Activation-dependent transcriptional regulation of the human fas promoter requires NF-kappa B p50-p65 recruitment

Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair r equired for immune homeostasis. Lymphocyte activation results in the upregu lation of Fas expression and the acquisition of sensitivity to FasL-mediate d apoptosis. Although Fas upregulation is central to the preservation of im munologic tolerance, little is known about the molecular machinery underlyi ng this process. To investigate the events involved in activation-induced F as upregulation, we have examined mRNA accumulation, fas promoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myri state acetate and ionomycin (P/I), pharmacological mimics of T-cell recepto r activation. Although resting Jurkat cells express Fas, Fas mRNA was induc ed approximately 10-fold in 2 h upon P/I stimulation. Using sequential dele tion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions -306 to -260 relative to the ATG) r equired for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both t he Spl and NF-kappa B transcription factors at positions -295 to -286. Elec trophoretic mobility shift assay (ER ISA) and supershift analyses of this r egion documented constitutive binding of Spl in unactivated nuclear extract s and inducible binding of p50-p65 NF-kappa B heterodimers after P/I activa tion. Sp1 and NF-kappa B transcription factor binding was shown to be mutua lly exclusive by EMSA displacement studies with purified recombinant Sp1 an d recombinant p50. The functional contribution of the kappa B-Sp1 composite site in P/I-inducible fas promoter activation was verified by using kappa B-Spl concatamers (-295 to -286) in a thymidine kinase promoter-driven repo rter construct and native promoter constructs in Jurkat cells overexpressin g I kappa B-alpha. Site-directed mutagenesis of the critical guanine nucleo tides in the kappa B-Sp1 element documented the essential role of this site in activation-dependent fas promoter induction.