Table of contents

NF kappa B and Nitric Oxide

To test the hypothesis that nitric oxide (NO) limits endothelial activation, cytokine-stimulated human endothelial cells were treated with several NO donors and their effects on the inducible expression of vascular cell adhesion molecule-1 (VCAM-1) were assessed. In a concentration-dependent manner, NO inhibited interleukin (IL)-1 alpha-stimulated VCAM-1 expression by 35-55%. This inhibition was paralleled by reduced monocyte adhesion to endothelial monolayers in nonstatic assays, was unaffected by cGMP analogues, and was quantitatively similar after stimulation by either IL-1 alpha, IL-1 beta, IL-4, tumor necrosis factor (TNF alpha), or bacterial lipopolysaccharide. NO also decreased the endothelial expression of other leukocyte adhesion molecules (E-selectin and to a lesser extent, intercellular adhesion molecule-1) and secretable cytokines (IL-6 and IL-8). Inhibition of endogenous NO production by L-N-monomethyl-arginine also induced the expression of VCAM-1, but did not augment cytokine-induced VCAM-1 expression. NO represses VCAM-1 gene transcription, in part, by inhibiting NF-kappa B. It is thought that NO's ability to limit endothelial activation and inhibit monocyte adhesion may contribute to some of its antiatherogenic and antiinflammatory properties within the vessel wall (De Caterina, 1995).

It has been suggested that the NF-kappaB transcription factor family may mediate expression of the gene encoding the cytokine-inducible form of nitric oxide synthase (iNOS). To establish if nitric oxide (NO) could in turn affect activity of NF-kappaB, the ability of NO-donor compounds to influence NF-kappaB DNA binding activity in vitro was investigated. NO-donor compounds sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) both inhibit the DNA binding activity of recombinant NF-kappaB p50 and p65 homodimers and of p50-p65 heterodimers. Inhibition of NF-kappaB p50 DNA binding by NO-donor compounds involves modification of the conserved redox-sensitive C62 residue, as a C62S p50 mutant is significantly more resistant to SNP-mediated inactivation. SNP can inhibit p50 DNA binding by mechanisms other than the formation of intersubunit disulphide bonds involving p50 residue C62. NO gas can modify C62 by S-nitrosylation. This study indicates that NO-donors can directly inhibit the DNA binding activity of NF-kappaB family proteins, suggesting that cellular NO provides another control mechanism for modulating the expression of NF-kappaB-responsive genes (Matthews, 1996).

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA is induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibits NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb is also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1+/+ and E2F1-/- murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity (Tanaka, 2002).

Hypothalamic NF-kappaB involved energy imbalance underlying obesity

Overnutrition is associated with chronic inflammation in metabolic tissues. Whether metabolic inflammation compromises the neural regulatory systems and therefore promotes overnutrition-associated diseases remains unexplored. This study shows that a mediator of metabolic inflammation, IKKbeta/NF-kappaB, normally remains inactive although enriched in hypothalamic neurons. Overnutrition atypically activates hypothalamic IKKbeta/NF-kappaB at least in part through elevated endoplasmic reticulum stress in the hypothalamus. While forced activation of hypothalamic IKKbeta/NF-kappaB interrupts central insulin/leptin signaling and actions, site- or cell-specific suppression of IKKbeta either broadly across the brain or locally within the mediobasal hypothalamus, or specifically in hypothalamic AGRP neurons significantly protects against obesity and glucose intolerance. The molecular mechanisms involved include regulation by IKKbeta/NF-kappaB of SOCS3, a core inhibitor of insulin and leptin signaling. These results show that the hypothalamic IKKbeta/NF-kappaB program is a general neural mechanism for energy imbalance underlying obesity and suggest that suppressing hypothalamic IKKbeta/NF-kappaB may represent a strategy to combat obesity and related diseases (Zhang, 2008).

Role of NF kappaB in limb morphogenesis

In Drosophila, the Dorsal protein establishes the embryonic dorso-ventral axis during development. The vertebrate homolog of Dorsal, nuclear factor-kappa B (NF-kappaB), is vital for the formation of the proximo-distal organizer of the developing limb bud known as the apical ectodermal ridge (AER). c-rel mRNA is first detected in the chick limb bud at stage 15/16, before the appearance of the AER. Expression remains strong within the distal compartment during limb bud outgrowth. As the digits form, c-rel mRNA levels begin to decrease within the mesenchyme, persisting only in regions adjacent to the cartilage anlage. By stage 34, c-rel message is no longer detected. Transcription of the NF-kappaB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-kappaB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Inhibition of NF-kappaB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-kappaB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation. It is thought thatthe function of the kappaB factors is to modulate Twist gene expression during development (Bushdid, 1998).

The development of the vertebrate limb serves as an amenable system for studying signaling pathways that lead to tissue patterning and proliferation. Limbs originate as a consequence of a differential growth of cells from the lateral plate mesoderm at specific axial levels. At the tip of the limb primordia the progress zone, a proliferating group of mesenchymal cells, induces the overlying ectoderm to differentiate into a specialized structure termed the apical ectodermal ridge. Subsequent limb outgrowth requires reciprocal signaling between the ridge and the progress zone. The Rel/NF-kappaB family of transcription factors is induced in response to several signals that lead to cell growth, differentiation, inflammatory responses, apoptosis and neoplastic transformation. In unstimulated cells, NF-kappaB is associated in the cytoplasm with an inhibitory protein, I-kappaB. In response to an external signal, I-kappaB is phosphorylated, ubiquitinated and degraded, releasing NF-kappaB to enter the nucleus and activate transcription. Rel/NF-kappaB genes are expressed in the progress zone of the developing chick limb bud. When the activity of Rel/NF-kappaB proteins is blocked by infection with viral vectors that produce transdominant-negative I-kappaBalpha proteins, limb outgrowth is arrested. It is shown that blocking Rel/NF-kappaB function downregulates Twist expression. These results indicate that Rel/NF-kappaB transcription factors play a role in vertebrate limb development (Kanegae, 1998).

NF kappa-B and lung morphogenesis

The role of NF-kappaB in directing the branching morphogenesis of the developing chick lung, a process which relies on epithelial-mesenchymal communication, has been investigated. High level expression of relA (and NFkappaB family member) is found in the mesenchyme surrounding the nonbranching structures of the lung but is not detected either in the mesenchyme surrounding the branching structures of the distal lung or in the developing lung epithelium. Specific inhibition of mesenchymal NF-kappaB in lung cultures results in increased epithelial budding. Conversely, expression of a trans-dominant activator of NF-kappaB in the lung mesenchyme represses budding. Ectopic expression of RelA is sufficient to inhibit the ability of the distal mesenchyme to induce epithelial bud formation. Cellular proliferation in the mesenchyme is inhibited by hyperactivation of NF-kappaB in the mesenchyme of lung cultures. Interestingly, increased NF-kappaB activity in the mesenchyme also decreases the proliferation of the associated epithelium, while inhibition of NF-kappaB activity increases cellular proliferation in lung cultures. Expression patterns of several genes that are known to influence lung branching morphogenesis are altered in response to changes in mesenchymal NF-kappaB activity, including fgf10, bmp-4, and tgf-beta1. Thus NF-kB represents the first transcription factor reported to function within the lung mesenchyme to limit growth and branching of the adjacent epithelium (Muraoka, 2000).

Role of NF kappa-B in B cells, T cells, and erythroid cells

NF-kappa B, a heterodimeric transcription factor composed of p50 and p65 subunits, can be activated in many cell types and is thought to regulate a wide variety of genes involved in immune function and development. Mice lacking the p50 subunit of NF-kappa B show no developmental abnormalities, but exhibit multifocal defects in immune responses involving B lymphocytes and nonspecific responses to infection. B cells do not proliferate in response to bacterial lipopolysaccharide and are defective in basal and specific antibody production. Mice lacking p50 are unable effectively to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae, but are more resistant to infection with murine encephalomyocarditis virus. These data support the role of NF-kappa B as a vital transcription factor for both specific and nonspecific immune responses, but do not indicate a developmental role for the factor (Sha, 1995).

NF-kappaB is a family of related, dimeric transcription factors that are readily activated in cells by signals associated with stress or pathogens. Although the Drosophila NF-kappaB homolog Dorsal is responsible for the develpment of all embryonic ventral structures, a similar role has not been shown to exist in mammals. In mammals, these factors are critical to host defense, as demonstrated previously with mice deficient in individual subunits of NF-kappaB. Mice deficient in both the p50 and p52 subunits of NF-kappaB have been generated to reveal critical functions that may be shared by these two highly homologous proteins. Unlike the respective single knockout mice, the p50/p52 double knockout mice fail to generate mature osteoclasts and B cells, apparently because of defects that track with these lineages in adoptive transfer experiments. These mice present markedly impaired thymic and splenic architectures and impaired macrophage functions. The blocks in osteoclast and B-cell maturation are unexpected. Lack of mature osteoclasts causes severe osteoperosis, a family of diseases characterized by impaired osteoclastic bone resorption. These findings now establish critical roles for NF-kappaB in development and expand its repertoire of roles in the physiology of differentiated hematopoietic cells (Franszoso, 1997).

In mice with an inactivated c-rel gene, whereas development of cells from all hemopoietic lineages appeared normal, humoral immunity was impaired and mature B and T cells were found to be unresponsive to most mitogenic stimuli. Phorbol ester and calcium ionophore costimulation, in contrast to certain membrane receptor-mediated signals, overcame the T cell-proliferative defect, demonstrating that T cell proliferation occurs by Rel-dependent and -independent mechanisms. The ability of exogenous interleukin-2 to restore T cell, but not B cell, proliferation indicates that Rel regulates the expression of different genes in B and T cells that are crucial for cell division and immune function (Kontgen, 1995).

To better understand the role of NF-kB in normal B cell physiology, purified population of resting B cells from p50/NF-kappa B knockout (p50-/-) mice was used to determine their ability to proliferate, secrete lg, express germ-line CH, RNA, and undergo lg isotype switching in vitro in response to a number of distinct stimuli. p50-/- B cells proliferate normally in response to dextran-anti-IgD Abs (alpha delta-dex) and membrane-bound, but not soluble, CD40 ligand (CD40), and they are virtually unresponsive to LPS when compared with control B cells. p50-/- B cells secrete markedly reduced lg in response to alpha delta-dex or mCD40L in the presence of IL-4 + IL-5, despite their relatively normal proliferative rates, whereas normal lg secretion is restored by the combination of alpha delta-dex and CD40L. p50-/- B cells expressed normal steady-state levels of germ-line CH gamma 3 and CH gamma epsilon RNA upon appropriate stimulation. Although p50-/- B cells undergo substantial switching to IgG1, a marked reduction in the switch to IgG3 and IgE, as IgA, is observed (Snapper, 1996).

B cell stimulation by CD40L and by anti-Ig antibody brings about nuclear expression of three transcripiton factors. Cross-linked CD40L induces nuclear expression of NF-kappa B, AP-1 and NF-AT with a time course and intensity similar to that produced by anti-Ig. Examination of NF-kappa B in more detail demonstrates that the CD40 mediates expression of DNA binding complexes correlated with induction of trans-activating activity which again attain similar levels following cross-linking of CD40 and surface Ig. Despite the marked similarity in transcription factor induction triggered through CD40 and Ig, differences in the intracellular signaling pathways utilized were apparent in that protein kinase C (See Drosophila PKC) depletion did not affect CD40 mediated induction of NF-kappa B even as induction by anti-Ig was abolished. These results suggest that a 'final common pathway' or convergence of transcription factor induction may exist for two distinct receptors, each of which is individually capable of triggering cell cycle progression, despite the use of separate intracellular signaling pathways that differ at the level of PKC. Although transcription factor induction was similar for CD40L and anti-Ig early on, subtle differences in expressed NF-kappa B and AP-1 nucleoprotein complexes were apparent at 24 h. Such differences may play a role in determining the variant effects on B cells of stimulation through these two receptors (Francis, 1995).

The transcription factor NF-kappaB is normally sequestered in the cytoplasm by members of the IkappaB family, including IkappaB alpha, IkappaB beta, and the recently cloned IkappaB epsilon. Upon cellular activation, these inhibitors are rapidly phosphorylated on two amino-terminal serines, then ubiquitinated, and degraded by the 26S proteasome, releasing a functional NF-kappaB. To determine the importance of IkappaB beta in NF-kappaB regulation in T cells, transgenic mice were generated expressing a constitutively active IkappaB beta mutant (mIkappaB beta) under the control of the lck promoter. The transgene contains the two critical N-terminal serine residues mutated to alanines and therefore no longer susceptible to degradation upon cell activation. mIkappaB beta is unable to totally displace IkappaB alpha from RelA-containing complexes, thus allowing a transient activation of NF-kappaB upon T-cell stimulation. However, mIkappaB beta completely blocks NF-kappaB activity after IkappaB alpha degradation. As a consequence of this inhibition, ikappabalpha expression is down-regulated, along with that of other NF-kappaB-regulated genes. These transgenic mice have a significant reduction in the peripheral T-cell population, especially CD8+ cells. The remaining T cells have impaired proliferation in response to phorbol 12-myristate 13-acetate plus phytohemagglutinin or calcium ionophore but not to anti-CD3/anti-CD28 costimulation. As a result of these alterations, transgenic animals present defects in immune responses, such as delayed-type hypersensitivity and the generation of specific antibodies against T-cell-dependent antigens. These results show that in nonstimulated T cells, IkappaB beta cannot efficiently displace IkappaB alpha bound to RelA-containing complexes and that persistent NF-kappaB activity is required for proper T-cell responses in vivo (Attar, 1998).

CD30 is a cell-surface receptor that can augment lymphocyte activation and survival through its ability to induce the transcription factor NF-kappaB. CD30, however, has also been implicated in the induction of apoptotic cell death of lymphocytes. One of the effects of CD30 signal transduction is to render cells sensitive to apoptosis induced by the type 1 tumor necrosis factor receptor (TNFR1). This sensitization is dependent on the TRAF-binding sites within the CD30 cytoplasmic domain. One of the proteins that binds to these sites is TRAF2, a signal transduction molecule that is also utilized by TNFR1 to mediate the activation of several downstream kinases and transcription factors. During CD30 signal transduction, binding of TRAF2 to the cytoplasmic domain of CD30 results in the rapid depletion of TRAF2 and the associated protein TRAF1 by proteolysis. These data suggest a model in which CD30 limits its own ability to transduce cell survival signals through signal-coupled depletion of TRAF2. Depletion of intracellular TRAF2 and its coassociated proteins also increases the sensitivity of the cell to undergoing apoptosis during activation of death-inducing receptors such as TNFR1. Consistent with this hypothesis, expression of a dominant-negative form of TRAF2 is found to potentiate TNFR1-mediated death. These studies provide a potential mechanism through which CD30, as well as other TRAF-binding members of the TNFR superfamily, can negatively regulate cell survival (Duckett, 1997).

The transcription factor NF-AT plays an essential role in the inducible transcription of several cytokine genes during T cell activation. The distal NF-AT site of the murine IL-2 promoter binds both NF-AT and AP-1 proteins, and thus represents a composite regulatory site that integrates Ca(2+)- and PKC-dependent signaling pathways in T cell activation. However, the individual contributions of the NF-AT and AP-1 components to promoter activity via this composite site have not been resolved, owing to the absence of a clearly defined AP-1 binding. There is an apparently analogous NF-AT/AP-1 composite site in the murine IL-4 promoter, which can be mutated to selectively block the recruitment of each component. The cooperative and coordinate involvement of both NF-AT and AP-1 is necessary for full activity of the NF-AT/AP-1 composite site, and, ultimately, the entire IL-4 promoter (Rooney, 1995).

Transformation of B-lineage precursors by the Abelson murine leukemia virus appears to arrest development at the pre-B stage. Abelson-transformed pre-B cell lines generally retain transcriptionally inactive, unrearranged immunoglobulin kappa alleles. Nontransformed pre-B cells expanded from mouse bone marrow efficiently transcribe unrearranged kappa alleles. In addition, they contain activated complexes of the NF-kappa B/Rel transcription factor family, in contrast with their Abelson-transformed counterparts. Using conditionally transformed pre-B cell lines, it hs been shown that the v-abl viral transforming protein, a tyrosine kinase (See Enabled), blocks germ-line kappa gene transcription and negatively regulates NF-kappa B/Rel activity. An active v-abl kinase specifically inhibits the NF-kappa B/Rel-dependent kappa intron enhancer, which is implicated in promoting both transcription and rearrangement of the kappa locus. v-abl inhibits the activated state of NF-kappa B/Rel complexes in a pre-B cell via a post-translational mechanism that results in increased stability of the inhibitory subunit I kappa B alpha. This analysis suggests a molecular pathway by which v-abl inhibits kappa locus transcription and rearrangement (Klug, 1994).

Induction of the adaptive immune response depends on the expression of co-stimulatory molecules and cytokines by antigen-presenting cells. The mechanisms that control the initial induction of these signals upon infection are poorly understood. It has been proposed that their expression is controlled by the non-clonal, or innate, component of immunity that preceded in evolution the development of an adaptive immune system in vertebrates. The cloning and characterization of a human homolog of the Drosophila Toll protein is reported. Toll has been shown to induce the innate immune response in adult Drosophila. Like Drosophila Toll, human Toll is a type I transmembrane protein with an extracellular domain consisting of a leucine-rich repeat (LRR) domain, and a cytoplasmic domain homologous to the cytoplasmic domain of the human interleukin (IL)-1 receptor. Both Drosophila Toll and the IL-1 receptor are known to signal through the NF-kappaB pathway. A constitutively active mutant of human Toll transfected into human cell lines can induce the activation of NF-kappaB and the expression of NF-kappaB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8, as well as the expression of the co-stimulatory molecule B7.1, which is required for the activation of naive T cells (Medzhitov, 1997).

During hexamethylene bisactamide (HMBA)-induced differentiation of murine erythroleukemia (MEL) cells erythroid genes are transcriptionally activated while c-myb and several other nuclear proto-oncogenes are down-regulated. Differentiation is inhibited by cAMP analogs and the adenyl cyclase-stimulating agent forskolin. These drugs prevent the late down-regulation of c-myb, which is known to be critical for MEL cell differentiation. Since the increase in c-myb mRNA levels is due to increased mRNA transcription, an examination was made of the transcription factors NF-kappaB and AP-1, which have been implicated in the regulation of c-myb expression. Binding of MEL cell nuclear proteins to a NF-kappaB recognition sequence in c-myb intron I is strongly induced by either 8-Br-cAMP or forskolin; induction is delayed and correlates with the up-regulation of c-myb. The cAMP-induced NF-kappaB complex contains p50 and RelB; in co-transfection assays, p50 and RelB transactivate a reporter construct containing the c-myb intronic NF-kappaB site upstream of a minimal promoter. 8-Br-cAMP and forskolin also increase the DNA binding activity of AP-1 complexes containing JunB, JunD and c-Fos in MEL cells that could cooperate with NF-kappaB. It is concluded that inhibition of MEL cell differentiation by pharmacological doses of cAMP can be explained by the up-regulation of c-myb, which is mediated, at least in part, by NF-kappaB p50/RelB heterodimers (Suhasini, 1997).

Dorsal homologs, cell growth, and the cell cycle

A tetracycline-regulated system was used to characterize the effects of c-Rel on cell proliferation. The expression of c-Rel in HeLa cells leads to growth arrest at the G1/S-phase transition, which correlated with its nuclear localization and the induction of endogenous IkappaB alpha expression. These changes are accompanied by a decrease in E2F DNA binding and the accumulation of the hypophosphorylated form of Rb. In vitro kinase assays show a reduction in Cdk2 kinase activity that correlates with elevated levels of p21WAF1 Cdk inhibitor and p53 tumor suppressor protein. While the steady-state levels of WAF1 transcripts are increased, pulse-chase analysis reveals a sharp increase in p53 protein stability. Importantly, the deletion of the C-terminal transactivation domains of c-Rel abolishes these effects. Together, these studies demonstrate that c-Rel can affect cell cycle control and suggest the involvement of the p21WAF1 and p53 cell cycle regulators (Bash, 1997).

Stratified epithelium contains a mitotically active basal layer of cells that first cease proliferating, then migrate outward and undergo terminal differentiation. The control of this process, which is abnormal in cutaneous neoplasia and inflammation, is not well understood. In normal epidermis, NF-kappaB proteins exist in the cytoplasm of basal cells and then localize in the nuclei of suprabasal cells, suggesting a role for NF-kappaB in the switch from proliferation to growth arrest and differentiation. The functional blockade of NF-kappaB by the expression of dominant-negative NF-kappaB inhibitory proteins in transgenic murine and human epidermis produce hyperplastic epithelium in vivo. Consistent with this, application of a pharmacologic inhibitor of NF-kappaB to intact skin induces epidermal hyperplasia. In contrast, overexpression of active p50 and p65 NF-kappaB subunits in transgenic epithelium produces hypoplasia and growth inhibition. These data suggest that spatially restricted NF-kappaB activation occurs in stratified epithelium and indicate that NF-kappaB activation in this tissue, in contrast to its role in other settings, is important for cellular growth inhibition (Seitz, 1998).

Table of contents

dorsal continued: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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