Recent Advances in Understanding NF-κB Regulation

Nuclear factor-kappa-B (NF-κB) is considered a central mediator of the immune response. Well over 100 genes are activated by this transcription factor and most of them are directly or indirectly proinflammatory (1,2). Increased NF-κB activity has been found in inflamed intestinal mucosa, and factors that are implicated in inflammatory bowel disease (IBD) such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and bacterial antigens (i.e., lipopolysaccharides [LPS]) are potent activators of NF-κB (3,–7). In addition, many current therapies for IBD act at least in part through the inhibition of NF-κB or through inhibition of signals that activate NF-κB (8,–13). Excellent reviews are available on the role of NF-κB in the intestine as well as the potential for therapeutic modalities targeting NF-κB in IBD (7,12,–15). This review will therefore attempt to focus on recent developments in the understanding of NF-κB activation as well as the signals that activate and inhibit NF-κB. The NF-κB proteins are a family of transcription factors comprised of homodimers and heterodimers of RelA (p65), RelB, cRel, NF-κB1 (p50), and NF-κB2 (p52). RelA and p50 constitute the majority of the subunits in cells, and RelA/p50 heterodimers are the most potent activator of NF-κB-dependent genes (2). For the purposes of this review, the RelA/p50 heterodimer will simply be referred to as NF-κB. NF-κB is maintained in an inactive state in the cytoplasm bound to I-κB. There are multiple isoforms of I-κB, including I-κBα, I-κBβ, I-κBε, I-κBγ, bcl-3, p105, and p100, but most of the signal-induced NF-κB activity is regulated by I-κBα (2). A variety of receptors, such as those for IL-1, LPS, bacterial or viral DNA, adhesion molecules, and the TNF superfamily will activate NF-κB (16). Although these receptors activate different proximate signaling pathways, they all converge at the level of the I kappa B kinase (IKK) complex (17,–20). The IKK complex consists of at least three subunits, IKKα, IKKβ, and IKKγ (21). IKKγ has no kinase activity but instead acts as a docking protein for IKK kinases or other signaling proteins (17,22). NF-κB activation by the IKK complex is dependent on serine phosphorylation of IKKβ, which may be accomplished by other kinases (Map3Ks or MEKKs) or through transautophosphorylation between IKK complex subunits (17). The role of each subunit of the IKK complex has been elucidated by gene targeting. IKKγ and IKKβ are essential for NF-κB responses to proinflammatory stimuli, as IKKγ−/− or IKKβ−/− cells do not activate NF-κB in response to TNF, IL-1, or LPS (23,–26). Surprisingly, IKKα−/− cells display relatively normal activation of NF-κB in response to TNF, IL-1, or LPS, demonstrating that this subunit is not required for those proinflammatory signals (27). It appears that IKKα may have at least three separate functions that are important for epidermal differentiation, B-cell maturation, and mammary gland development (28,–30). Activation of the IKK complex results in the phosphorylation of I-κB. Phosphorylated I-κB is then polyubiquitinated by a multiprotein complex, E3-SCFβ-TrCP, and is thus targeted for degradation by the 26S proteosome (2,31). This degradation of ubiquitinated I-κB frees NF-κB to translocate to the nucleus, where it can induce the transactivation of target genes (Fig. 1). Activated NF-κB induces the rapid synthesis of I-κBα, which binds and inactivates NF-κB and translocates it back to the cytoplasm. This rapid negative regulation of NF-κB is important since NF-κB transactivates genes for a large number of potent inflammatory proteins. Activation and negative regulation of NF-κB. A: Activating stimuli act on the IKK complex, resulting in the phosphorylation of IKKβ. The IKK complex phosphorylates I-κB, targeting it for ubiquitination and degradation by the proteosome. NF-κB then translocates to the nucleus where it binds to consensus sites on the DNA and transactivates gene transcription. Other factors such as PKA and GSK-3β are not required for NF-κB translocation but are necessary for NF-κB induction of gene transcription. Acetylation of NF-κB may prolong its residency in the nucleus. B: NF-κB rapidly induces the production of I-κB, which binds, inactivates, and returns NF-κB to the cytosol. Negative regulation also involves the deacetylation of NF-κB to allow binding to I-κB. A20 is rapidly induced by NF-κB and is critical for the negative regulation of some proximate IKK activating signals. NF, nuclear factor; IKK, I kappa B kinase. Phosphorylation and acetylation of NF-κB also control its activity within the nucleus (Fig. 1). Protein kinase A potently increases the gene-transactivating activity of NF-κB by phosphorylating RelA, which facilitates NF-κB association with the transcriptional coactivator CBP/p300 (2). In addition, cells lacking glycogen synthase kinase-3β (GSK-3β) display cytokine-driven translocation of NF-κB without subsequent transactivation of NF-κB-dependent genes (32). Recently the direct and reversible acetylation of NF-κB has been described as a regulatory mechanism for NF-κB activity (33). Chen et al. found that the RelA subunit of NF-κB is acetylated in the nucleus and that this prevents binding and inhibition by I-κBα. The subsequent deacetylation of NF-κB by histone deacetylase 3 (HDAC3) allows effective I-κB binding and the nuclear export and termination of NF-κB activity (Fig. 1). In support of this, the HDAC inhibitor trichostatin caused dramatically elevated NF-κB activation and DNA binding in TNF-stimulated 293T cells (33). These findings are in contrast to the report by Inan et al. of trichostatin effects in HT-29 epithelial cells (34). In those studies, trichostatin and butyrate inhibited the induction of NF-κB DNA binding by TNF. It was proposed that butyrate, like trichostatin, may be acting as an NF-κB inhibitor by blocking histone deacetylases, as butyrate has long been known to induce histone hyperacetylation in the nucleus (35,36). These results may not necessarily be incongruous since the report by Chen et al. examined acetylation of TNF-induced RelA/p50 heterodimers, whereas Inan et al. examined the constitutive p50 homodimers typical of intestinal cells. In addition, the effects of butyrate that lead to NF-κB inhibition may be divergent from deacetylase activity. In any case, the observation that butyrate can reduce NF-κB activity in epithelial cells suggests a possible mechanism for the beneficial effects of butyrate on intestinal inflammation (37). There are two receptors for TNF, TNFRI (tumor necrosis factor receptor) (p55) and TNFRII (p75), that have different expression patterns and cytoplasmic signaling domains (38). While TNFRI is clearly central to the proinflammatory response to TNF, the biologic role of TNFRII is less well understood, partly because this receptor may not respond well to soluble TNF in vitro (39). Binding of TNF to TNFRI (Fig. 2) elicits the recruitment of the adapter protein TRADD to the cytoplasmic domain of the receptor (40). TRADD can then recruit RIP and TRAF2 leading to the activation of the IKK complex (41,42). The mechanism whereby these proximate receptor events lead to IKK activity is not entirely known, but RIP is essential, while TRAF2 is dispensable for TNFRI-induced NF-κB activation. RIP−/− cells do not activate IKK and thus have no NF-κB activation in response to TNF, whereas both TNF-induced IKK and NF-κB activation are preserved in TRAF2−/− cells (43,44). Kinases capable of phosphorylating IKK, such as NIK and MEKK1, have been examined for their role in TNFR-induced NF-κB activation (45,46). NIK is a potent IKK activator first identified from yeast two hybrid screens of TRAF-interacting proteins. Over-expression of NIK or MEKK1 in cells activates IKK, and kinase-defective NIK or MEKK1 mutants block TNF- and IL-1-induced IKK and NF-κB (17). However, targeted deletions of NIK and MEKK1 have demonstrated that these kinases are dispensable for TNF- or IL-1-induced IKK activation (47,48). The alymphoplasia (aly) mutation of mouse has been localized to a single amino acid substitution in NIK that renders it unable to bind TRAF and activate IKK (49). This mutation does not disrupt TNF or IL-1-induced NF-κB but does block lymphotoxin-induced NF-κB (49). This may explain why the phenotype of LTβ−/− mice (i.e., altered PP organogenesis) resembles that of aly/aly mice. MEKK1−/− cells display normal NF-κB activation in response to TNF, but are defective in their ability to respond to stresses that alter cell shape or cytoskeletal organization (47). MEKK3 can also phosphorylate IKK in vitro and MEKK3−/− cells have dramatically reduced NF-κB response to TNF (50). Thus MEKK3 may be the kinase that transmits signals from the TNFR to the IKK complex. Although there are several kinases that can phosphorylate IKK, leading to NF-κB activation, IKK activation can also occur through oligomerization without the participation of IKK kinases (Fig. 2) (17,51,–53). The Tax transactivator protein of human T cell to IKK activation in cells. Tax directly binds to IKKγ oligomerization of IKK which phosphorylate each other and thus activate NF-κB This of transautophosphorylation of IKK may also for the activation of IKK by since RIP can bind to and oligomerization of IKK by RIP to NF-κB activity TNF, IL-1, and LPS signaling A: TNF binding to TNFRI the association of the protein which then RIP and TRAF2 to the receptor complex. Activation of IKK may occur through the activation of IKK kinases or the direct oligomerization of IKK A20 TNFRI signaling by an mechanism that may A20 association with TRAF2 or TNFRI induces TRADD and activates A20 TNF-induced cell through its association with B: binds to which then and to the LPS binds to the recruitment of and to the These receptor induce the association of resulting in the activation of the IKK kinase which phosphorylates and activates the IKK complex. TNF, tumor necrosis factor; LPS, IKK, I kappa B In to activating TNFRI can also induce cell (Fig. This is because the adapter protein TRADD can bind to through its domain can bind to through association of domains leading to activation by a mechanism that may within the proximate receptor complex activation then to cell through dependent However, TNF does not most cells because the activation of NF-κB by TNF prevents TNF-induced cell This is demonstrated dramatically by the phenotype of which from cell in the This is by TNF, as and mice deletions of of the NF-κB signaling such as IKKγ−/− and IKKβ−/− also from In addition, RIP−/− cells, which do not activate NF-κB in response to TNF, are to TNF-induced cell of RIP and IKKβ renders cells to TNF-induced cell It is not known NF-κB activation prevents TNF-induced cell but of proteins have some (38). and first demonstrated that and TRAF2 with TNFRII in a it was found that and TRAF2 with to also in a and TRAF2 are with inhibitor of proteins and both and found to with TNFRI TNF In addition, both and are of genes The proteins can directly bind and inhibit it has been that the of and within the TNFRI signaling complex can directly bind or block activation and TNF-induced In support of this, TRAF2−/− cells are capable of activating are to TNF-induced cell mice have defective negative regulation of TNF-induced NF-κB and to TNF-induced but effects of on cell have not been described Other genes such as and are also induced by NF-κB and may for some of the effects of NF-κB activation. mice have no cell phenotype but have of and to a mechanism The role of proximate TNFRI signaling in the of TNF-induced cell gene targeting and of other potential proteins. TNFRI can also through the kinase to phosphorylate and transcription of target This TNFRI signaling is by TRAF2 through its association with TRADD from TRAF2−/− mice do not activate in response to TNF Recently two have that TNF activation of cells results in activation of This that NF-κB-dependent genes are required for the negative regulation TNF-induced In these found potential and that the role of NF-κB on the inhibition of in cells may from TNF-induced It is not TNF-induced to cell in these or or inhibit TNF-induced However, mice have no cell and in signaling in cells have not been described activates cells through (Fig. 2) signaling in response to LPS also which binds to LPS and a cell is of a family of first identified by their to receptor There are different identified in that bind to antigens and are considered critical for the immune response to for several of the have been described and viral and bacterial DNA are by a domain and a cytoplasmic signaling a domain The receptor family also has cytoplasmic TNF, and LPS activate NF-κB through the IKK complex in an and (17). TNF, and LPS receptors are not dependent on as RIP−/− cells have normal and NF-κB activation these receptors on other adapter and signaling that and (Fig. 2) is an adapter protein that a domain and a of or with these receptors through The of then to the complex. then with and activates by an However, the kinase activity of is not necessary for NF-κB activation and mice are to LPS, and cells from these mice dramatically and IL-1-induced inflammatory responses this, it be that cells, some to activate NF-κB in response to the induction of NF-κB-dependent genes was in cells, there was but binding of NF-κB to its consensus This suggests that is not required for the activation of IKK, degradation of I-κBα, and translocation of NF-κB to the nucleus, but that it is required for the transcriptional activity of NF-κB. This may the of LPS It has been that both and can act as LPS receptors mice do not respond to LPS cells, cells display no NF-κB DNA binding in response to LPS in cell is required for both IKK activation and NF-κB DNA binding The LPS response of cells may also an adapter different from that can for the activation of In this a protein has been homodimers or heterodimers with and activate NF-κB (Fig. This of but not which both Although may for the ability of cells to in response to LPS, it is not this lead to the NF-κB nuclear binding without subsequent NF-κB target gene activation in The translocation of NF-κB to the nucleus the activation of IKK and degradation of I-κB, but transcriptional activity also the phosphorylation of NF-κB Thus may be dispensable for IKK activation and NF-κB DNA but essential for NF-κB It to be is required for the phosphorylation of NF-κB. and family do not directly with the IKK complex but instead their to IKK through (Fig. 2) is of described factors that signaling by many TNF superfamily cells display dramatically reduced NF-κB activation in response to LPS and but not TNF activation of IKK is not well understood, but recent in vitro of and IKK have some have two and that are essential for IKK activity. is comprised of two ubiquitination proteins and These ubiquitination of proteins. the of ubiquitination that does not lead to degradation but to the regulation of target protein ubiquitination is rapidly as an important of protein like the regulation of proteins by activation of IKK ubiquitination is ubiquitinated by its complex with and IKK can be directly activated by ubiquitination in but it is not known ubiquitination of IKK is in signal-induced NF-κB activation in the essential for activation of IKK, a and two proteins and The kinase is activated by association with ubiquitinated This can phosphorylate IKKβ, leading to the activation of NF-κB (Fig. In this may be the IKK kinase the between and IKK activation in and NF-κB activation the most recent in the of IBD is the that in the gene (Fig. are to disease In et al. identified the first for localized the of et al. and et al. identified within the gene for in the that to been identified by et al. as an family This family is by proteins an recruitment domain a central binding domain and a regulatory domain In the of the domain consists of that the oligomerization of the binds from the and allows to through the association of This the which can recruit and activate leading to cell like has a central and but a different regulatory of The of proteins is from the of the domain of The may a role as the of as of this the activation of NF-κB is cells can induce both and NF-κB activation in cells. The ability of to cells is preserved in cells, demonstrating that is of NF-κB activation activation of NF-κB is by the association of the domain of with the domain of known as or It is that like RIP or the Tax can bind to IKKγ and the transautophosphorylation of IKKβ. The oligomerization of through its may therefore leading to transautophosphorylation of IKK and activation of NF-κB Activation of NF-κB by LPS or some other binds to the of This oligomerization of through its with domains and oligomerization of IKK them to activate by of in to of these are amino acid that to and to A mutation a at a that the amino of three occur on the of a of cells renders them to but not LPS, whereas of the with does not cells to nuclear factor; IKK, I kappa B LPS, was through a for genes proteins to the of in multiple expression is to the and to of the The of is to a a central and an with two domains (Fig. of induces NF-κB activation that is dependent on both of both activates NF-κB. These like those of can with and activate NF-κB through the oligomerization of IKK of proteins lacking the dramatically NF-κB activation, to the NF-κB activation of activation of NF-κB may therefore be regulated by the to the of activation by its can activate through this regulatory and that a signaling mechanism proteins through the that they have demonstrated that LPS induces NF-κB activation in 293T cells or In they that this LPS was and and that bind LPS directly in vitro proteins are this suggests that they act as receptors for of found with by et al. and et al. (Fig. single amino acid and while a a mutation leading to a three found on the of a the amino acid The mutation is to the amino the of the of This be to have on the of the However, this mutation was NF-κB activation was This a mutation leading to reduced LPS proinflammatory of cells may lead to with the that is an inflammatory However, several potential have been It is possible that this mutation the of to such that activation or bacterial of the can of the of the mutation have been in and it be to effects in such cells. However, such are clearly to the understanding of since LPS may activate signaling (i.e., in the described mutation of may reduce LPS but may also NF-κB activation by an or This be with the observation that NF-κB is activated in of inflamed intestinal it be that the described in et al. the at but not the of It that the of with may a different from the There is some as to is a LPS receptor For the role of as a LPS receptor is to with the role of as the receptor for LPS in mice and are to LPS to their expression of and was to be the LPS but this was to be to other bacterial (i.e., found in most of LPS of LPS are required to the response in cells, and this may other bacterial in such proteins display activation of NF-κB activation in response to LPS in or cells but not in cells It possible that is in the signals from the LPS of is to an LPS this not the observation that is activating NF-κB in response to in the cell LPS may not any directly but instead may signals from events the to responses to of mice may be required to the of the with or a gene for has been These findings that the to is at least in part as in an protein found in The signaling leading to NF-κB activation have been in the and is known NF-κB activity is The central of NF-κB regulation is that NF-κB is inhibited by I-κBα, activated by I-κBα and then inhibited by of I-κBα (Fig. 1). The synthesis of I-κBα is dramatically and rapidly induced by this an and effective negative to control inflammatory A critical role for I-κBα in this was demonstrated by the and NF-κB activation of mice of IKKγ and IKKβ have the essential role for these I-κB kinases in TNF-induced NF-κB activation (17). IKK is activated by proteins through phosphorylation by other kinases or by IKK and this TNF is dependent on RIP is known the negative regulation of proximate in the TNFR signaling It is that some mechanism to IKK as I-κB phosphorylation and degradation with of TNF a has been described as a critical negative of TNF-induced NF-κB activation (Fig. 2) A20 was first identified by and as a rapidly TNF-induced gene in human cells The of A20 are within of TNF and are by A20 is induced as rapidly as the for I-κB. The of A20 multiple NF-κB consensus A20 transcription to NF-κB activation with this, multiple other stimuli have been found to A20 including LPS, IL-1, and the viral proteins and A20 is identified gene of cells by NF-κB activating of A20 NF-κB activation in response to TNF, IL-1, LPS, and multiple other signals but not NF-κB activation Tax activation of NF-κB appears to through direct oligomerization of IKK, and A20 is not to block this of NF-κB activation. Thus it that A20 activating signals proximate to the IKK with this, A20 NF-κB activity induced by of and but does not block NF-κB activation of NIK of A20 does not cells to TNF to they do not activate NF-κB This is because A20 also TNF-induced cell This A20 in a as most that block TNF-induced NF-κB cells to two hybrid and have that A20 can with a number of proteins in cells, including and several viral protein binding proteins including Tax binding and Thus A20 binds to a number of proteins that can be implicated in NF-κB activation the role of A20 in mice These mice inflammation and at a and cells are unable to TNF-induced NF-κB activation, demonstrating a critical role for A20 as a negative of TNF-induced NF-κB. The of I-κB in cells but I-κB does not because it is and cells display TNF-induced IKK activity. Thus A20 appears to be critical for the proximate negative regulation of TNF signals and is an essential of the regulated NF-κB response (Fig. Although A20 can inhibit and NF-κB activation, cells not NF-κB activation in response to This suggests that A20 does not block all of IKK, or inhibit IKK but proximate TNFR signals. in most cells, signals are regulated in a to TNF. Other A20 like proteins such as and may to inhibit proximate signaling from the receptor The regulation of TNF-induced cell was also in mice activation of cells to TNF. This that A20 cells TNF-induced and that A20 is necessary for TNF NF-κB-dependent genes are The ability to cell and NF-κB may be a of association with proximate signaling proteins such as or IKKγ (Fig. 2) of the of these proximate signaling proteins in cells will to the of A20 mice and have TNF and NF-κB This suggests that mice will be important for the of human IBD where both TNF and NF-κB have been It is possible that A20 may activation of NF-κB. an will an immune response by the activation of NF-κB-dependent This may be by direct effects of bacterial acting through or indirectly through the of inflammatory by In case, receptor activation induces the phosphorylation of I-κB, which to its ubiquitination and degradation by the proteosome. and have a and of this by in intestinal epithelial cells of cells with of or the production of NF-κB-dependent induced by subsequent to This of within of and to other proinflammatory of including TNF. also reduced the TNF induction of NF-κB-dependent and I-κBα as well as TNF induction of a NF-κB TNF-induced I-κBα degradation and the nuclear translocation of NF-κB. These effects to epithelial cell as they not in cells or Surprisingly, phosphorylation of I-κB and in response to all stimuli in with cells with not or their I-κB. may the NF-κB activation to reduce inflammatory responses in the intestinal these can be to intestinal by other or of it may to a role of as of to these findings support of possible of The activation of NF-κB by multiple proinflammatory signals through phosphorylation of I-κB, leading to I-κB ubiquitination and degradation by the proteosome. This NF-κB to translocate to the nucleus where it binds to DNA and transactivates a of genes with the immune response. This NF-κB-dependent gene transactivation is regulated by phosphorylation and acetylation of NF-κB. signals the docking protein IKKγ and the kinase activity of IKKβ to activate NF-κB. The IKK complex can be activated by phosphorylation through IKK kinases or may be activated by proinflammatory effects of TNF are by which signals through RIP to activate TNF can also cells, but activation of NF-κB prevents TNF-induced cell in most cells. The LPS and the both activate IKK through a mechanism that involves ubiquitination of and activation of the kinase signaling such as and are by and and may the protein proteins with and can activate NF-κB through oligomerization and transautophosphorylation of in the gene the protein have been with to is in and may signals from LPS, but this is a of some this, the association of with the regulation of NF-κB in immune cells in this with targeted of A20 have the critical role for this protein as a negative of TNF-induced NF-κB activation. A20 appears to block proximate TNFR that lead to IKK activation. In addition, A20 cells TNF-induced cell mice the ability of A20 to TNF signals may be important in Although a proinflammatory the of the are This may be to the ability of some to block ubiquitination of I-κB and NF-κB from the nucleus in intestinal epithelial cells. This of NF-κB may the possible effects of or the of described in this review was by of and a development from the of the and the and the