Transcription factors NF - IL 6 and NF - KB synergistically activate transcription of the inflammatory cytokines , interleukin 6 and interleukin 8 ( C / EBP / protein - protein interaction / acute - phase reaction ) TAIJI MATSUSAKA

Single binding sites for transcription factors NF-IL6 and NF-iB are present in the promoter of the interleukin (IL) 6 gene. Previous studies of internally deleted promoter mutants demonstrated that these two sites are important for the transcriptional regulation of this gene. In this report, we describe the synergistic activation of the IL-6 promoter by transcription factors NF-IL6 and NF-KB. Cotransfection of NF-IL6 with the NF-cB p65 subunit resulted in strong synergistic activation of an IL-6 promoter-reporter construct. Both the NF-IL6 and NFcB binding sites in the IL-6 promoter were required for synergistic activation. Similar synergistic activation was observed in the IL-8 promoter, which also contains both NF-IL6 and NF-cB binding sites. Furthermore, we demonstrated that NF-IL6 and the NF-KB p65 subunit directly associated via the basic leucine-zipper domain of NF-IL6 and the Rel homology domain of p65. Since the promoters of many other genes involved in the inflammatory and acute-phase responses also contain binding sites for NF-IL6 and NF-cB, the cooperation between these two factors may have an important role in these responses. We also discuss the possible interplay between various viral gene products and these two factors in the process of viral infection and constitutive cytokine production. We have shown (1) that the transcriptional activation of the interleukin (IL) 6 gene by IL-1 was dependent on a 14-bp palindromic sequence located at position -150 and that an IL-1-inducible nuclear factor, termed NF-IL6, bound to this site. A cDNA encoding NF-IL6 was cloned and found to be a member of the C/EBP transcription factor family, which belongs to the larger family of basic leucine-zipper (b-Zip) transcription factors (2). This protein has recently been identified in several different species and designated AGP/ EBP, LAP, IL-6DBP, rNFIL-6, C/EBP,B, CRP2, or NF-M (3-9). NF-IL6 not only activates the IL-6 gene but also is responsible for the regulation of genes encoding other inflammatory cytokines, acute-phase proteins, albumin, c-Fos, several adipocyte-specific proteins, and viral proteins. On the other hand, several groups have reported that the NF-KB binding site located between bp -72 and -63 was important for the activation of the IL-6 gene by IL-1, tumor necrosis factor a (TNF-a) or lipopolysaccharide (LPS) (1012). NF-KB was identified initially as a nuclear factor that binds to an enhancer element, called a KB motif, of the immunoglobulin K light chain (13). NF-KB is thought to be involved in the expression of several viruses and many cellular inducible genes encoding cytokines, immunoregulaThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tory receptors, and acute-phase proteins. The major form of NF-KB is a heterodimer composed of 50and 65-kDa subunits (p50 and p65, respectively), both of which belong to the Rel family. NF-KB is constitutively present in the nuclei of mature B cells. However, in most other cells NF-KB is present in cytoplasm. Various stimuli, including mitogens, cytokines, viruses, and bacteria, cause NF-KB to translocate into the nucleus and bind to the specific binding site (for review, see refs. 14 and 15). Because many immune response genes and acute-phase response genes contain both the NF-IL6 and the NF-KB sites, it is highly possible that cooperative interactions between NF-IL6 and NF-KB play an important role in the expression of these genes. In this report, we demonstrate the synergism between NF-IL6 and NF-KB in the transcription of the genes for the inflammatory cytokines, IL-6 and IL-8. MATERIALS AND METHODS Cell Culture. The human monocytic cell line U937 was cultured in RPMI 1640 medium supplemented with 10%6 (vol/vol) fetal calf serum. The murine embryonic carcinoma cell line P19 was cultured in minimal essential medium (a modification) supplemented with 10% fetal calf serum. Plasmid Constructions. A cDNA clone encoding the human NF-KB p5O subunit was obtained by the PCR technique and cloned into BCMGneo. Three mutants of NF-IL6 [ASpINFIL6, NF-IL6(S288A), and ASplASacNFIL6] were as described (16). These mutant NF-IL6 cDNAs and wild-type NFI-L6 cDNA were cloned into pEF-BOS. The IL-6 promoter-luciferase plasmid was as described (17). Two kinds of mutant IL-6 promoter-luciferase plasmids were constructed. In one the NF-KB site was disrupted by converting GGGATTTTCC to AATATTTTCC, and in the other NF-IL6 site was disrupted by converting ACATTGCACAATCT to ACACTACAAACTCT. The IL-8-luciferase gene was prepared by inserting the IL-8 gene from positions -94 to +44 into the luciferase reporter gene. As an internal control plasmid, elongation factor (Ef-f3galactosidase was used. Electrophoretic Mobility Shift Assays (EMSAs) and Luciferase Assays. Double-stranded oligonucleotides spanning the NF-KB site of the IL-6 gene (GGGATTTTCC) were synthesized, annealed, and labeled. Nuclear extracts from U937 cells with or without LPS stimulation were prepared as described (16). EMSAs were carried out by the protocol ofT. Fujita et al. (18). When recombinant protein was used, bovine Abbreviations: IL, interleukin; CMV, cytomegalovirus; LPS, lipopolysaccharide; TNF, tumor necrosis factor; b-ZIP, basic leucinezipper; EMSA, electrophoretic mobility shift assay; MBP, maltose binding protein; EF, elongation factor. §To whom reprint requests should be addressed.