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-κB 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-κB motif, the IL-6 NF-κB motif efficiently bound c-Rel, c-Rel/NFKB1, and the recently identified inducible T-cell factor NF-MATp35. However, the IL-6 NF-κB sequence together with the IL-8 CD28RE and HIV-1 NF-κB sequence differed from the IL-2 CD28RE in the binding of NF-κB/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-κB/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-κB motif conferred similar responsiveness as the genuine IL-6 NF-κB motif in the transcriptional activation of the IL-6 promoter upon CD28 costimulation. In contrast, replacement of the IL-6 NF-κB 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-κB binding capacity and the ability to confer CD28 costimulatory responsiveness toward a heterologous promoter.
This study was supported by the Netherlands Organization of Scientific Research (NWO) Grant 900-507-156.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.