Abstract
Constitutively expressed Fas ligand in the cornea, Sertoli cell of the testes, Paneth cell of the intestines, and Clara cell of the airway protect surrounding parenchymal tissue by inducing apoptosis of Fas(+) immune cells during inflammatory reactions. Indeed, the action of corneal Fas ligand has been suggested to facilitate successful allogeneic cornea transplantation. To study the transcriptional regulation of Fas ligand in the eye, we employed an immortalized mouse corneal endothelial cell line (C3H3) that constitutively expresses Fas ligand. By deletion analysis of the mouse Fas ligand promoter, gel mobility shift assays, and site-directed mutagenesis, we found that a TCCT motif located -299 base pairs upstream from the transcriptional start site served as a major positive regulatory cis-element in C3H3 cells. In contrast, this element was not required for Fas ligand transcriptional activity in Sertoli cells and airway epithelial cells. By UV cross-linking analysis, we found that an approximately 30-kDa corneal nuclear protein binds to the Fas ligand promoter TCCT box and, thus, likely plays an important role in Fas ligand expression in corneal endothelial cells.
Highlights
Fas (APO-1, CD95) is a type I cell surface protein belonging to the tumor necrosis factor/nerve growth factor receptor family that transduces a cell death signal upon binding to Fas ligand (FasL)1 [1]
To further study the production of FasL in corneal cell culture, C3H3 cell protein lysates were examined by Western analysis
These results indicate that the C3H3 cell line can serve as a suitable in vitro system to investigate the basis for constitutive FasL expression in the cornea
Summary
FasL, Fas ligand; PCR, polymerase chain reaction; ds, double-stranded; NFAT, nuclear factor for activated T cells; NF-B, nuclear factor B; SRF, serum response factor; GR, glucocorticoid receptor; bp, base pair(s); CE, concatenated oligonucleotide. Corneal graft cells expressing FasL induce apoptosis of infiltrating host immune cells, thereby improving corneal allograft survival [11, 16, 17]. These findings suggest that alterations in the expression of FasL may be an important element controlling the outcome of corneal allografting. We sought to characterize the molecular basis for constitutive FasL gene expression in cornea cells To meet this goal, we characterized mouse FasL promoter activity and found that action of ϳ30-kDa nuclear protein at a TCCT box (Ϫ292 to Ϫ302) is involved in the constitutive expression of FasL in corneal endothelial cells
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