Celebrating 25 years of NF‐κB research

Video podcast available - go via www.immunologicalreviews.com to watch interview with Guest Editor Sankar Ghosh This article introduces a series of reviews covering NF-κB appearing in Volume 246 of Immunological Reviews. In the 25 years since its discovery, the nuclear factor-κB (NF-κB) family of transcription factors has been the subject of intensive research in virtually all areas of biomedical science. Research into the function and regulation of this single transcription factor family has been at the forefront of studies in immunology, cancer biology, and molecular pathology, and has progressed hand in hand with conceptual and technological advances in molecular biology, genetic engineering, systems biology, and computational biology. The progress made in elucidating the triggers of NF-κB activation, the components of the NF-κB pathway, and characterization of the physiological and pathophysiological functions of NF-κB have been remarkable. This anniversary therefore represents an excellent opportunity to revisit the work that forms the foundation upon which so much effort and progress has been built. The articles in this volume of Immunological Reviews cover much of what is known about the NF-κB pathway and its basic function. Yet each review also draws attention to the myriad and often surprisingly fundamental issues that remain unresolved. Finally, in marking this important landmark, most reviewers have shared a personal and historical narrative documenting their connection to their area of study in NF-κB. These accounts offer a unique view of the early days of NF-κB research, when so much of the fundamental underpinnings of the pathway were laid down in short order, and provide particular insight into how this field was enriched through the expertise of scientific immigrants from disparate areas of biology, further highlighting the amazing diversity of biological systems influenced by this single transcription factor family. In this introductory article, we lay down some of the most basic and fundamental concepts in order to assist the uninitiated in gaining access to the more detailed discussions that characterize the rest of this issue. Thus, this represents a severely abridged introduction to the NF-κB signaling pathway and its biological function. Where appropriate, we point the reader towards reviews elsewhere in this issue from which they may gain more detailed insight and historical context to the various facets of NF-κB. However, in keeping with the format of this issue, I (S. G.) begin by offering an account of my own involvement with NF-κB. I (S. G.) joined David Baltimore's laboratory in January 1989 after having completed my graduate studies with Dr. Umadas Maitra at Albert Einstein College of Medicine. As a graduate student, I had biochemically purified and characterized eukaryotic translation initiation factors. When I arrived in David's laboratory, NF-κB was one of the most exciting areas of research in the laboratory (of course along with other exciting areas such as RAG proteins, helix-loop-helix proteins, etc). Following the discovery of NF-κB by Ranjan Sen in 1986 (1), a number of postdoctoral fellows in the laboratory were pursuing the biology of this transcription factor (Michael Lenardo, Patrick Baeuerle, Gary Nabel, and Jaqueline Pierce). A major breakthrough in understanding how NF-κB is regulated, in particular its inducibility in response to signals such as phorbol-esters, was the discovery by Patrick Baeuerle of the latent, cytoplasmic state of NF-κB in unstimulated cells, followed by the identification of the inhibitor of NF-κB (IκB) (2). Despite the rapid elucidation of the mechanism of NF-κB regulation, further progress was difficult without knowing the identity of the genes encoding these proteins. Therefore, efforts were underway in David's laboratory, as well as in many others, to clone the NF-κB genes. However, for reasons that were unclear at the time, traditional approaches using binding to oligonucleotide columns yielded amounts of purified protein that were insufficient for protein sequencing. To take a fresh approach at the problem, I decided to try and purify NF-κB and IκB from tissue extracts. To choose the right organ, I made extracts of all mouse organs and analyzed the presence of NF-κB DNA-binding activity in detergent (deoxycholate)-treated extracts to reveal the total amount of NF-κB. This simple analysis revealed that the strongest signal was in lungs, probably because of the presence of resident macrophages. To scale up the purification, I decided to use rabbit lungs, which could be purchased from Pel-Freez, a company that otherwise sold rabbit meat to supermarkets. I began by first purifying the NF-κB:IκB complex, dissociating the two components, and then purifying them individually. The IκB activity was assayed by its ability to inhibit NF-κB DNA-binding activity, and I soon found that IκBα was a 37 kDa protein that could inhibit DNA-binding by NF-κB (3). At that time one of the best characterized inducers of NF-κB activity were phorbol-esters, which were known to function by activating protein kinase C (PKC). I therefore purified PKCs and showed that they could phosphorylate IκBα and that the phosphorylated IκB could no longer inhibit NF-κB DNA-binding (3). It seemed to be a clear explanation of how IκB might be regulated, but as we know now, it is not how NF-κB is activated in cells. While we have not revisited these early experiments, it is likely that phosphorylation of IκB somehow alters its conformation, thereby making it unable to block NF-κB DNA-binding. Unlike IκBα, the final steps of purification of the dissociated NF-κB was using affinity purification of κB oligonucleotide-coupled resins. Previous studies had suggested that NF-κB bound with very high affinity to its cognate binding site, and therefore would elute from the resin only at high salt: thus separating bona fide NF-κB from other non-specific DNA-binding proteins. However, my experiments indicated that NF-κB eluted along with many other contaminating DNA-binding proteins. To overcome this problem, I created another column with a mutated κB-site oligonucleotide and went through a sequential purification, first through the wildtype oligo column, where NF-κB eluted at high salt, followed by its purification through the mutant oligo column, where NF-κB now eluted at low salt, but most contaminating DNA-binding proteins continued to elute at high salt. Using this approach, I was able to obtain a large amount of highly purified NF-κB that contained the p50 and p65 subunits. I sent a portion of my purified p50 sample to a protein sequencing facility at another institution, and after 2 months was informed that they had been unable to obtain any sequence. I embarked on a frantic search for another protein sequencing facility and fortunately became aware of Paul Tempst at Harvard, who had recently established his protein sequencing laboratory after completing his postdoctoral training at Lee Hood's laboratory. Paul agreed to sequence the sample, and in about a month, he gave me nearly 250 amino acids of sequence out of a 440-amino acid protein! I immediately began the process of cloning the gene from a mouse λgt11 cDNA library. Although we were unaware of any specific competition, I felt I had to get the cloning and characterization done as soon as possible. Between March 1990 and July 1990 we succeeded in cloning the 4.5 kB cDNA, completely sequencing it, and demonstrating that it was encoded as a precursor of 105 kDa (4). The mad rush was justified in hindsight, as it turned out Alain Israel's laboratory had also sequenced the p105 gene and had submitted their paper one day earlier to the same journal (5)! Besides the sequence of p50, Paul Tempst had also given me sequences from p65 (some of these sequences were published in the p50 paper to show the similarity between the proteins), and another postdoctoral fellow, Garry Nolan, who had joined the laboratory went on to clone p65 (6). However, the situation in the laboratory had changed in the meantime. David had announced earlier in the spring that he would join Rockefeller University to become the President beginning July 1, 1990. While initially I had expected to move with him to New York, the publication of the p50 paper suddenly raised the possibility that I would be able to start my own laboratory far sooner that I had expected. As I had also purified sufficient amounts of IκBα, it was my goal to clone IκB as soon as I had finished my job interviews. Unfortunately Paul Tempst moved from Harvard to Sloan-Kettering that winter, and my purified IκBα sample remained unsequenced in his laboratory until May 1991. Upon receiving the sequences, I geared up to clone IκBα; however, Nathan Davis, a student from Henry Bose's laboratory, with whom I had been collaborating on characterizing the chicken IκB, pp40, informed me that he had succeeded in cloning the pp40 gene. It was immediately clear from the sequence that pp40 was the chicken homolog of rabbit IκBα. We rushed our article with the mammalian sequence and description of the chicken pp40 clone, and publication of our study (7), along with Al Baldwin's article (8) clearly established the nature of IκB proteins. I began my laboratory at Yale in July 1991 and continued to work on NF-κB. However, this was also the time when the field had exploded, and as a young starting laboratory, I was unable to participate in many of the discoveries other than as an onlooker. The project that I focused on was the purification and cloning of IκBβ, but interestingly the first independent paper from my laboratory was on something quite different. The concept that NF-κB was a key regulator of inflammation was just beginning to be established, and a very talented student in the laboratory, Elizabeth Kopp, became interested in testing whether known anti-inflammatory drugs might act through NF-κB. We were debating between testing glucocorticoids and non-steroidal anti-inflammatory drugs when we realized that the well-known effect of aspirin on cyclooxygenases, which involved the transfer of acetyl group of aspirin to cyclooxygenase, did not explain the anti-inflammatory effect of salicylates, which lacked the acetyl group. We went on to show that high dose salicylates and aspirin can inhibit NF-κB, and it is now accepted to be the mechanism by which salicylates exert their anti-inflammatory functions (9). In parallel, we did clone IκBβ and showed that it behaved differently from IκBα, although many details of its regulation of NF-κB remained unclear (10). For a variety of reasons, the mice lacking IκBβ remained uncharacterized, and it was only recently that analysis of these mice revealed the surprising role of IκBβ in both inhibiting and facilitating inflammatory gene expression (11). It was from our efforts to biochemically purify and study IκBβ containing complexes that we realized that regulation of gene expression by NF-κB involved post-translational modifications (PTMs) of NF-κB itself. We have continued to explore the regulation of the transcriptional activity of NF-κB, using a combination of biochemical and genetic approaches (12–14). Finally, we have also explored the potential therapeutic utility of disrupting the IκB kinase (IKK)-NF-κB essential modulator (NEMO) interaction using NBD-peptides, and many labs, including our own, have demonstrated the utility of this strategy in numerous animal models of inflammatory, autoimmune, and oncological disease (15, 16). However, extending this approach to patients remains the ultimate goal, and we are exploring strategies to enhance the efficacy of the NBD-peptides that we hope will allow translation of NBD-peptides to the clinic in the near future. The mammalian NF-κB family consists of p65 (RelA), c-Rel, RelB, p50/p105 (NF-κB1), and p52/p100 (NF-κB2). These proteins exist in the resting state as homodimers or heterodimers primarily bound to IκB family proteins. The NF-κB family proteins are characterized by the presence of a conserved N-terminal 300 amino acid Rel homology domain (RHD) that is responsible for dimerization, interaction with IκBs and binding to DNA (reviewed in 17). IκB proteins act by constraining the function of NF-κB dimers and are defined by the presence of five to seven C-terminal ankyrin repeats. Mammalian cells contain eight established IκB family members: IκBα, IκBβ, IκBε, IκBζ, BCL-3, IkB-NS and the precursor proteins p100 and p105 (reviewed in 18). An additional potential IκB family protein, IκBη, has recently been identified (19). The typical IκB proteins function to prevent DNA binding by NF-κB dimers. For example, IκBα or IκBβ bound to p65/p50 or p65/c-Rel dimers mask the nuclear localization sequence (NLS) of p65, which despite the presence of an accessible p50 NLS results in steady state cytoplasmic localization (reviewed in 17). Release of NF-κB dimers from typical IκB proteins depends upon the action of the IκB kinase complex (reviewed in 20). Upon phosphorylation by IKKs, IκB proteins are recognized and ubiquitinated by members of the Skp1-Culin-Roc1/Rbx1/Hrt-1-F-box (SCF or SCRF) family of ubiquitin ligases (reviewed in 21). βTrCP, the receptor subunit of the SCF family ubiquitin ligase machinery, binds directly to the phosphorylated degron sequence (DS*GXXS*) on IκBs. Recognition by the ubiquitin ligase machinery leads to the polyubiquitination and subsequent degradation of IκBs or processing in the case of p100 by the proteasome. Freed NF-κB dimers translocate to the nucleus and bind to specific sequences, termed κB sites, in the promoter or enhancer regions of target genes (reviewed in 22, 23). Activated NF-κB can then be downregulated through multiple mechanisms (reviewed in 24, 25) including the well-characterized feedback pathway whereby newly synthesized IκBα restores steady state cytoplasmic localization of NF-κB dimers. Aspects of this basic signaling scheme and even some parts of the signaling pathways leading to NF-κB activation are well conserved in terms of mechanism and biological functions, perhaps for as much as a billion years of evolutionary history (reviewed in 26). NF-κB signaling is generally considered to occur through either the canonical or noncanonical pathways. The so called canonical, or classical, pathway of NF-κB activation is triggered by numerous extracellular and intracellular signals, prototypically proinflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), Toll-like receptors (TLRs), and antigen receptors. While most of these canonical signaling events emanate from cell surface receptors, various intracellular signals, including recognition of intracellular pathogens (reviewed in 27, 28), as well as more direct cell stresses including reactive oxygen species and DNA double strand breaks (reviewed in 29), also activate NF-κB. Whether initiated from an extracellular or intracellular source, signaling in the canonical pathways generally occurs through the recruitment of adapter proteins containing homotypic and heterotypic protein:protein interaction motifs such as receptor interacting protein (RIP) homotypic interaction motifs (RHIMs), death domains (DDs), and Toll IL-1 receptor homology domains (TIRs). For example caspase activation and recruitment domain (CARD)-containing proteins are implicated in the formation of signaling platforms in multiple classical NF-κB signaling pathways (reviewed in 30). The resulting oligomeric structures may be crucial for signaling to NF-κB, although this has not been directly proven in vivo for the NF-κB pathway. It is thought that in conjunction with non-degradative ubiquitination of multiple signaling intermediates (reviewed in 27), these complexes serve as platforms for the recruitment and activation of the kinases responsible for phosphorylation of IκB, the IκB kinase (IKK) complex. IKKα, IKKβ, or both complexed with the regulatory protein NEMO (IKKγ) are recruited to receptor/adapter protein complexes and either directly via trans-autophosphorylation or through the induction of an IKK kinase, such as TAK1, are activated. Thus, it is hypothesized that induction of the canonical NF-κB pathway is an aggregative signaling process in which formation of a multiprotein complex leads to kinase activation, either IKK or upstream kinase, through proximity-induced trans-autophosphorylation. The noncanonical pathway may be described by comparison as a dissociative signaling mechanism. Receptor signaling leads to IKKα activation through disruption of a protein complex that of the IKKα activating kinase, kinase of this complex upon binding to members of the such as activating factor to the family receptor and leads to of which IKKα leading to phosphorylation of p100 (reviewed in of IκB is a that is initiated upon specific phosphorylation by activated The IKK complex has an molecular of kDa and to be of a of IKKα and and the regulatory subunit NEMO (reviewed in 20). that this kinase complex is as an of dimers (reviewed in 17). The canonical NF-κB signaling pathway results in phosphorylation of IκBα on and and IκBβ on and and IKKα can to canonical pathway activation, although in many is sufficient in 20). The noncanonical pathway depends only on the IKKα, independent of and IKKα then in p100 and its processing to (reviewed in A pathway is by the degradation of the precursor protein p105 degradation results in activation of the kinase activation of extracellular kinase (reviewed in In the years since the identification of the IKK complex, numerous in to IκB proteins have been These serve to the NF-κB pathway with other signaling pathways cell and cell (reviewed in Following degradation of IκB, the NF-κB is able to bind promoter and enhancer regions containing κB with the sequence - any and although κB more sequences have been described (reviewed in 23). The structures of NF-κB dimers bound to the κB enhancer reveal how both domains that up the the DNA of the κB The domain is primarily responsible for sequence of NF-κB, the domain the RelB, c-Rel, and p65 contain a domain towards the that is for by these proteins. of and p50 and have no ability to In binding of or p50 homodimers to κB of resting cells leads to of gene expression IκB proteins of binding to NF-κB dimers can either this function as in the case of and BCL-3, transcriptional activation (reviewed in 18). The on p65, c-Rel, and transcription in through the recruitment of transcriptional such as the or (reviewed in of transcriptional is through post-translational modifications (PTMs) NF-κB proteins. For p65, including and have been described (reviewed in 22, A understanding of the and of the various p65 is years and work on other Rel proteins is As a our understanding of fundamental of transcriptional regulation by NF-κB is in its The reviews in this volume by and some of the that in to the initiation and of gene by NF-κB. of the functions of NF-κB, IκB, and IKK proteins are in in this issue. and Sen and provide detailed reviews of NF-κB in cell and function. reviews the progress in the in understanding the role of NF-κB in biology and some of the many suggested by NF-κB and and and provide detailed analysis of NF-κB in and inflammation and review the of NF-κB and the and take between NF-κB and and we provide a of some of the biological functions of these proteins. of the mammalian IκB, and IKK family members have been in As many of these of NF-κB and IκB proteins, use of tissue specific has been a in years to These efforts have additional on some of the of NF-κB pathway components, although additional work is in this The role of pathway components in the and response of cells (reviewed in as well as in (reviewed in is in in this The reviews from the of and also provide detailed discussions of the IKK complex and IκB family The first genetic studies the NF-κB pathway revealed the crucial role of p65 in from signaling (reviewed in a in NF-κB pathway that was in and NEMO (reviewed in 20). lacking p65 to at day The was to be the of signaling in the as mice lacking p65 to either or the and revealed the role of p65 in In p50 mice not show demonstrating p50 is not essential for induction of but cells lacking p50/p105 not to the role of the classical p65/p50 in signaling pathways. lacking have in their and the of the noncanonical signaling pathway in The is with a role for in with to the of organs The protein forms homodimers and heterodimers with p50 and Although mice to a response and in both and is to that some is between and As a mice lacking the which gene expression but can a more These have and of functions in activation of of in conjunction with p50 also a role for in cell work also an essential role for in the of regulatory cells (reviewed in is unique in that it not and with and p50 and not with any IκB other than p100 heterodimers not bind other they are generally thought to nuclear localization However, dimers may be by p100 or p105 in typical p100 and p105 complexes Although may with p65 the physiological role of this is not clear (reviewed in 17). of results in inflammation as well as in lacking have to in cells and cell function This is when p50 is also that p50 and in the regulation of genes that inflammation cells, although in their and in response to various The of in is well established likely to in signaling to is also likely responsible for the cell as this process depends upon and While the role of the noncanonical pathway has been best in the context of the canonical pathway is also activated by these same family members and clearly also to and Thus, not only NF-κB multiple essential in the specific steps of and activation it is also crucial for the of the which and activation is by the ability of specific NF-κB dimers to with members of the IκB family. For example, the classical NF-κB is although not by IκBα. Therefore, mice but not p65/p50 DNA binding activity, demonstrating that additional mechanisms can the activity of this IκBα is known to participate in a feedback whereby newly synthesized IκBα the activity of although how this NF-κB from κB sites, it in is not known in of the in the of IκBα, the of NF-κB activation in response to is In one IκBβ, which otherwise not participate in the of NF-κB activity, of the IκBα the of NF-κB activation and are This is not to that IκBα and IκBβ are they clearly are analysis of cell in which multiple of IκBs have been out that a portion of the of IκBα, IκBβ, and are the of in their degradation and transcriptional regulation of their The of the typical IκB proteins, from their transcriptional regulation, that to their functions remain to be NF-κB family proteins, our understanding of the functions of IκBs remains At the signaling has been progress in out how IκBs the transcriptional A portion of this work has been the of efforts in of the NF-κB pathway, and is by However, have particular on the functions of IκB proteins, remains much to be about the biological of IκBs. In the ability of IκB proteins to and inhibit NF-κB transcriptional to and the formation of NF-κB and the of to with other signaling pathways to be in vivo to more the functions of the IκB family in 18). The is by no to be it to a basic of NF-κB biology, as a regulator of genes and crucial of and function. signals from a of physiological and to an of target genes NF-κB can to a of and For of these and many other of NF-κB regulation and biology, we the reader to the that in this volume of Immunological Reviews. factor-κB (NF-κB) is a regulator of the transcriptional to a variety of physiological and from cytokines to stresses including reactive oxygen species and DNA breaks to of antigen receptors to activation of NF-κB. and can also to activation of NF-κB. NF-κB transcriptional a variety of genes These transcriptional have biological and numerous of and as well as Reviews on NF-κB are in a and many have in this anniversary this of we would to attention to the two unique of the articles in this issue of Immunological Reviews. As each review in the field is and that be in in order to As not only the of how this field become but also in the that often have important biological are Therefore, the personal and historical in this volume are this process of on the history of the field has also many of the to fundamental that remain The in the reviews in this volume how much remains to be done to the regulation and function of NF-κB. The mechanisms of IKK activation, IκB transcriptional activation, and all are Yet perhaps the most of these upon in of the reviews in this issue, is the of NF-κB as a therapeutic Where these 25 years have progress in the identification of NF-κB pathway components and functions, the research in the to progress in elucidating and this for the of that can target the myriad characterized by NF-κB We would to the to this who despite numerous other were able to the articles that this in the is by from the and to and The have no of to