NF-κB and Rel Proteins in Innate Immunity

Elizabeth B. Kopp*; Sankar Ghosh†    * Department of Cell Biology and Biochemistry and Section of Immunobiology, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520
† Department of Molecular Biophysics and Biochemistry and Section of Immunobiology, Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520

I Introduction

Vertebrates respond to infection through a combination of adaptive or acquired immunity and innate or natural immunity. The principal feature of acquired immunity is the generation of receptors on B and T cells that can distinguish between self and nonself, and hence protect the organism from infectious agents such as bacteria or viruses. By contrast, the hallmarks of innate immunity consist of physical barriers and the ability to generate a battery of cytokines upon nonspecific recognition of conserved structures on infectious agents such as bacterial lipopolysaccharides (LPS) (Colten and Ravetch, 1992). The cytokines help to mount an inflammatory response and to recruit specialized cells, such as natural killer cells, to the site of infection (Abbas et al., 1991). A particularly interesting question is whether the rapid induction in the synthesis of these cytokines is coordinated by some common element. Work carried out over the past several years has identified such an element in a transcription factor known commonly as NF-κB. NF-κB is critical for the inducible expression of many genes involved in the immune and inflammatory responses including IL-1, IL-2, IL-2Rα, IL-6, IL-8, TNF-α, TNF-β, β-IFN, GM-CSF, and serum amyloid A protein. In addition, NF-κB has been conserved through evolution. A number of recent reviews have described various aspects of NF-κB function in great detail (Grilli et al., 1993; Baeuerle and Henkel, 1994); therefore, this report will instead focus primarily on the role of NF-κB as a unifying element in the body’s response to infection and injury and thus as an important mediator of natural immunity in vertebrates.

II Description of NF-κB and IκB

NF-κB was first characterized in mature B and plasma cells as a nuclear protein that binds specifically to a 10-bp sequence in the κ intronic enhancer (Sen and Baltimore, 1986a,b). The correlation between the activity of this transcription factor and the expression of the κ gene suggested that it might be a critical regulator for the tissue and developmental stage-specific expression of this gene (Sen and Baltimore, 1986b; Atchison and Perry, 1987; Lenardo et al., 1987). However, the finding that NF-κB existed in virtually all cells and could be induced by treatment with agents, such as LPS or PMA, indicated that it had a significantly broader role. Subsequent studies revealed that a wide variety of inducible genes contained NF-κB-responsive sites in their promoters and enhancers, thus indicating a general role for NF-κB as a rapid response transcription factor in different cells (reviewed in detail recently in Grilli et al., 1993; Baeuerle and Henkel, 1994). In most cells, with the exception of mature B cells, macrophages, and some neurons (Kaltschmidt et al., 1994), NF-κB remains in the cytoplasm by being bound to an inhibitory protein called IκB (Baeuerle and Baltimore, 1988a,b; Baeuerle et al., 1988; Gilmore and Morin, 1993). Treatment of cells with various inducers leads to the dissociation of the cytoplasmic complex and the translocation of free NF-κB to the nucleus (Baeuerle et al., 1988). Therefore, NF-κB serves as a signal transducer by carrying information from external agents directly to the nucleus.

NF-κB is classically described as a heterodimer of p50 and p65 subunits; however, the cloning of the genes encoding these subunits revealed that they were members of a much larger family of proteins known as the rel family of transcription factors (Bours et al., 1990; Ghosh et al., 1990; Kieran et al., 1990; Meyer et al., 1991; Nolan et al., 1991; Ruben et al., 1991; Blank et al., 1992). Now, NF-κB is often more loosely described as a homo- or heterodimer of rel subunits. In addition to p50 and p65, the rel family currently includes p52, rel-B, the oncogene v-rel, the corresponding protooncogene c-rel, and the Drosophila morphogen dorsal (Stephens et al., 1983; Wilhelmsen et al., 1984; Steward, 1987; Brownell et al., 1989; Neri et al., 1991; Schmid et al., 1991; Bours et al., 1992; Mercurio et al., 1992; Ruben et al., 1991; Ryseck et al., 1992).

A conserved, N-terminal, 300-amino-acid segment termed the rel homology (RH) domain is responsible for the DNA binding, dimerization, activation, and IκB interactions of the rel proteins. It is currently believed that the selection of rel partners in an NF-κB dimer imparts transcriptional activity (or inactivity) on the complex. Thus, the combination of p50 with p65 or c-rel is transcriptionally active as are p65 homodimer and p65/c-rel, whereas p52 and p50 homodimers are transcriptionally inactive and can repress κB-dependent transcription (Ballard et al., 1992; Lernbecher et al., 1993; Brown et al., 1994; Hansen et al., 1994). A PCR-assisted selection of binding sites using recombinant proteins demonstrated that the combination of subunits confers distinct specificities for DNA sequence (Kunsch et al., 1992). The 10-bp consensus binding sequence is GGGGYNNCCY, where each rel protein subunit contacts one-half of the binding site (Urban and Baeuerle, 1990; Urban et al., 1991; see also Baeuerle and Henkel, 1994). The three-dimensional crystal structure of the NF-κB p50 dimer bound to a symmetric binding sequence confirms the basic principles previously established from mutagenesis studies (G. Ghosh et al., unpublished observations). The crystal structure has also revealed a novel DNA binding motif (G. Ghosh et al., unpublished observations).

The p50 and p52 proteins are unique in that both of these molecules are derived from larger precursor proteins which are probably cleaved via a novel proteolytic processing mechanism (Fan et al., 1991). The mRNAs for p50 and p52 code for proteins of 105 and 100 kDa, respectively (Bours et al., 1990; Ghosh et al., 1990; Kieran et al., 1990; Meyer et al., 1991; Neri et al., 1991; Schmid et al., 1991; Bours et al., 1992; Mercurio et al., 1992). The N-terminal region of p105 and p100 yields the p50 and p52 molecules. The C-terminal region resembles the NF-κB inhibitory molecule, IκB, in that it contains repeats of a sequence motif known as ankyrin repeats. Indeed, intact p105 and p100 do not bind DNA and do not enter the nucleus because the C-terminal region folds back and masks the nuclear localization signal and the DNA-binding domain present in the N-terminus (Beg et al., 1992; Blank et al., 1992; Hatada et al., 1992; Henkel et al., 1992; Liou et al., 1992).

As previously mentioned, NF-κB is retained in the cytoplasm of most cells in an inactive form by binding to an inhibitor protein known as IκB (Baeuerle and Baltimore, 1988a,b). Like NF-κB, IκB is a member of a much larger group of proteins. The distinguishing feature of these proteins is the presence of multiple conserved ankyrin repeats which are thought to interact with the rel domain of NF-κB (Davis et al., 1991; Haskill et al., 1991; Inoue et al., 1992b; Franzoso et al., 1992; Gilmore and Morin, 1993; Naumann et al., 1993). The various members of the IκB family have preferences for specific combinations of rel proteins, and the number and spacing of ankyrin repeats appears to determine this specificity (Beg and Baldwin, 1993; Hatada et al., 1993; Naumann et al., 1993). Currently, the IκB family consists of IκB-α, IκB-β, IκB-γ, and Bcl-3 (Ohno...