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Characterization of a novel cis-element that regulates Fas ligand expression in corneal endothelial cells
J Zhang1, B Ma, A Marshak-Rothstein
1The Pulmonary Center and the Department of Biochemistry, Boston, MA 02118, USA. jiezhang@bu.edu
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.
Insights
Fas ligand in the cornea plays a key role in immune cell regulation and transplant success. A specific DNA sequence (TCCT motif) and a corneal protein are crucial for its expression in eye endothelial cells.
Area of Science:
- Immunology
- Molecular Biology
- Ophthalmology
Background:
- Fas ligand (FasL) is constitutively expressed in immune-privileged sites like the cornea.
- Corneal FasL induces apoptosis in Fas(+) immune cells, protecting surrounding tissue and aiding transplant success.
- Understanding FasL transcriptional regulation in the cornea is vital for ocular immunology and transplantation.
Purpose of the Study:
- To investigate the transcriptional regulation of Fas ligand in corneal endothelial cells.
- To identify key regulatory elements and protein factors involved in corneal FasL expression.
Main Methods:
- Deletion analysis of the mouse Fas ligand promoter in an immortalized corneal endothelial cell line (C3H3).
- Gel mobility shift assays and site-directed mutagenesis to identify regulatory motifs.
- UV cross-linking analysis to detect protein-DNA interactions.
Main Results:
- A TCCT motif, located 299 base pairs upstream, was identified as a major positive regulatory cis-element in corneal endothelial cells.
- This TCCT motif was not essential for FasL transcription in Sertoli or airway epithelial cells.
- A ~30-kDa corneal nuclear protein was found to bind to the Fas ligand promoter TCCT box.
Conclusions:
- A specific TCCT motif and a ~30-kDa corneal nuclear protein are critical for Fas ligand transcriptional regulation in corneal endothelial cells.
- These findings highlight a cornea-specific mechanism for controlling Fas ligand expression.
- This knowledge could inform strategies for improving cornea transplantation outcomes.