NF-IL6 and NF-κB in Cytokine Gene Regulation*

Shizuo Akira*; Tadamitsu Kishimoto†    * Department of Biochemistry, Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan
† Osaka University Medical School, Department of Medicine III, Suita, Osaka 565, Japan

I Introduction

The immune system is regulated through a complicated network modulated by a variety of cytokines and their cognate receptors. Transcriptional activation of inflammatory response genes, such as the genes for cytokines, their receptors, cell adhesion molecules, and acute phase proteins, is regulated by a specific assembly of transcription factors on the enhancers and promoters of these genes. Accumulating evidence indicates that a relatively small number of transcription factors play a critical role in achieving the high level of orchestration required for the complex gene expression involved in the immune response. These include the NF-κB, NF-IL6, CREB/ATF, Jun–Fos, STAT, and NF-AT families of transcription factors. On the other hand, dysfunctional regulation of these transcription factors may induce immunologically mediated diseases. In this review, we highlight how protein–protein interactions between transcription factors may modulate the activation of the cytokine genes. Particular attention is directed to two important families of transcription factors, NF-IL6 and NF-κB.

II NF-IL6

A STRUCTURE AND FUNCTION OF NF-IL6

NF-IL6 was originally identified as a nuclear factor binding to a 14-bp palindromic sequence (ACATTGCACAATCT) within an IL-1 responsive element in the human IL-6 gene (Isshiki et al., 1990). Cloning the cDNA encoding human NF-IL6 revealed that it has a high degree of homology with C/EBP in the carboxy-terminal basic and leucine zipper domains, responsible for DNA binding and dimerization, respectively (Akita et al., 1990). NF-IL6 recognizes the same nucleotide sequences as C/EBP. Both proteins bind to a variety of the divergent nucleotide sequences with different affinities, and the consensus sequence is T(T/G)NNGNNAA(T/G). NF-IL6 homologs in other species have been cloned under the names AGP/EBP (Chang et al., 1990), LAP (Descombes et al., 1990), IL-6DBP (Poli et al., 1990), rNFIL-6 (Metz and Ziff, 1991), C/EBPβ (Cao et al., 1991), CRP2 (Williams et al., 1991), and NF-M (Katz et al., 1993). The NF-IL6 gene is intronless, and it produces two proteins—LAP (liver-enriched transcriptional activator protein, equivalent to NF-IL6) and LIP (liver inhibitory protein)—by the alternative use of two AUG initiation codons within the same open reading frame (Descombes and Schibler, 1991). LIP contains DNA binding and dimerization domains but is devoid of the N-terminal transcriptional activation domain and therefore behaves as an antagonist of LAP-induced transcription. The ratio of these two forms varies depending on the cell type and on the developmental stage and can be altered to activation dominance by IL-6 or other extracellular signals such as retinoic acid (Descombes and Schibler, 1991; Hsu et al., 1994).

B ACTIVATION OF NF-IL6 THROUGH PHOSPHORYLATION

NF-IL6 activity is regulated by phosphorylation. Transient expression of a series of site-directed mutants of NF-IL6 and subsequent phosphopeptide mapping identified three phosphoiylated residues: Ser-231 and Thr-235, both located within the serine-rich domain (SRD) adjoining bZIP, and Ser-325 that is located within the leucine zipper. The amino acid sequence immediately surrounding Thr-235 (SSPPGTPSP) coincides with the consensus for MAP kinase recognition. In fact, a synthetic peptide containing Thr-235 was phosphoiylated in vitro by purified MAP kinases. When vectors expressing NF-IL6 or oncogenic p21ras were cotransfected with an IL-6 promoter–luciferase gene reporter construct, simultaneous expression of both NF-IL6 and oncogenic p21ras resulted in a dramatic synergistic stimulation of the reporter gene. Two dimensional phosphopeptide mapping showed that oncogenic p21ras expression markedly augmented Thr-235 phosphorylation. Furthermore, the substitution of Ala for Thr-235 resulted in the loss of ras-dependent NF-IL6 activation. These results demonstrate that NF-IL6 is activated through phosphorylation of Thr-235 by a ras-dependent MAP kinase cascade (Nakajima et al., 1993). The molecular mechanisms of NF-IL6 transcriptional activation through phosphorylation on Thr-235 have been clarified (Kowen-Leutz et al., 1994). NF-M, the chicken homolog of NF-IL6, is a critical transcription factor required for the expression of chicken myelomonocytic growth factor (cMGF), which is distantly related to G-CSF and IL-6 (Burk et al., 1993; Katz et al., 1993). In v-myb- or v-myc-transformed cells, NF-M is the target of activated kinase oncogenes, which induce cMGF gene expression, resulting in autocrine growth stimulation. Analyses of functional domains of NF-M demonstrated that the amino-terminal domains contribute to the transactivating function of the protein, whereas the internal portion (amino acids 116–229) between bZip and the amino-terminal activating domains inhibits the transcriptional activation. A consensus sequence for MAP kinase located within the inhibitory domain is conserved between avian and mammalian NF-IL6. NF-M was activated by deleting the entire region that harbors the MAP kinase site or by a point mutation at the MAP kinase site that mimics the negative charge of a phosphate residue. These results suggest that the inhibitory regions within NF-IL6 mask the transactivation domain and prevent its interaction with the basic transcription machinery.

NF-IL6 is also phosphorylated within the leucine zipper in response to increased intracellular calcium concentrations via the activation of a calcium–calmodulin-dependent kinase (Wegner et al., 1992). In addition, the cAMP-mediated phosphorylation of NF-IL6 is associated with nuclear translocation and transcriptional activation (Metz and Ziff, 1991). NF-IL6 is activated through phosphorylation of the N-terminal domain by PKC (Trautwein et al., 1993). Thus, NF-IL6 is, in fact, activated via multiple signaling pathways (Fig. 1).

C C/EBP FAMILY MEMBERS

Following NF-IL6, several other C/EBP family members have been molecularly cloned. Currently, there are five known members of the C/EBP family (Fig. 2). These include C/EBP (Landschulz et al., 1988), NF-IL6, Ig/EBP (also referred to as GPE-1-BP and C/EBPγ) (Roman et al., 1990; Nishizawa et al., 1991), NF-IL6β (C/EBPδ) (Kinoshita et al., 1992; Kageyama et al., 1991; Cao et al., 1991; Williams et al., 1991), and CHOP-10 (gadd153) (Ron and Habener, 1992). The genes encoding these proteins map to different loci of different chromosomes; C/EBP maps to human chromosome 19q13.1 (mouse chromosome 7) (Birkenmeier et al., 1989), NF-IL6 to human chromosome 20q13.1 (mouse chromosome 2) (Hendricks-Taylor et al., 1992), NF-IL6β to human chromosome 8q11 (mouse chromosome 16) (Cleutjens et al., 1993), and CHOP-10 to human chromosome 12q13.1–q13.2 (Park et al., 1992).

C/EBP is expressed in adipose, liver, and placental tissues that play vital roles in energy metabolism (McKnight et al., 1989). C/EBP mRNA increases markedly during the differentiation of 3 T3-L1 preadipocytes to adipocytes (Birkenmeier et al., 1989). C/EBP can transactivate the promoters of adipocyte-specific genes, a fatty acid binding protein 422 (aP2), stearoyl-CoA desaturase 1 (SCD1), and insulin-responsive glucose transporter-4 (GLUT4) (Christy et al., 1989). C/EBP is an important regulatory factor in adipocyte differentiation. Premature activation of a constitutively expressed, but inactive, C/EBP–estrogen receptor fusion protein causes premature expression of aP2 mRNA relative to die normal differentiation program (Umek et al., 1991). Other studies have also presented similar results (Lin and Lane, 1992; Freytag and Geddes, 1992). The ectopic expression of C/EBP by retroviral vectors induces adipogenesis directly in otherwise nonadipogenic NIH-3 T3 cells (Freytag et al., 1994). In contrast, antisense C/EBP RNA suppresses several adipose-specific mRNAs such as aP2, SCD1, and GLUT4, as well as triglyceride accumulation during the differentiation of 3 T3-L1 preadipocytes (Samuelsson et al., 1991; Lin and Lane, 1992).

C/EBP may also be involved in controlling the expression of genes of which the products are critical to fiver function...