A regulatory element in the CD95 (APO-1/Fas) ligand promoter is essential for responsiveness to TCR-mediated activation

Abstract

Expression of the CD95 (APO-1/Fas) ligand (CD95L) in activated T cells is a major cause of T cell activation-induced apoptosis. To study the molecular mechanisms of transcriptional control of CD95L expression in T cells, we investigated the human CD95L promoter in Jurkat T cells. Deletion studies revealed that the CD95L proximal promoter sequence from -220 to the transcription start site is essential for T cell stimulation-induced expression of CD95L. In this study, we discovered a novel regulatory element located at -120 of the CD95L promoter which contains DNA binding sites for SP-1 and a yet unknown inducible factor. Mutation analysis demonstrated that binding of the inducible factor to the -120 region is crucial for the biological function of the CD95L promoter upon T cell stimulation. The DNA sequence at -120 also contains two DNA motifs homologous to the binding site for NF-AT. NF-AT does not directly bind to this element. However, cotransfection studies with an NF-AT expression vector showed that NF-AT may confer a strong inducible activity to the CD95L promoter at this regulatory region. Our data also show that the immunosuppressive agent cyclosporin A down-regulates CD95L transcription by inhibiting the function of this positive regulatory element.

The Regulation of CD95 (Fas) Ligand Expression in Primary T Cells: Induction of Promoter Activation in CD95LP-Luc Transgenic Mice

“…Several transcription factors have been described that show a divergence in expression patterns or activity between differentiated Th1 vs Th2 populations. We and others previously reported that the transcription factor NF-AT contributes to CD95L promoter activation in Jurkat T cells, and other transcription factors, such as Egr-2 and -3 and NF-B, participate in transcriptional regulation of the promoter as well (15)(16)(17)(18)(19)(20)(21)(22)(23). Therefore, these proteins constitute likely candidates for regulating CD95L promoter activity in Th1 and Th2 populations.…”

“…Before the development of transgenic mice, the activity of the reporter construct was assessed by transient transfection into the Jurkat and Sertoli TM4 cell lines. We and others have shown that transcriptional activation of the CD95L promoter is induced in Jurkat T cells following ligation of the TCR (15)(16)(17)(18)(19)(20)(21)(22) and is constitutive in the testicular Sertoli TM4 cell line (15,28). As predicted, transient transfection of the 6.1-kb CD95LP-pBSA-Luc construct produced minimal luciferase activity in resting Jurkat T cells and enhanced activity (10-to 15-fold increases over unstimulated controls) in response to plate-bound anti-TCR stimulation (data not shown).…”

“…Using the Jurkat T cell line as a model system, we and others have demonstrated a role for NF-AT in mediating transcriptional activation of the CD95L promoter (15)(16)(17)(18). Other transcription factors, such as NF-B, Sp1, and Egr-2 and -3, also contribute to inducible CD95L promoter activity (19 -22).…”

“…Thus, most studies have measured either CD95L mRNA levels, generally by RT-PCR, or protein expression, indirectly through bioassays. Studies of the CD95L promoter have relied almost exclusively on transient or stable transfections of promoter reporter constructs into T cell lines (15)(16)(17)(18)(19)(20)(21)(22)(23)25).…”

“…This segment of the promoter extends in a 5Ј direction from the translational start site and encompasses all previously identified response elements (15)(16)(17)(18)(19)(20)(21)(22)(23). In the present study we have used this strain, designated CD95LP-Luc, to examine the regulation of CD95L promoter induction of luciferase activity (CD95L/luciferase) in a variety of primary murine T cell populations.…”