed with Matrigel in a three-dimensional lattice and the effects of SP1 over-expression on the matrix integrity will be monitored. We will also determine whether the abnormality increased SP1 in KC is due to increased mRNA transcript, altered mRNA stability, or decreased protein turnover, and will examine the possible upstream scenarios leading to the SP1 up-regulation in KC corneas. Cytokines and aberrant developmental regulation are speculated to be possible contributing factors for the SP1 gene abnormality in KC. The modulation of the SP1 expression by cytokines, particularly interleukin-I and its receptor will be examined. The SP1 expression and the DNA binding activity at developmental stages will be studied using mouse eyes from embryonic stages to adults. The expression pattern of enzyme and inhibitor genes such as alpha1-PI will be evaluated in parallel. Finally, differentially expressed mRNAs in the epithelium and stroma KC corneas will be identified using differential display and subtractive hybridization techniques. Comparisons between KC, age-similar normal human corneal tissues, and other diseased controls will be made. Special attention will be paid to genes of relevance to SP1 expression and/or maturation and differentiation. The results should held identify potential candidate genes in both the stroma and epithelium of KC corneas and provide a direction for future genetic studies. We hope to gain further insights into the molecular events associated with KC conditions and to better illustrate the fundamental mechanisms operative in normal corneas.
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