Table of contents

Rel protein structure and evolution

The crystal structure of human NF-kappaB p52 in its specific complex with the natural kappaB DNA binding site MHC H-2 has been solved at 2.1 Å resolution. Whereas the overall structure resembles that of the NF-kappaB p50-DNA complex, pronounced differences are observed within the 'insert region'. This sequence segment differs in length between different Rel proteins. Compared with NF-kappaB p50, the compact alpha-helical insert region element is rotated away from the core of the N-terminal domain, opening up a mainly polar cleft. The insert region presents potential interaction surfaces to other proteins. The high resolution of the structure reveals many water molecules that mediate interactions in the protein-DNA interface. Additional complexity in Rel protein-DNA interaction comes from an extended interfacial water cavity that connects residues at the edge of the dimer interface to the central DNA bases. The observed water network might account for differences in binding specificity between NF-kappaB p52 and NF-kappaB p50 homodimers (Cramer, 1997).

The NF-kappaB p50/p65 heterodimer is the classical member of the Rel family of transcription factors that regulate diverse cellular functions such as immune response, cell growth, and development. Other mammalian Rel family members, including the proteins p52, proto-oncoprotein c-Rel, and RelB, all have amino-terminal Rel-homology regions (RHRs). The RHR is responsible for the dimerization, DNA binding and cytosolic localization of these proteins by virtue of complex formation with inhibitor kappaB proteins. Signal-induced removal of kappaB inhibitors allows translocation of dimers to the cell nucleus and transcriptional regulation of kappaB DNA-containing genes. NF-kappaB specifically recognizes kappaB DNA elements with a consensus sequence of 5'-GGGRNYYYCC-3' (R is an unspecified purine; Y is an unspecified pyrimidine, and N is any nucleotide). This paper describes the crystal structure at 2.9 A resolution of the p50/p65 heterodimer, bound to the kappaB DNA of the intronic enhancer of the immunoglobulin light-chain gene. The structure reveals a 5-base-pair 5' subsite for p50, and a 4-base-pair 3' subsite for p65. This structure indicates why the p50/p65 heterodimer interface is stronger than that of either homodimer. A comparison of this structure with those of other Rel dimers reveals that both subunits adopt variable conformations in a DNA-sequence-dependent manner. These results explain the different behaviour of the p50/p65 heterodimer with heterologous promoters (Chen, 1998).

Gambif1 from Anopheles gambiae is a member of the Rel family and a close homolog of the morphogen Dorsal, which establishes dorsoventral polarity in the Drosophila embryo. The crystal structure is presented of the N-terminal specificity domain of Gambif1 bound to DNA. This first structure of an insect Rel protein-DNA complex shows that Gambif1 binds a GGG half-site element using a stack of three arginine sidechains. Differences in affinity to Dorsal binding sites in target gene promoters are predicted to arise from base changes in these GGG elements. An arginine that is conserved in class II Rel proteins (members of which contain a transcription activation domain) contacts the outermost guanines of the DNA site. This previously unseen specific contact contributes strongly to the DNA-binding affinity and might be responsible for differences in specificity between Rel proteins of class I and II. Thus, the Gambif1-DNA complex structure illustrates how differences in Dorsal affinity to binding sites in developmental gene promoters are achieved. Comparison with other Rel-DNA complex structures leads to a general model for DNA recognition by Rel proteins (Cramer, 1999).

The recent sequencing of several complete genomes has made it possible to track the evolution of large gene families by their genomic structure. Following the large-scale association of exons encoding domains with well defined functions in invertebrates could be useful in predicting the function in mammals of complex multidomain proteins produced by accretion of domains. With this objective, the genomic structure of the 14 genes in invertebrates and vertebrates that contain rel domains has been examined. The sequence encoding the rel domain is defined by intronic boundaries and has been recombined with at least three structurally and functionally distinct genomic sequences to generate coding sequences for: (1) the rel/Dorsal/NFkappaB proteins that are retained in the cytoplasm by IkB-like proteins; (2) the NFATc proteins that sense calcium signals and undergo cytoplasmic-to-nuclear translocation in response to dephosphorylation by calcineurin; and (3) the TonEBP tonicity-responsive proteins. Remarkably, a single exon in each NFATc family member encodes the entire Ca2+/calcineurin sensing region, including nuclear import/export, calcineurin-binding, and substrate regions. The Rel/Dorsal proteins and the TonEBP proteins are present in Drosophila but not Caenorhabditis elegans. However, the calcium-responsive NFATc proteins are present only in vertebrates, suggesting that the NFATc family is dedicated to functions specific to vertebrates such as a recombinational immune response, cardiovascular development, and vertebrate-specific aspects of the development and function of the nervous system (Graef, 2001).

The positions of introns in genes coding for rel domain proteins are highly conserved, with introns positioned to either side of the sequence encoding the rel domain. The exceptions to this are informative: the sequences encoding the rel domain in Relish, Dif, Dorsal, and Rel B lack an intron 5' to the coding region. If the ancestral gene contained an intron demarcating the N-terminal coding region in these genes, this intron must have been lost before the formation of Rel B, Dorsal, Dif, and Relish, because the other vertebrate genes all have retained this intron. Alternatively, if the ancestral gene lacked an intron demarcating the N-terminal coding region of the rel domain, it must have been inserted after the Relb, dif, dorsal, and relish genes had originated from the ancestral gene. By either scenario, Rel B is the closest vertebrate relative of Dorsal, Dif, and Relish. Introns could not have been randomly lost or inserted, because a number of studies have shown that their positions are highly conserved within gene families. The sequence encoding the C terminus of the rel domain is also bounded by introns for each of the proteins except Relish. Indeed, the conserved proline codon at the C terminus of all rel domains occurs within five amino acid codons of the C-terminal intronic insertion (Graef, 2001).

The most distinctive structural feature of the rel domain is the division of the dimerization and specificity domains. Remarkably, in all vertebrate rel domain-containing genes, an intron precisely separates the sequences encoding the dimerization and the DNA specificity domains within the rel domain. Again, the exceptions are informative, in that no insect gene other than Drosophila TonEBP has this intron insertion site between the recognition and dimerization domains. One possible explanation is that the ancestral gene contained an intron at this position that was lost. However, several lines of evidence bode against intron loss, particularly because there is no evidence of processing and reinsertion of the insect rel domains. A more likely scenario is that the ancestral gene gained an intron separating the sequences encoding the dimerization and specificity domains, which then allowed the rel domain to successfully recombine and disseminate in vertebrates (Graef, 2001).

In the p100, p105, and Relish proteins, a cytoplasmic retention domain is a distinct region in each protein and is characterized by the presence of ankyrin repeats. This region is processed and eventually degraded to allow translocation to the nucleus. This cis-acting cytoplasmic retention function in Relish is encoded by a single exon, which in vertebrate p105 is divided into 13 different exons and a large but as yet undetermined number of exons in the p100 gene. Cytoplasmic retention can also be provided by the cactus or IkB proteins, which have sequence similarity to p105, p100, and Relish outside the rel domain (Graef, 2001).

A rel domain related to the one found in the NFATc proteins was recently reported in the mammalian TonEBP or NFAT5. This protein is encoded by a single mammalian gene and is transcriptionally regulated by osmotic stress. A gene related to mammalian TonEBP was found in Drosophila (Misexpression Suppressor of Ras 1). This gene also has a large exon 5' to the coding sequence for the rel domain, but the protein contains neither the ankyrin repeats of the p105/Relish proteins nor the translocation domain of the NFATc family. The Drosophila protein shares some features of the human TonEBP protein outside the rel domain, including the glutamine-rich regions. The mammalian gene has been partially sequenced and found to encode a rel domain with its sequence divided by introns at sites that correspond to those present in the NFATc genes. However, outside of the rel domain, the genomic structure of TonEBP is unrelated to NFATc family members. Most definitively, TonEBP lacks the translocation exon, indicating that it is not functionally related to the NFATc proteins (Graef, 2001).

The NF-kappaB family members p65 (RelA) and c-Rel recognize similar DNA sequences, yet the phenotypes of mutant mice suggest that these proteins regulate distinct sets of genes. This study demonstrates that 46 unique residues within an 86-residue segment of the Rel homology region (RHR) of c-Rel are responsible for the c-Rel requirement for Il12b gene induction by lipopolysaccharide in bone marrow-derived macrophages. These same residues are responsible for the c-Rel requirement for Il12a induction in dendritic cells, and in both instances, no evidence of c-Rel-specific coactivator interactions was found. Although the residues of c-Rel and p65 that contact specific bases and the DNA backbone within nuclear factor-kappaB (NF-kappaB) recognition sequences are identical, homodimers of c-Rel and of a chimeric p65 protein containing the critical c-Rel residues bind with high affinity to a broader range of NF-kappaB recognition sequences than did wild-type p65 homodimers. These results demonstrate that the unique functions of closely related transcription factor family members can be dictated by differences in the range of DNA sequences recognized at high affinity, despite having similar binding site consensus sequences and DNA contact residues (Sanjabi, 2005).

Invertebrate Dorsal homologs and vertebrate Rel: Roles in early development

In the long-germ insect Drosophila melanogaster, dorsoventral polarity is induced by localized Toll-receptor activation which leads to the formation of a nuclear gradient of the rel/NF-kappaB protein Dorsal. Peak levels of nuclear Dorsal are found in a ventral stripe spanning the entire length of the blastoderm embryo allowing all segments and their dorsoventral subdivisions to be synchronously specified before gastrulation. A nuclear Dorsal protein gradient of similar anteroposterior extension exists in the short-germ beetle, Tribolium castaneum, which forms most segments from a posterior growth zone after gastrulation. Tc-dl mRNA accumulates in the oocytes of mid and late stage egg chambers suggesting that Tc-dl, like Drosophila DL mRNA is maternally provided. In contrast to Drosophila, (1) nuclear accumulation is first uniform and then becomes progressively restricted to a narrow ventral stripe; (2) gradient refinement is accompanied by changes in the zygotic expression of the Tribolium Toll-receptor suggesting feedback regulation and, (3) the gradient only transiently overlaps with the expression of a potential target, the Tribolium twist homolog, and does not repress Tribolium decapentaplegic. The Tribolium Toll homolog however, differs from Drosophila Toll by being transcribed only zygotically. No nuclear Dorsal is seen in the cells of the growth zone of Tribolium embryos, indicating that here dorsoventral patterning occurs by a different mechanism. However, Dorsal is up-regulated and transiently forms a nuclear gradient in the serosa, a protective extraembryonic cell layer ultimately covering the whole embryo (Chen, 2000).

Although the regulatory behaviour of DV patterning found in perturbation experiments suggests reduced dependence on a maternal prepattern, it is also possible that interactions between zygotic DV patterning genes play an important role. In this context, it is interesting that the Tc-dl not only differs from the Drosophila Dl gradient, but also the relationship of the gradients to their respective target genes and hence to the cell fates along the DV axis appears to be different. This is apparent for both the mesoderm and the ectoderm. In Drosophila, a ventral stripe of high nuclear Dorsal in the trunk region of the blastoderm embryo is congruent with the mesodermal anlagen since it defines the lateral expansion of twi expression which, together with sna, promotes ventral furrow formation. Drosophila Dl remains present in twi- and sna-expressing cells until the mesoderm has invaginated. In Tribolium, in contrast, the early weak Tc-twi expression domain, which is even along the AP axis and coincides with the highest levels of nuclear Tc-dl, is rapidly replaced by a domain with strong AP asymmetry by becoming repressed anteriorly and broadened toward the posterior pole. When this expression pattern is fully developed, nuclear Tc-dl has disappeared from the germ rudiment. However, this final Tc-twi domain corresponds to the presumptive mesoderm since it presages the position and shape of the ventral furrow. This implies that the shape of the gradient does not fully determine the mesodermal anlagen and that Tc-twi transcription becomes independent of activation by Tc-dl at late blastoderm (Chen, 2000).

The connection between Tc-dl and the patterning of the ectoderm is even more indirect. In Drosophila low levels of nuclear Dorsal activate the expression of type II target genes which are required for the formation of neuroectoderm. Simultaneously, Dorsal represses the type III target genes of the Dpp group confining their expression to the dorsal side, which gives rise to the amnioserosa and to the non-neurogenic ectoderm. No fate maps have been constructed for the Tribolium blastoderm embryo so far. However, the serosa cells seem to derive from a broad anterior domain that is slightly tilted to the dorsal side. The serosa is likely to be homologous to the amnioserosa of Drosophila. zen and dpp are expressed in both tissues. While in Drosophila these are dorsal expression domains, in Tribolium the zen and dpp domains initially have the shapes of anterior caps, which are symmetric with regard to the DV axis, even though the Tc-dl gradient has already formed. Only later Tc-zen shifts to the dorsal side, and Tc-dpp shifts to the ventral side. The latter is remarkable since it indicates that a target gene, which is repressed by Dorsal in Drosophila, might be activated by Dorsal in Tribolium. In Drosophila, zen and dpp are also expressed in terminal caps, which are not influenced by Dorsal. However, no function has so far been attributed to these terminal caps since all patterning functions seem to reside in the dorsal expression domains. It is tempting to suggest that the terminal caps are evolutionary remnants of short-germ development, where the extraembryonic tissues are derived from anterior rather than dorsally located regions of the blastoderm (Chen, 2000).

While the region giving rise to the serosa can be located by following the course of embryonic development, this is harder with regard to the subdivisions of the ectoderm. Therefore, it is not yet clear how early Tc-dpp expression relates to the later Tc-dpp domain corresponding to the dorsal ectoderm. There is some indication that the dorsal ectoderm expression is independently initiated in more posterior regions of the germband and is thus not continuous with the early Tc-dl associated domain (Chen, 2000).

In summary, a comparison of spatial and temporal aspects of Tc-dl gradient formation with respect to both the expression pattern of potential target genes and the likely position of the blastoderm anlagen suggests that Dorsal has a less direct role in cell fate determination in Tribolium than it has in Drosophila. It is quite possible that its major role in the germ anlage is the initiation of Tc-twi expression while the patterning of the embryonic ectoderm is a secondary consequence of mesoderm formation initiated by Tc-twi (Chen, 2000).

Mesodermal development is a multistep process in which cells become increasingly specialized to form specific tissue types. In Drosophila and mammals, proper segregation and patterning of the mesoderm involves the bHLH factor Twist. The activity of a Twist-related factor, CeTwist, was investigated during Caenorhabditis elegans mesoderm development. Within the bHLH domain, CeTwist shares 59%-63% identity to published Twist family members in other species. Outside of the bHLH domain, there is no obvious homology between CeTwist and other Twist family members. Embryonic mesoderm in C. elegans derives from a number of distinct founder cells that are specified during the early lineages; in contrast, a single blast cell (M) is responsible for all nongonadal mesoderm formation during postembryonic development. Using immunofluorescence and reporter fusions, the activity pattern of the gene encoding CeTwist was determined. No activity is observed during specification of mesodermal lineages in the early embryo; instead, the gene is active within the M lineage and in a number of mesodermal cells with nonstriated muscle fates. Analysis of sequences involved in CeTwist regulation suggests that a protein of the HOX family might be an upstream component in this regulatory pathway. The homeotic selector factor MAB-5 plays an essential role in the early activation of CeTwist. MAB-5 protein is prominent in the M mesoblast and in descendants of M for at least four cell divisions; MAB-5 expression is maintained in the sex myoblats and their undifferentiated progeny while expression trails off in M-lineage cells that differentiate as striated muscle. MAB-5 activation cannot, however, account for the entire CeTwist activity pattern, and it is proposed that additional mesoderm-specific components (perhaps interacting with an upstream NF-kappaB/Dorsal control element) cooperate with MAB-5 in the initial activation of CeTwist. The 315-bp promoter-proximal element is required for CeTwist promoter activity in undifferentiated cells of the M lineage. Deletion of 21 bp from the 5' end of the promoter-proximal element results in a complete loss of activity. Within this 21-bp region, the promoter-proximal M lineage element contains a single putative NF-kappaB/Dorsal-binding site and a GAGA motif. Targeted mutations in either site abolish the activity of the 315-bp minimal promoter in the entire M lineage. Interestingly, alteration of a DNA sequence 3' of the NF-kappaB/GAGA motif region also result in inactivation of the hlh-8 promoter. The sequences in this region showed high homology to binding sites for the Antennapedia class of homeodomains. The 315-bp promoter-proximal element also contains two putative CeTwist-binding sites. In vivo ectopic CeTwist + CeE/DA coexpression experiments reveal no evidence of CeTwist autoregulation (Harfe, 1998).

Homologs of the Drosophila genes dorsal and snail have been cloned from the glossiphoniid leech Helobdella robusta. Sequences from one dorsal-class gene (Hro-dl) and two snail-class genes (Hro-sna1 and Hro-sna2) were identified. Polyclonal antibodies were raised against the most conserved domains of HRO-DL and HRO-SNA1. Nuclear staining appeared for both proteins in mid-embryogenesis, in mesodermal and ectodermal precursors. During segmentation, segmentally iterated stripes of cells with strong HRO-DL staining appeared. The stripes of HRO-DL staining were first concentrated in the cytoplasm of cells, and later in the nuclei. Around this time, HRO-SNA levels also appeared in nuclei in segmentally iterated stripes. The localization of HRO-DL and HRO-SNA proteins raise the possibility that these genes are part of a conserved genetic pathway that, instead of specifying the dorsoventral axis and the mesoderm as in flies, might play a role in the diversification of cell types within segment primordia during leech development (Goldstein, 2001).

It is unlikely that the HRO-DL and HRO-SNA proteins act in dorso-ventral axis specification, since neither show patterns of localization consistent with this possibility; for example, neither appear to be localized in a pattern expected for a spiralian dorsoventral determinant. It remains possible that they function in mesoderm specification, although since the proteins are expressed in both mesodermal and ectodermal precursors, this seems unlikely. The results suggest that the role of Dorsal-class proteins in specification of the dorsoventral axis and the mesoderm might have been present in the common ancestor to leeches and flies and was lost in the lineage leading to leeches, or it might not have been present in the common ancestor and was gained in the lineage leading to flies. Preliminary tests of an innate immunity function in leeches -- determining whether bacterial infection can cause cytoplasmic HRO-DL to become nuclear, as Dorsal does in flies -- have produced negative results. Specific HRO-SNA expression in the nervous system has not been observed. It is concluded therefore that none of the developmental functions known for these genes in flies have been found yet in leeches. Widespread expression appears transiently in leech development, in all the micromeres and teloblast derivatives between stage 6b and late stage 8. How these genes might function in these cells, or whether they have a function during this period, remains unclear. The most suggestive parts of the leech expression patterns are the stripes of nuclear staining that arise during segmentation: this pattern raises the possibility that these genes are part of a genetic pathway which, instead of specifying the dorsoventral axis and the mesoderm as in flies, might play a role in the diversification of cell types within segment primordia during leech development. Segmentally iterated stripes of staining have also been seen in Xenopus embryos for the dorsal homolog Xrel1. With the caveat in mind that it is not know if or how these striped patterns of Dorsal-class proteins might function in either leech or Xenopus embryos, these results at least raise the possibility that this may be a feature of a Dorsal pattern that was present in the ancient urbilaterians but was lost in the lineage leading to flies. Alternatively, Dorsal-class proteins might not have been expressed in iterated stripes in the common ancestor of these organisms, and might have independently acquired at least superficially similar patterns in the lineages leading to the leeches and frogs. Snail-class genes have been proposed to have an ancestral function in cell migration; whether HRO-DL activates snail in a segmentally iterated population of cells which will later migrate remains to be determined (Goldstein, 2001).

The Xenopus homolog (XrelA) of NF-kappaB and Dorsal is expressed in oocytes and early embryos. The mRNA is relatively evenly expressed over the whole embryo during the early embryonic stages, but the protein becomes localized to the nuclei of animal and equatorial cells at the midblastula stages. Wild type XrelA has a role in dorsoventral patterning. XrelA overexpression in the dorsal side of embryos reduces dorsal development and attenuates in vitro dorsal morphogenetic movements. XrelA alters normal dorsoanterior patterning by altering gastrulation movements, specifically delaying blastopore lip formation, attenuating convergent extension, and reducing notochord formation. Overexpression in dorsal regions of Xenopus glycogen synthase kinase-3ß, the homolog of Drosophila shaggy/zeste white 3, reduces dorsoanterior development in a dominant manner, while overexpression of dominant negative Xgsk-3ß mutants in ventral regions induces a complete secondary axis. XrelA strongly reduces axis duplication caused by overexpression of a dominant negative mutant of Xgsk-3ß (Kao, 1996).

Since there is a strong similarity between the NF-kappaB/Rel family members and the Drosophila protein DORSAL, the pattern of NF-kappaB activity was studied during mouse development. Two lacZ reporter constructs, each driven by promoter elements that are dependent on the presence of nuclear NF-kappaB/Rel activity, were used to produce transgenic mice. The analysis of these mice does not identify nuclear NF-kappaB/Rel activity in early development prior to implantation or during the gastrulation processes. Earliest expression of the lacZ transgene is detected on day E12.5. Before birth lacZ expression is seen in discrete regions of the rhombencephalon of the developing brain, in the spinal medulla, in some of the blood vessels and in the thymus. After birth, the NF-kappaB/Rel activity in the thymus remains but nuclear activity is also found in the bone marrow, in the spleen and in the capsule of the lymph nodes. In the central nervous system, drastic changes in NF-kappaB/Rel activity occur in the first 3 weeks after birth, when the cortex and the cerebellum reach functional and morphological maturity. Considering the results of the p50, p65, relB and c-rel knock-out mice and these present findings, it is thought that the NF-kappaB/Rel proteins known so far are probably not implicated in processes of early development and differentiation of the different tissues, but rather in maintaining their function once matured (Schmidt-Ullrich, (1996).

The Rel/NF-kappaB gene family encodes a large group of transcriptional activators involved in myriad differentiation events, including embryonic development. Xrel3, a Xenopus Rel/NF-kappaB-related gene, is expressed in the forebrain, dorsal aspect of the mid- and hind-brain, the otocysts and notochord of neurula and larval stage embryos. Overexpression of Xrel3 causes formation of embryonic tumors. Xrel3-induced tumors and animal caps from embryos injected with Xrel3 RNA express Otx2, Shh and Gli1. Heterodimerization of a C-terminally deleted mutant of Xrel3 with wild-type Xrel3 inhibits in vitro binding of wild-type Xrel3 to Rel/NF-kappaB consensus DNA sequences. This dominant interference mutant disrupts Shh, Gli1 and Otx2 mRNA patterning and inhibits anterior development when expressed in the dorsal side of zygotes: anterior development is rescued by co-injecting wild-type Xrel3 mRNA. In chick development, Rel activates Shh signaling, which is required for normal limb formation -- Shh, Gli1 and Otx2 encode important neural patterning elements in vertebrates. The activation of these genes in tumors by Xrel3 overexpression and the inhibition of their expression and head development by a dominant interference mutant of Xrel3 indicates that Rel/NF-kappaB is required for activation of these genes and for anterior neural patterning in Xenopus (Lake, 2001).

The rel/NF-kappaB transcription factor Dorsal controls dorsoventral (DV) axis formation in Drosophila. A stable nuclear gradient of Dorsal directly regulates ~50 target genes. In Tribolium castaneum (Tc), a beetle with an ancestral type of embryogenesis, the Dorsal nuclear gradient is not stable, but rapidly shrinks and disappears. Negative feedback accounts for this dynamic behavior: Tc-Dorsal and one of its target genes activate transcription of the IkB homolog Tc-cactus, terminating Dorsal function. Despite its transient role, Tc-Dorsal is strictly required to initiate DV polarity, as in Drosophila. However, unlike in Drosophila, embryos lacking Tc-Dorsal display a periodic pattern of DV cell fates along the AP axis, indicating that a self-organizing ectodermal patterning system operates independently of mesoderm or maternal DV polarity cues. The results also elucidate how extraembryonic tissues are organized in short-germ embryos, and how patterning information is transmitted from the early embryo to the growth zone (da Fonsaca, 2008).

Tc-Toll transcription appears to start evenly along the DV axis at the syncytial blastoderm stage but is rapidly enhanced at the ventral side, where higher levels of nuclear Tc-Dorsal accumulate. This positive feedback between Toll expression and nuclear import of Dorsal could explain an initiation of DV axis formation at ectopic positions of the embryonic blastoderm, a situation which has been observed upon experimental manipulations in beetles and various hemimetabolous insects. During normal development, however, ectopic axis formation has to be prevented, and this can be achieved by coupling positive feedback control to inhibitory processes. Linking self-enhancement to limiting mechanisms provides a general condition for pattern formation as has been shown by mathematical modeling. The Tc-Dorsal-dependent transcriptional activation of Tc-cact might provide the mechanism counterbalancing the positive feedback between Tc-Toll and Tc-Dorsal (da Fonsaca, 2008).

Within the limits of detection, Tc-cact expression appears to be restricted to the ventral side of the embryo. However, the knockdown of Tc-cact leads to nuclear import of Tc-Dorsal also at the dorsal side. To explain this long-range requirement of Tc-cact, one might speculate that detection of Tc-cact transcripts is not sensitive enough or that Tc-Cact protein is able to diffuse within the cytoplasm from ventral toward dorsal. Irrespective of the mechanism, a long-range action of Tc-Cact would meet an important prediction for pattern formation by reaction-diffusion systems, namely that the inhibitor should spread faster and thus act less locally than the activator (da Fonsaca, 2008).

Besides its potential role in pattern formation, Tc-cact activation seems also to be involved in the temporal control of the Dorsal gradient. During late blastoderm stages Tc-cact activation by Tc-dorsal is replaced through activation by Tc-twi. The Tc-twi knockdown phenotype shows that this shift is relevant to prevent Dorsal from accumulating in ventral nuclei during gastrulation. Thus, it seems that in Tribolium a Dorsal target gene is involved in terminating Dorsal function (da Fonsaca, 2008).

Collectively, these observations indicate that major evolutionary changes have occurred regarding Tc-Dorsal gradient formation and the network of downstream target genes. Nevertheless, traces of the feedback mechanisms uncovered in Tribolium have been preserved in the Drosophila lineage. Recently, zygotic enhancers of Dm-cactus and Dm-Toll were identified by ChIP-on-chip experiments and bioinformatics approaches. These enhancers contain Dm-Dorsal and Dm-twist binding sites and are active in the prospective mesoderm of Drosophila. However, the analysis of mutant phenotypes precludes an important function of these enhancers in DV patterning or cell type specification. A weak stabilizing function may explain why they were retained in evolution (da Fonsaca, 2008).

On an even larger evolutionary scale it is interesting to note that negative feedback control is a hallmark of NF-κB-mediated signaling. Like in Tribolium, the transcription of the Cactus homolog I-κB is activated by NF-κB in vertebrates both in the mesoderm and during innate immune response. The ensuing negative feedback loop can cause oscillatory signaling outputs or termination of signaling. Even an involvement of twist in negative feedback regulation of NF-κB has been demonstrated in vertebrate mesoderm cells. It has been proposed that the twi-NF-κB interactions represent an evolutionarily conserved regulatory module. The Dorsal/NF-κB- and twi-dependent activation of Tc-cact might be a relic of mesodermal and innate immune functions the pathway had in the common ancestor of vertebrates and arthropods. According to this scenario, the ancestral feedback mechanisms were adjusted to the needs of spatial patterning after the pathway was adopted for DV axis formation (da Fonsaca, 2008).

Classical fragmentation experiments have suggested two routes for pattern regulation along the DV axis: an early route which takes place before gastrulation and a later one which can be initiated after mesoderm internalization. Evidence has been provided for late autonomous patterning within the ectoderm that depends on the Dpp/Sog system and additional inhibitory processes. Tc-Toll knockdown embryos add additional support for this assumption. They show pattern duplications of ectodermal DV cell fates along the AP axis. This remarkable phenotype is not just restricted to the abdominal segments derived from the growth zone, but it occurs also within the anterior (thoracic) segments. Thus, it is unlikely to reflect a specific mechanism that operates only in the growth zone (da Fonsaca, 2008).

The modulation of Dpp activity underlying the periodic cell fate changes is likely to be due to periodic transcription of Tc-dpp and inhibition of Tc-Dpp diffusion or signaling along the AP axis. Since Tc-sog is not re-expressed in Tc-Toll1 RNAi embryos, the expression of other Dpp inhibitors was analyzed. Tc-bambi showed periodic expression in the same domains as Tc-dpp and thus might provide the inhibitory function. The fact that Tc-dpp is transcribed in regions of high pMAD activity suggests positive feedback control which is counterbalanced by Tc-bambi. Thus, the interaction might be similar to that described for Tc-Toll and Tc-cact (da Fonsaca, 2008).

The unusual orientation of the ectodermal patterning process might depend on the early AP asymmetry of Dpp signaling in Tc-Toll1 RNAi embryos. After Tc-Toll1 RNAi, Tc-dpp is expressed along the symmetric border between serosa and germ rudiment and in the posterior pit region (data not shown). These regions also have high levels of pMAD. Thus, the ectodermal patterning process is initiated with AP asymmetric boundary conditions after Tc-Toll RNAi. In WT embryos this process is oriented along the DV axis through the Toll-dependent activation of Tc-sog at the ventral side, which leads to a Dpp signaling gradient with peak levels along the dorsal midline (da Fonsaca, 2008).

Experiments clearly demonstrate that Tc-Dorsal is essential for establishing all aspects of normal DV polarity in Tribolium, including DV polarity of the growth zone from which the abdominal segments emerge. Thus, although DV patterning in the growth zone starts after gastrulation, when the Tc-Dorsal gradient has vanished, it is not independent of Tc-Dorsal. It is suggested that there are two ways by which early DV polarity is transmitted to the growth zone. First, distinct inner and outer cell layers are formed during gastrulation. The observation that the majority of the mesenchymal layer cells are absent in Tc-Toll RNAi embryos strongly suggests that the mesenchymal cells in the growth-zone are derived from cells internalized by ventral furrow formation in the early embryo. These cells cannot be resupplied by a growth zone-specific process of cell internalization. Thus, gastrulation-like mechanisms do not continue in the growth zone of Tribolium, as has been suggested for the tail-bud of vertebrate embryos. Second, DV patterning in the growth zone does not only depend on the generation of two separate cell layers. The ectoderm needs also to be patterned, a process which mainly depends on Dpp signaling. To a certain degree this process takes place in a Toll knockdown embryo. However, the orientation of the resulting pattern is incorrect. It is assumed that during WT development DV polarity is first established within the anterior (gnathal and thoracic) segments. Subsequently, this pattern is used as a template for the DV pattern of the abdominal segments emerging from the growth zone. This would require a process of forward-induction from differentiated to nondifferentiated tissues. Since there is no DV polarity in Tc-Toll RNAi early embryos, forward-induction cannot operate (da Fonsaca, 2008).

The loss of Toll signaling in Tribolium leads to phenotypes that are similar to those produced by the loss of the Dpp inhibitor sog. In both situations the ectoderm lacks normal polarity, the amnion and the CNS are largely deleted (the CNS is completely absent after Tc-sog RNAi and reduced to narrow periodic stripes after Tc-Toll RNAi), and the embryos form long tube-like structures. This situation is strikingly different in Drosophila. There, loss of Toll signaling leads to completely dorsalized embryos, while loss of sog causes only minor deletions in the CNS and subtle ectodermal patterning defects. These differences are due to the fact that Toll signaling in Drosophila provides functions which the Dpp/Sog system fulfils in Tribolium. For example, in Drosophila Dorsal represses dpp and activates brinker, an inhibitor of Dpp target genes, within the presumptive neuroectoderm and thereby specifies the CNS through mechanisms which act independently from and parallel to sog. These mechanisms do not exist in Tribolium. Apparently, the Dorsal gradient has a less direct role with regard to cell-type specification in Tribolium than in Drosophila, and DV patterning in Tribolium relies to higher degree on the Dpp/Sog system. Since the Dpp/Sog (BMP/Chordin) system is involved in DV axis formation in all bilaterian animals investigated so far, this is likely to represent the ancestral mode of DV axis formation. It is suggested that the trend observed by comparing Drosophila and Tribolium applies to other insect orders and that the functional shift between Dpp and Toll signaling with regard to DV axis formation will be even more prominent in basal hemimetabolous insects. Thus, the study of more basal insects groups might reveal the evolutionary path of how Toll signaling was co-opted for DV axis formation (da Fonsaca, 2008).

Components of the dorsal-ventral pathway also contribute to anterior-posterior patterning in honeybee embryos (Apis mellifera)

A key early step in embryogenesis is the establishment of the major body axes; the dorsal-ventral (DV) and anterior-posterior (AP) axes. Determination of these axes in some insects requires the function of different sets of signalling pathways for each axis. Patterning across the DV axis requires interaction between the Toll and Dpp/TGF-beta pathways, whereas patterning across the AP axis requires gradients of Bicoid/Orthodenticle proteins and the actions of a hierarchy of gene transcription factors. This study examined the expression and function of Toll and Dpp signalling during honeybee embryogenesis to assess to the role of these genes in DV patterning. Pathway components that are required for dorsal specification in Drosophila are expressed in an AP-restricted pattern in the honeybee embryo, including Dpp and its receptor Tkv. Components of the Toll pathway are expressed in a more conserved pattern along the ventral axis of the embryo. Late-stage embryos from RNA interference (RNAi) knockdown of Toll and Dpp pathways had both DV and AP patterning defects, confirmed by staining with Am-sna, Am-zen, Am-eve, and Am-twi at earlier stages. Two orthologues of dorsal were observed in the honeybee genome, with one being expressed during embryogenesis and having a minor role in axis patterning, as determined by RNAi and the other expressed during oogenesis. This study has found that early acting pathways (Toll and Dpp) are involved not only in DV patterning but also AP patterning in honeybee embryogenesis. Changes to the expression patterns and function of these genes may reflect evolutionary changes in the placement of the extra-embryonic membranes during embryogenesis with respect to the AP and DV axes (Wilson, 2014).

Co-expression of Dorsal and Rel2 negatively regulates antimicrobial peptide expression in the tobacco hornworm Manduca sexta

Nuclear factor κB (NF-κB) plays an essential role in regulation of innate immunity. In mammals, NF-κB factors can form homodimers and heterodimers to activate gene expression. In insects, three NF-κB factors, Dorsal, Dif and Relish, have been identified to activate antimicrobial peptide (AMP) gene expression. However, it is not clear whether Dorsal (or Dif) and Relish can form heterodimers. This study reports the identification and functional analysis of a Dorsal homologue (MsDorsal) and two Relish short isoforms (MsRel2A and MsRel2B) from the tobacco hornworm, Manduca sexta. Both MsRel2A and MsRel2B contain only a Rel homology domain (RHD) and lack the ankyrin-repeat inhibitory domain. Overexpression of the RHD domains of MsDorsal and MsRel2 in Drosophila S2 and Spodoptera frugiperda Sf9 cells can activate AMP gene promoters from M. sexta and D. melanogaster. This study confirmed the interaction between MsDorsal-RHD and MsRel2-RHD, and suggest that Dorsal and Rel2 may form heterodimers. More importantly, co-expression of MsDorsal-RHD with MsRel2-RHD suppressed activation of several M. sexta AMP gene promoters. These results suggest that the short MsRel2 isoforms may form heterodimers with MsDorsal as a novel mechanism to prevent over-activation of antimicrobial peptides (Zhong, 2016).

Translation of NF-kappaB and proteolysis

The NFkappaB1 gene encodes two functionally distinct proteins termed p50 and p105. p50 corresponds to the N terminus of p105; along with p65 (RelA) it forms the prototypical NF-kappaB transcription factor complex. In contrast, p105 functions as a Rel-specific inhibitor (IKappaB) and has been proposed to be the precursor of p50. p50 is generated by a unique cotranslational processing event (an event occuring during translation) involving the 26S proteasome. Cotranslational folding of sequences near the C terminus of p50 abrogates proteasome processing and leads to p105 production. These results indicate that p105 is not the precursor of p50 and reveal a novel mechanism of gene regulation that ensures the balanced production and independent function of the p50 and p105 proteins. To this point, function of the 26S proteasome has been thought to involve the complete destruction of target proteins. In contrast, cotranslational processing by the proteasome is here shown to yield p50. Additional work will be required to determine whether co-translational processing of proteins by protease-chaperones is a widespread process (Lin, 1998).

The 26S proteasome plays a critical role in the generation of the p50 subunit of the NF-kappaB complex, as demonstrated by both biochemical and genetic experiments. Both p50 and the larger p105 protein are products of the NFKB1 gene. The p50 polypeptide coincides with the N-terminal portion of p105 and spans the ~300- residue Rel homology domain (RHD). The C-terminal portion of the p105 protein contains multiple ankyrin repeats, a hallmark of the IkappaB family of cytoplasmic Rel inhibitors. In this regard, p105 has been shown to function as an IkappaB. p50 is generated principally during translation of the NFKB1 mRNA and p50 and p105 production may be balanced by the transient folding state of the p105 nascent polypeptide. Homeostasis of p105 and p50 appears to be physiologically important since transgenic mice expressing p50 but not p105 exhibit severe inflammation. In contrast, NFKB1 knockout mice lacking expression of both p50 and p105 do not display such inflammatory changes and only manifest minor defects in B cell function. These in vivo results indicate that p105 likely plays an important role in regulating p50 function -- this property of p105 apparently is not compensated for by the other IkappaBs. The cotranslational processing of the NFKB1 gene product leads to a natural balance of these two proteins, and thus ensures their distinct biological functions within the cell. The intracellular ratio of these two proteins can also be regulated by signal-induced kinases that trigger the complete degradation of p105, a process that may facilitate p50 homodimer formation (Lin, 2000 and references therein).

The Rel homology domain in p50 undergoes cotranslational dimerization and this interaction is required for efficient production of p50. This coupling of dimerization and proteasome processing during translation uniquely generates p50-p105 heterodimers. Accordingly, after the primary cotranslational event, additional posttranslational steps regulate p50 homodimer formation and the intracellular ratio of p50 and p105. This cellular strategy places p50 under the control of the p105 inhibitor early in its biogenesis, thereby regulating the pool of p50 homodimers within the cell (Lin, 2000).

Processing of the nf-kappab2 gene product p100 to generate p52 is an important step in NF-kappa B regulation. This step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-kappaB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nf-kappab2 knockout mice exhibit similar immune deficiencies (Xiao, 2001).

The proteasome degrades some proteins, such as transcription factors Cubitus interruptus (Ci) and NF-kappaB, to generate biologically active protein fragments. This study identifies and characterizes the signals in the substrate proteins that cause this processing. The minimum signal consists of a simple sequence preceding a tightly folded domain in the direction of proteasome movement. The strength of the processing signal depends primarily on the complexity of the simple sequence rather than on amino acid identity, the resistance of the folded domain to unraveling by the proteasome and the spacing between the simple sequence and folded domain. Two unrelated transcription factors, Ci and NF-kappaB, use this mechanism to undergo partial degradation by the proteasome in vivo. These findings suggest that the mechanism is conserved evolutionarily and that processing signals may be widespread in regulatory proteins (Tian, 2005).

Proteasomal proteolysis controls the cellular concentrations of hundreds of regulatory proteins. Normally, the proteasome degrades its substrates completely into small peptides by sequentially running along their polypeptide chain and hydrolyzing the peptide bonds approximately every 8 residues. The proteasome can also function as a processing enzyme that produces functional protein fragments from larger precursors by partial degradation. Processing occurs when the proteasome encounters a stop signal during its sequential hydrolysis of a substrate protein. The stop signal consists of two components: a sequence of low compositional complexity followed by a tightly folded domain in the direction of proteasome movement. Glycine-alanine repeat regions in the Epstein-Barr virus nuclear antigen-1 are known to protect the protein from proteasomal degradation. This study finds that many different simple sequences can cause partial degradation, but only in combination with a tightly folded domain and at the appropriate spacing (Tian, 2005).

Partial degradation can modulate the function of regulatory proteins, as shown for p105 and Ci, and provides a simple mechanism for directly switching a signaling pathway from an active state to a repressed state and vice versa. Processing can also produce more subtle changes in activity. The amount of fragment formed during the degradation of a protein depends on the strength of the processing signal. More fragment is formed the less complex the simple sequenceand the more stable the folded domain. For example, the low-complexity region of Ci is not as simple as the glycine-rich region in p105. Therefore, less Ci repressor is formed in the absence of Hh signaling, and a reporter target gene is less tightly repressed than possible if the low-complexity regions of Ci were replaced with the p105 glycine-rich region. The ratio of Ci activator to Ci repressor in turn determines the activity of the Ci target genes, and, therefore, processing efficiency affects the shape of the Ci activity gradient at the distal edge of Hh signaling. The second component of the processing signal is the susceptibility of folded domains to unraveling by the proteasome, which depends on the stability of the local structure first encountered by the proteasome. A very stable domain, such as methotrexate-stabilized dihydrofolate reductase, can lead to fragment formation without a neighboring simple sequence. It may be possible to modulate the strength of a processing signal in the cell by modifying the simple sequence, for example by phosphorylation, or by adjusting the stability of the folded domain, for example by ligand binding (Tian, 2005).

Processing signal function depends on the direction of degradation, because the proteasome has to encounter the simple sequence before the folded domain and because the susceptibilities of folded domains to unraveling from the N or C terminus can differ. This polarity provides a simple mechanism for the degradation of the protein fragments when they are no longer needed. In the eye, the protein-ubiquitin ligase Cul3 targets Ci for complete degradation using unknown ubiquitination sites. It is predicted that Cul3 ubiquitinates in the N-terminal region of Ci, which would lead to its complete degradation by the proteasome unimpaired by the processing signal (Tian, 2005).

Other examples of protein processing by the proteasome probably exist. The NF-kappaB subunit p52 is synthesized as the larger precursor p100, which is homologous to p105 and presumably processed by the same mechanism. Vertebrates have three Ci homologs, Gli1, Gli2 and Gli3, but only the latter two are processed to a smaller form, probably in a proteasome-dependent manner. Consistent with this observation, only Gli2 and Gli3 seem to have a processing signal and Gli1 may not be ubiquitinated. Processing could also exist in proteins unrelated to Ci or NF-kappaB and does not have to be limited to transcription factors. Regions of low compositional complexity are common and found in half of all predicted eukaryotic proteins, but to form a processing signal, a simple sequence must be positioned adjacent to a tightly folded domain at the appropriate spacing. Standard sequence alignments cannot detect the processing signals, because folded domains can be formed by unrelated sequences and the function of the simple sequence does not depend on the identity of the repeated amino acids (Tian, 2005).

The ubiquitin-proteasome system is also involved in the activation of the two membrane-bound yeast transcription factors Spt23 and Mga2 by the release of N-terminal fragments of these proteins from the membrane. Processing requires folded domains that are homologous to the Rel-homology domain of p105, but the proteins do not contain simple sequences at the expected places. Notably, p105 processing in yeast also does not depend on the presence of a simple sequence. However, because Spt23 and Mga2 are membrane anchored and degradation seems to proceed from an internal loop, it is also possible that the processing is more complicated than the simple mechanism described in this study (Tian, 2005).

The biochemical mechanism by which the processing signal causes partial degradation is not known. Prokaryotic ATP-dependent proteases can release their substrate when they reach protein domains that are hard to unfold. In all the substrates described in this study, the ubiquitination sites had been degraded by the time the proteasome reached the folded domain in their substrate. Thus, the protease was associated with its substrate only through the part of the substrate that was about to be degraded, which contained the simple sequence. The spacing requirement between simple sequence and folded domain for processing differs with the direction of proteasome movement, and this disparity may indicate that the proteasome interacts with its substrates differently depending on the direction of degradation. The simple sequences could then lead to processing if they reduced the affinity of the substrate for the proteasome. In apparent agreement with this proposal, simple sequences cannot serve as efficient degradation initiation sites. Once the substrate is released from the proteasome, it has escaped proteasomal degradation because the ubiquitination site has already been removed. Thus, according to this model, protein processing occurs when a tightly folded domain located at the entrance to the degradation channel stalls the progression of the proteasome along the polypeptide chain and a simple sequence in the channel accelerates the release of the folded domain and the remaining protein from the proteasome (Tian, 2005).

In summary, a combination of a simple sequence followed by a folded domain in the direction of proteasome movement inhibits proteasome progression; this results in the accumulation of a partially degraded fragment. Proteasomal processing by partial degradation has an essential function in at least two unrelated cellular signaling pathways. The processing mechanism is conserved between flies and humans, and more examples of this process probably exist (Tian, 2005).

Transcriptional Regulation of NFkappaB

Stimulation of cells with inducers of NF-kappaB (a Rel protein related to Drosophila Dorsal) such as LPS and IL-1 leads to the degradation of IkappaB-alpha and IkappaB-ß proteins (homologs of Drosophila Cactus) and translocation of NF-kappaB to the nucleus. Besides the physical partitioning of inactive NF-kappaB to the cytosol, the transcriptional activity of NF-kappaB is regulated through phosphorylation of NF-kappaB p65 by protein kinase A (PKA). The catalytic subunit of PKA (PKAc) is maintained in an inactive state through association with IkappaB-alpha or IkappaB-ß in an NFkappa-B-IkappaB-PKAc complex. Signals that cause the degradation of IkappaB result in activation of PKAc in a cAMP-independent manner and the subsequent phosphorylation of p65. Therefore, this pathway represents a novel mechanism for the cAMP-independent activation of PKA and the regulation of NF-kappa B activity (Zhong, 1997).

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) selectively bind to distinct members of the Trk family of tyrosine kinase receptors, but all three bind with similar affinities to the neurotrophin receptor p75 (p75NTR). The biological significance of neurotrophin binding to p75NTR in cells that also express Trk receptors has been difficult to ascertain. In the absence of TrkA, NGF binding to p75NGR activates the transcription factor nuclear factor kappa B (NF-kappa B) in rat Schwann cells. This activation is not observed in Schwann cells isolated from mice that lacked p75NTR. The effect was selective for NGF; NF-kappa B was not activated by BDNF or NT-3 (Carter, 1996).

In resting T lymphocytes, the transcription factor NF-kappaB is sequestered in the cytoplasm via interactions with members of the I kappa B family of inhibitors, including IkappaBalpha and IkappaBbeta. During normal T-cell activation, IkappaBalpha is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional NF-kappaB. In contrast to its transient pattern of nuclear induction during an immune response, NF-kappaB is constitutively activated in cells expressing the Tax transforming protein of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that HTLV-1 Tax targets IkappaBalpha to the ubiquitin-proteasome pathway. In addition to acting on IkappaBalpha, Tax stimulates the turnover of IkappaBbeta via a related targeting mechanism. Like IkappaBalpha, Tax-mediated breakdown of IkappaBbeta in transfected T lymphocytes can be blocked either by cell-permeable proteasome inhibitors or by mutation Of IkappaBbeta at two serine residues present within its N-terminal region. Despite the dual specificity of HTLV-1 Tax for IkappaBalpha and IkappaBbeta at the protein level, Tax selectively stimulates NF-kappaB-directed transcription of the IkappaBalpha gene. Consequently, IkappaBbeta protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with IkappaBbeta provide a potential mechanism for the constitutive activation of NF-kappaB in Tax-expressing cells (McKinsey, 1996).

The Rho family of small GTPases includes critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus. Other family members are the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. A novel signaling pathway is activated by the Rho proteins; this pathway may be responsible for biological activities carried out by Rho proteins, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor KB (NF-KB) by a mechanism that involves phosphorylation of IKappaBalpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, independent of the involvement of Ras GTPase and the Raf-1 kinase. Activation of NF-KB by TNFalpha depends on CDC42 and RhoA because this activity is drastically inhibited by CDC42 and RhoA dominant-negative mutants. In contrast, activation of NF-KB by UV light is not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-KB-dependent transcription (Perona, 1997).

Tumor Necrosis Factor (TNF) is one of the most potent physiological inducers of the nuclear transcription factor NF-kappa B. In light of the pivotal role of NF-kappa B in the development of immune responses and activation of HIV replication, the identification of TNF signal transduction pathways involved in NF-kappa B activation is of particular interest. The TNF signal transduction pathway-mediating NF-kappa B activation involves two phospholipases, a phosphatidylcholine-specific phospholipase C (PC-PLC) and an endosomal acidic sphingomyelinase (aSMase). The aSMase activation by TNF is secondary to the generation of 1,2-diacylglycerol (DAG) produced by a TNF-responsive PC-PLC. SMase and its product ceramide induce degradation of the NF-kappa B inhibitor I kappa B as well as NF-kappa B activation. Besides endosomal acidic SMase, TNF also rapidly activates a plasmamembrane-associated neural SMase (nSMase), that, however is not involved in TNF-induced NF-kappa B activation. NSMase and aSMase are activated by different cytoplasmic domains of the 55 kDa TNF-receptor and are coupled to select pathways of TNF signaling. Ceramide generated by nSMase directs the activation of proline-directed serine/threonine protein kinases and phospholipase A2 and ceramide produced by aSMase triggers the activation of NF-kappa B. No apparent crosstalk was detected between nSMase and aSMase pathways, indicating that ceramide action depends on the topology of its production (Schutze, 1995).

Tumor necrosis factor alpha (TNF-alpha) and gamma interferon (IFN-gamma) are required for an effective immune response to bacterial infection. These cytokines synergize in a variety of biological responses, including the induction of cytokine, cell adhesion, and inducible nitrous oxide synthase gene expression. Typically, the synergistic effect on gene expression is due to the independent activation of nuclear factor kappaB (NF-kappaB) by TNF-alpha and of signal transducers and activators of transcription or IFN-regulatory factor 1 by IFNs, allowing these transcription factors to bind their unique promoter sites. However, since activation of NF-kappaB by TNF-alpha is often transient and would not activate long-term kappaB-dependent transcription effectively, the effects of IFN-gamma on TNF-alpha-induced NF-kappaB activity were explored. IFN-gamma, which typically does not activate NF-kappaB, synergistically enhances TNF-alpha-induced NF-kappaB nuclear translocation via a mechanism that involves the induced degradation of I kappaBbeta and that apparently requires tyrosine kinase activity in preneuronal cells but not in endothelial cells. Correspondingly, cotreatment of cells with TNF-alpha and IFN-gamma leads to persistent activation of NF-kappaB and to potent activation of kappaB-dependent gene expression, which may explain, at least in part, the synergy observed between these cytokines, as well as their involvement in the generation of an effective immune response (Cheshire, 1997).

Activation of the transcription factor NF-kappaB is a paradigm for signal transduction through the ubiquitin-proteasome pathway: ubiquitin-dependent degradation of the transcriptional inhibitor IkappaB in response to cell stimulation. A major issue in this context is the nature of the recognition signal and the targeting enzyme involved in the proteolytic process. Following a stimulus-dependent phosphorylation, and while associated with NF-kappaB, IkappaB is targeted by a specific ubiquitin-ligase via direct recognition of the signal-dependent phosphorylation site: phosphopeptides corresponding to this site specifically inhibit ubiquitin conjugation of IkappaB and its subsequent degradation. The ligase recognition signal is functionally conserved between IkappaBalpha and IkappaBbeta, and does not involve the nearby ubiquitination site. Microinjection of the inhibitory peptides into stimulated cells abolish NF-kappaB activation in response to TNFalpha and the consequent expression of E-selectin, an NF-kappaB-dependent cell-adhesion molecule. Inhibition of NF-kappaB function by specific blocking of ubiquitin ligase activity provides a novel approach for intervening in cellular processes via regulation of unique proteolytic events (Yaron, 1997).

Specific cyclin-dependent kinases are found to regulate transcriptional activation by NF-kappaB transcription factor through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65), a subunit of NF-kappaB, interacts with an amino-terminal region of p300 that is distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex (See Drosophila cyclin E). The CDK inhibitor p21 or a dominant negative Cdk2, which inhibits p300-associated cyclin E-Cdk2 activity, stimulates NF-kappaB-dependent gene expression, which is also enhanced by expression of p300 in the presence of p21 (See Drosophila Dacapo). The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression (Perkins, 1997).

Nitric oxide inhibits the activation of transcription by NFkappaB, a transcription factor implicated in regulation of immunologic NOS (iNOS) (See Drosophila Nitric oxide synthase). Neuronal NOS (nNOS) is the predominant isoform constitutively expressed in glia. NO derived from nNOS in glia inhibits transcriptional activation by the transcription factor NFkappaB, as evidenced by the fact that NOS inhibitors enhance transcriptional activation by NFkappaB. In astrocytes, an NO scavenger dramatically induces the NFkappaB-dependent enzyme iNOS, supporting the physiologic relevence of endogenous NO regulation of NFkappaB. These data suggest that nNOS-generated NO in astrocytes regulates NFkappaB activity and consequently iNOS expression, and indicate a novel regulatory role for nNOS in tonically suppressing central nervous system, NFkappaB-regulated genes (Togashi, 1997).

Transforming growth factor beta (TGF-beta) is the prototype of a large superfamily of signaling molecules involved in the regulation of cell growth and differentiation. In certain patients infected with human immunodeficiency virus type 1 (HIV-1), increased levels of TGF-beta promote the production of virus and also impair the host immune system. In an effort to understand the signaling events linking TGF-beta action and HIV production, evidence is provided that TGF-beta can stimulate transcription from the HIV-1 long terminal repeat (LTR) promoter through NF-kappaB binding sites in both HaCaT and 300.19 pre-B cells. When introduced into a minimal promoter, NF-kappaB binding sites support nearly 30-fold activation from the luciferase reporter upon TGF-beta treatment. Electrophoretic mobility shift assay indicates that in HaCaT cells, p50-p65 heterodimeric NF-kappaB is a major factor, binding to the NF-kappaB site. Coexpression of Gal4-p65 chimeric proteins supports TGF-beta ligand-dependent gene expression from a luciferase reporter gene driven by Gal4 DNA binding sites. NF-kappaB activity present in HaCaT cells is not affected by TGF-beta treatment as judged by the unchanged DNA binding activity and concentrations of p50 and p65 proteins. Consistently, steady-state levels of IkappaB alpha and IkappaB beta proteins are not changed by TGF-beta treatment. These results demonstrate that TGF-beta is able to stimulate transcription from the HIV-1 LTR promoter by activating NF-kappaB through a mechanism distinct from the classic NF-kappaB activation mechanism involving the degradation of IkappaB proteins (Li, 1998).

NF-kappaB2 (p100/p52), a member of the NF-kappaB/Rel family of transcription factors, is involved in the regulation of a variety of genes important for immune function. The NF-kappaB2 gene is regulated both postively and negatively. Two kappaB elements within the NF-kappaB2 promoter mediate tumor necrosis factor alpha-inducible transactivation. In addition, there exists a transcriptional repression in the absence of NF-kappaB. To identify a DNA binding activity responsible for this transcriptional repression, a nuclear complex, named Rep-kappaB has been partially purified. Detailed examination of Rep-kappaB-DNA interaction reveals the sequence requirements for binding are almost identical to those of recombination signal binding protein Jkappa (RBP-Jkappa), the mammalian homolog of the protein encoded by Drosophila suppressor of hairless [Su(H)]. In electromobility shift assays, Rep-kappaB binding activity is recognized by an antibody directed against RBP-Jkappa. Human RBP-Jkappa represses basal as well as RelA (p65)-stimulated NF-kappaB2 promoter activity. Studies in Drosophila melanogaster have shown that Su(H) is implicated in the Notch signaling pathway, which regulate cell fate decisions. In transient-transfection assays, it has been shown that truncated Notch-1 strongly induces NF-kappaB2 promoter activity. In summary, these data clearly demonstrate that Rep-kappaB is closely related or identical to RBP-Jkappa. RBP-Jkappa is a strong transcriptional repressor of NF-kappaB2. This repression can be overcome by activated Notch-1, suggesting that NF-kappaB2 is a novel putative Notch target gene (Oswald, 1998).

Mutation of NF-kappaB

NF-kappaB inhibition promotes epidermal tumorigenesis; however, whether this reflects an underlying role in homeostasis or a special case confined to neoplasia is unknown. Embryonic lethality of mice lacking NF-kappaB RelA has hindered efforts to address this. Therefore developmentally mature RelA-/- skin was generated. RelA-/- epidermis displays hyperplasia without abnormal differentiation, inflammation, or apoptosis. Hyperproliferation is TNFR1-dependent because Tnfr1 deletion normalizes cell division. TNFR1-dependent JNK activation occurs in RelA-/- epidermis, and JNK inhibition abolishes hyperproliferation due to RelA deficiency. Thus, RelA antagonizes TNFR1-JNK proliferative signals in epidermis and plays a nonredundant role in restraining epidermal growth (Zhang, 2004).

Table of contents

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

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