Costimulation by B7-1 and LFA-3 Targets Distinct Nuclear Factors That Bind to the Interleukin-2 Promoter: B7-1 Negatively Regulates LFA-3-Induced NF-AT DNA Binding

Abstract

We have characterized the regulation of nuclear factors involved in transcriptional control of the interleukin-2 (IL-2) promoter-enhancer activity in Jurkat T cells stimulated with superantigen presented on HLA-DR transfectants combined with the ligands LFA-3 (CD58) and B7-1 (CD80). Gel shift analyses showed that NF-AT was strongly induced in LFA-3-costimulated Jurkat T cells, suggesting that NF-AT is a key target nuclear factor for the CD2-LFA-3 pathway. Studies using HLA-DR-B7-1-LFA-3 triple transfectants showed that the LFA-3-induced NF-AT DNA binding activity was negatively regulated by B7-1 costimulation. In contrast, induction of a CD28 response complex containing only c-Rel proteins was seen after B7-1 costimulation. Both LFA-3 costimulation and B7-1 costimulation induced the AP-1 and NF-B nuclear factors. Distinct compositions of the NF-AT complexes were seen in B7-1-and LFA-3-costimulated cells. LFA-3 induced primarily Jun-D, Fra-1, and Fra-2, while B7-1 induced June-D-Fos complexes. In contrast, AP-1 and NF-B complexes induced in B7-1-and LFA-3-costimulated T cells showed similar contents. Transient transfection of Jurkat T cells with a construct encoding the IL-2 enhancer-promoter region (position ؊500 to ؉60) linked to a luciferase reporter gene revealed that B7-1 costimulation was required to induce strong transcriptional activity. Combined B7-1-LFA-3 costimulation resulted in a synergistic increase in IL-2 transcriptional activity. Multimers of the AP-1, NF-AT, NF-B, and CD28 response elements showed distinct kinetics and activity after LFA-3 and B7-1 costimulation and revealed that B7-1 and LFA-3 converge to superinduce transcriptional activity of the AP-1, NF-AT, and CD28 response elements. Transcriptional studies with an IL-2 enhancerpromoter carrying a mutation in the CD28 response element site revealed that the activity was reduced by 80% after B7-1 and B7-1-LFA-3 costimulation whereas the transcriptional activity induced by LFA-3 was unaffected. Our data strongly suggest a selectivity in induction of nuclear factors by the CD2-LFA-3 and CD28-B7-1 pathways. This selectivity may contribute to regulation of the levels of IL-2 induced by LFA-3 and B7-1 costimulation and favor autocrine and paracrine T-cell responses, respectively.

Overexpression of EGR-1 modulates the activity of NF-κB and AP-1 in prostate carcinoma PC-3 and LNCaP cell lines

“…To address the effect of overexpression of Egr-1 on the activity of other genes, we examined whether overexpressed EGR-1 alone or in combination with cisplatin or TPA resulted in transcriptional activation of AP-1 and NF-κB in vitro luciferase assay. Thus we assayed the activity of the consensus region for AP-1 and NF-κB (34,35), using the pGL-2 luciferase gene reporter construct which contains a tandem repeated of 4xAP-1 and 2xNF-κB-binding site, by determining the luciferase activity in LNCaP ( Fig. 2A and B) and PC-3 ( Fig.…”

“…The AP-1 and NF-κB reporter plasmid driven by the rat prolactin minimal promoter (-36 to +37) under the control of four copies of the human AP-1 site (49) was kindly provided by M. Rincon and R.A-Flavell (section of immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT). 5'-TCGATT GAGTCAGGGTAA-3' and the two copies of the NF-κB binding site of the human Ig κ light chain enhancer 5'-GGGA CTTTCC-3' (33)(34)(35).…”

“…Lysates were spun down for 1 min, and the total supernatants were analyzed using Luciferase Reagent (Promega) and measured in a luminometer (MicroLumat LB 96 P, Berthold) for 5 sec. Background measurement was subtracted from each duplicate, and experimental values are expressed either as recorded light units, luciferase activity or as relative activity compared to extracts from unstimulated cells (34,35).…”

A p56 -independent Pathway of CD2 Signaling Involves Jun Kinase

“…Furthermore, in the absence of p56 lck , anti-CD2 stimulation is accompanied by the enhanced binding of the c-Jun/c-Fos heterodimers to the consensus AP-1 sequence leading to IL-2 production. A previous study performed by stimulating Jurkat cells with superantigen pulsed HLA-DR transfectants revealed differences in the composition of the NF-AT⅐AP-1 complexes following costimulation with either LFA-3 or B7 (87). These differences were due to the dimerization of JunD with different members of the Fos family (Fra1 and Fra2), indicating a selective induction of certain nuclear factors depending on the costimulatory pathway.…”

“…In addition, the coordinate action of TCR engagement and the B7 versus LFA-3 costimulatory signal could differentially induce Fos family proteins. In the study by Parra et al (87), AP-1 and NF-B complexes binding to their respective site in the IL-2 promoter revealed no differences after either type of costimulation. By stimulation via CD2 alone, we found not only JunD but also c-Jun induced and heterodimerizing with c-Fos in JCaM1.6s.S3 (Fig.…”