Functional Disparity of Distinct CD28 Response Elements toward Mitogenic Responses

Azem Civil,Irma Rensink,Lucien A Aarden,Cornelis L Verweij

doi:10.1074/jbc.274.48.34369

Azem Civil, Irma Rensink + Show 2 more

Open Access

https://doi.org/10.1074/jbc.274.48.34369

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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.

Highlights

Activation of T cells through the antigen-specific T-cell receptor in combination with a costimulatory signal results in the coordinate expression of a number of cytokine genes [1]
CD28REs—Using electrophoretic mobility shift assay we compared the ability of the IL-6 NF-␬B sequence, the human immunodeficiency virus-1 (HIV-1) NF-␬B sequence, and known CD28 response elements, i.e. IL-2 CD28RE and IL-8 CD28RE, to form complexes with recombinant NF-␬B/Rel proteins expressed in COS cells
Several reports have demonstrated that CD28 signal transduction increases the transcriptional activity of the IL-2, GM-CSF, IL-3, IL-8, and interferon-␥ promoter 3- to 6-fold (6 –10)

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructions—The wild-type IL-6 promoter-luciferase plasmid (wt-pIL6luc) and a variant IL-6 promoter construct containing mutant NF-␬B sequence (5Ј-AATATTTTCC-3Ј) (pIL6(Ϫ␬B)luc) were kind gifts of Dr Shizuo Akira and have been described elsewhere [28]. The NF-␬B site (TGGGATTTTCCC) in the IL-6 promoter was replaced by the HIV-1 NF-␬B site (GGGGACTTTCCG), the IL-8CD28RE site (GTGGAATTTCCT), or the IL-2CD28RE site (TTGGAATTTCTT). These constructs were designated as pIL6(HIV-1NF␬B)luc, pIL6(IL8CD28RE)luc, and pIL6 (IL2CD28RE)luc, respectively. Binding reactions containing 4 ␮g of nuclear protein, 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, and 2.5 ␮g of poly(dI-dC) were incubated for 10 min at room temperature before incubation with about 10,000 –50,000 cpm of a 32P-labeled double-stranded probe for 30 min at room temperature in a total volume of 15 ␮l. The mutant oligonucleotide has the sequence 5Ј-GACTCAATTTCACC-3Ј (GGG to CTC substitution). The following probes were used (thymidines tagged with an asterisk were substituted by bromodeoxyuridine): IL-6 NF-␬B

SEQUENCES I

RESULTS

DISCUSSION