title: Functional disparity of distinct CD28 response elements toward mitogenic responses.
abstract: Activation of T cells through the antigen-specific T-cell receptor in combination with a costimulatory signal results in efficient cytokine gene transcription. The CD28-induced signal represents a major costimulatory signal for T cells. A CD28 response element, named CD28RE, was first identified in the interleukin-2 (IL-2) promoter region. Here we demonstrate that the NF-kappaB sequence in the IL-6 promoter functions as a CD28 response element. Mutations in this sequence rendered the IL-6 promoter unresponsive to CD28 costimulation. Moreover, this element could replace the IL-2 CD28RE in conferring CD28 responsiveness to the IL-2 promoter. In analogy to the known CD28 response elements IL-2 CD28RE, IL-8 CD28RE, and the human immunodeficiency virus-1 (HIV-1) NF-kappaB motif, the IL-6 NF-kappaB motif efficiently bound c-Rel, c-Rel/NFKB1, and the recently identified inducible T-cell factor NF-MATp35. However, the IL-6 NF-kappaB sequence together with the IL-8 CD28RE and HIV-1 NF-kappaB sequence differed from the IL-2 CD28RE in the binding of NF-kappaB/Rel family proteins. Although the IL-2 CD28RE exerted selective binding with c-Rel and c-Rel/NFKB1, the other CD28REs allowed efficient binding of a wide range of NF-kappaB/Rel family proteins. The difference in binding specificity correlated with the capacity of the distinct CD28 response elements to function in the context of the IL-6 promoter in response to T-cell activation. Domain swapping experiments revealed that the IL-8 CD28RE and HIV-1 NF-kappaB motif conferred similar responsiveness as the genuine IL-6 NF-kappaB motif in the transcriptional activation of the IL-6 promoter upon CD28 costimulation. In contrast, replacement of the IL-6 NF-kappaB sequence by the IL-2 CD28RE motif strongly reduced the responsiveness of the IL-6 promoter. These data indicate that despite the sequence similarity, two different classes of CD28 responsive elements exist that differ in their NF-kappaB binding capacity and the ability to confer CD28 costimulatory responsiveness toward a heterologous promoter.
authors.affiliation: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Department of Autoimmune Diseases, 1066 CX Amsterdam, The Netherlands.
authors.forename: A
authors.initials: A
authors.lastname: Civil
authors.forename: I
authors.initials: I
authors.lastname: Rensink
authors.forename: L A
authors.initials: LA
authors.lastname: Aarden
authors.forename: C L
authors.initials: CL
authors.lastname: Verweij
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism*; D017252:CD3 Complex / Q000276:immunology; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003432:Cross-Linking Reagents; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D008934:Mitogens / Q000494:pharmacology*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D014466:Ultraviolet Rays
chemical_list: D000906:Antibodies; D018106:CD28 Antigens; D017252:CD3 Complex; D003432:Cross-Linking Reagents; D015850:Interleukin-6; D008934:Mitogens; D016328:NF-kappa B; D011993:Recombinant Fusion Proteins; D004247:DNA; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase