NF-kappaB and the innate immune response.

In the innate immune reaction, microbial pathogens activate phylogenetically conserved cellular signal transduction pathways that regulate the ubiquitous nuclear factor-kappaB (NFkappaB). NF-kappaB has pleiotropic functions in immunity; however, it is also critical for development and cellular survival. Many aspects of how the different pathways utilize a common kinase complex that ultimately activates NF-kappaB have been clarified by gene inactivation and biochemical analysis.

Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha.

p105 (NFKB1) acts in a dual way as a cytoplasmic IkappaB molecule and as the source of the NF-kappaB p50 subunit upon processing. p105 can form various heterodimers with other NF-kappaB subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IkappaB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKKbeta phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSGPsi motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IkappaBalpha, -beta, and -epsilon. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules betaTrCP1 and betaTrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKKgamma, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing.

NF-kappa B precursor p100 inhibits nuclear translocation and DNA binding of NF-kappa B/rel-factors.

The NF-kappa B precursor p100 (lyt-10, p97, p98) generates after proteolytic processing a 52 kDa subunit, which can bind to kappa B-motifs. A deregulated form of the p100 gene, which is structurally altered by a t(10;14) translocation, has a potential oncogenic role in certain human B cell lymphomas. In this study p100 was analysed for its ability to interact with its own processing product p52, with p50, the product of the NF-kappa B precursor p105, and with other NF-kappa B/rel-proteins. As demonstrated by a combination of Western blot analysis, band shift analysis and indirect immunofluorescence labelling of transfected cells, p100 itself was localized in the cytoplasm and indiscriminately retained each co-expressed NF-kappa B subunit. Thereby it simultaneously inhibited their DNA binding activities. Thus, a major function of p100 is, like p105, to associate with subunits of the rel multigene family in the cytoplasm in an I kappa B-like fashion. The similarity between p100 and p105 is also reflected by equivalent protein interactions of their processing products: like NF-kappa B-p50, also NF-kappa B-p52 heteromerised promiscuously with all rel-factors tested. Moreover, p52 efficiently interacts with the candidate oncogene product Bcl-3 and also binds to the basic-leucine zipper protein NF-IL6.

Common structural constituents confer I kappa B activity to NF-kappa B p105 and I kappa B/MAD-3.

The vertebrate NF-kappa B/c-rel inhibitors MAD-3/I kappa B alpha, I kappa B gamma/pdI and bcl-3 all share a conserved ankyrin repeat domain (ARD) consisting of six complete repeats, a short acidic motif and/or an incomplete seventh repeat. We present here a detailed analysis of the domain in p105/pdI and MAD-3/I kappa B involved in inhibition of DNA binding and in protein interaction with rel factors. We demonstrate that in both cases an acidic region and six ankyrin-like repeats are sufficient and required for protein interaction with the rel factors. However, for p105/pdI to achieve the high affinity needed to suppress DNA binding, an incomplete seventh repeat is required in addition. Both pdI and MAD-3 associate with rel proteins by forming heterotrimeric complexes, as shown by native gel analysis and by cross-linking. Furthermore, we demonstrate that deletion of only three amino acids in the first repeat converts the subunit specificity of the p105 ARD into that of MAD-3/I kappa B. We conclude that functionally the ARD in these molecules has a modular structure, with different subregions determining the specificity for the NF-kappa B subunits p50 and p65.

Interaction and functional interference of C/EBPbeta with octamer factors in immunoglobulin gene transcription.

The ubiquitous transcription factor C/EBPbeta functions as an activator or inhibitor depending on the ratios of three isoforms translated from in-frame AUG. We have identified C/EBP binding sites in both light and heavy chain immunoglobulin (Ig) promoters. Of the two C/EBP sites present in the light chain promoter, the upstream site is essential for promoter function. Mutation of this element drastically decreases promoter activity, despite the presence of an intact octamer element. Both light and heavy chain promoters were activated or inhibited by C/EBPbeta isoforms in transfected cells according to the transactivation ability of these isoforms. Endogenous IgM mRNA and protein were repressed by the inhibitory form, C/EBPbeta-3, indicating a general role of C/EBPbeta in the regulation of Ig genes. We show that C/EBPbeta-3 forms ternary complexes with Oct-1 and Oct-2 on heavy and light chain promoters, and also interacts with both octamer-binding proteins in the absence of DNA. This suggests that interference of Oct-1/Oct-2 function by C/EBPbeta-3 may account for the observed repression. Inhibition by C/EBPbeta-3 occurs not only through a C/EBP site, but also through the octamer element, as shown by co-transfection experiments with heterologous promoter constructs. Thus, C/EBPbeta regulates Ig promoter transcription by modulating octamer factor activity.

Effects of growth and insulin treatment on the levels of insulin receptors and their mRNA in Hep G2 cells.

We have studied the variations in the number of insulin receptor and insulin receptor mRNA levels in (Hep G2) cells in response to growth and insulin treatment. The levels of insulin receptors are relatively low in growing cells. After approximately 5 days in culture, if cells are not refed they cease to divide and the number of receptors/cell increases, reaching 4 times the initial values by the 9th day. Refeeding the cells completely prevented both growth arrest and the increase in insulin receptor number. Insulin added daily to cells at 0.33 microM caused receptor down-regulation but did not prevent a 3-fold increase in binding with growth arrest. Pulse-chase studies of metabolically labeled ([35S]methionine) cells showed that the receptor degradation rate (apparent t 1/2, 18-20 h) was comparable in rapidly growing versus growth-arrested cells. The increased receptor level in non-refed cells is not due to generation of a soluble factor by confluent cells, nor is it caused by depletion of insulin, glucose, or insulin-like growth factor I from the culture medium. The levels of insulin receptor mRNA measured on Northern blots increased in growth-arrested cells in parallel to the increase in receptor number. The mRNA value begins to increase from the 3rd day in culture and by the 9th day reaches a level 6.0 times that on the 3rd day. Chronic insulin-induced receptor down-regulation did not alter insulin receptor mRNA levels at any time point studied. These data demonstrate that the increase in insulin receptor number/cell in growth-arrested cells is paralleled by an increase in insulin receptor mRNA content with no change in the receptor degradation rates. This suggests that the increase in the number of insulin receptors is due to enhanced receptor synthesis due to increased receptor mRNA content. Conversely, down-regulation of the insulin receptor does not affect the level of insulin receptor mRNA and thus must be due to increased receptor degradation.

Efficient transcription of an immunoglobulin kappa promoter requires specific sequence elements overlapping with and downstream of the transcriptional start site.

The expression of immunoglobulin (Ig) genes depends on tissue-specific elements in the promoter and enhancer regions of light chain and heavy chain genes. In contrast to the complex modular character of Ig enhancers, the promoters appear to be simple, depending primarily on a conserved TATA box and octamer elements. We have analyzed the role of proximal sequences for Igkappa promoter function. Igkappa promoter transcription critically depends on initiator-like sequences and on a downstream element located at +24 to +39 relative to the start site. Replacement of these sequences resulted in strong reduction of promoter activity. In vitro, these elements were found to be more effective in extracts of lymphoid than of non-lymphoid origin. Deletion of the downstream and initiation site regions had a comparable effect on promoter activity to obliteration of the TATA box or octamer element. The downstream sequence was bound by two nuclear proteins, identical to the previously identified Ig-specific C5 and C6 complexes. Whereas C5 is found in HeLa cells and in lymphoid cells, C6 is lymphoid specific. Thus, further specific sequences in addition to the previously characterized elements, the octamer and the TATA box, are required for efficient kappa promoter expression in B lymphocytes.

The I kappa B kinase (IKK) complex is tripartite and contains IKK gamma but not IKAP as a regular component.

A critical step in the activation of NF-kappa B is the phosphorylation of I kappa Bs by the I kappa B kinase (IKK) complex. IKK alpha and IKK beta are the two catalytic subunits of the IKK complex and two additional molecules, IKK gamma/NEMO and IKAP, have been described as further integral members. We have analyzed the function of both proteins for IKK complex composition and NF-kappa B signaling. IKAP and IKK gamma belong to distinct cellular complexes. Quantitative association of IKK gamma was observed with IKK alpha and IKK beta. In contrast IKAP was complexed with several distinct polypeptides. Overexpression of either IKK gamma or IKAP blocked tumor necrosis factor alpha induction of an NF-kappa B-dependent reporter construct, but IKAP in addition affected several NF-kappa B-independent promoters. Whereas specific down-regulation of IKK gamma protein levels by antisense oligonucleotides significantly reduced cytokine-mediated activation of the IKK complex and subsequent NF-kappa B activation, a similar reduction of IKAP protein levels had no effect on NF-kappa B signaling. Using solely IKK alpha, IKK beta, and IKK gamma, we could reconstitute a complex whose apparent molecular weight is comparable to that of the endogenous IKK complex. We conclude that while IKK gamma is a stoichiometric component of the IKK complex, obligatory for NF-kappa B signaling, IKAP is not associated with IKKs and plays no specific role in cytokine-induced NF-kappa B activation.

The ankyrin repeat domains of the NF-kappa B precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-kappa B DNA binding.

The inducible pleiotropic transcription factor NF-kappa B is composed of two subunits, p50 and p65. The p50 subunit is encoded on the N-terminal half of a 105-kDa open reading frame and contains a rel-like domain. To date, no function has been described for the C-terminal portion. We show here that the C-terminal half of p105, when expressed as a separate molecule, binds to p50 and can rapidly disrupt protein-DNA complexes of p50 or native NF-kappa B. Deletion analysis of this precursor-derived inhibitor activity indicated a domain containing ankyrin-like repeats as necessary for inhibition. The protooncogene bcl-3, which contains seven ankyrin repeats, can equally inhibit p50 DNA binding. These observations identify bcl-3 as an inhibitor of NF-kappa B and strongly suggest that the ankyrin repeats in these factors are involved in protein-protein interactions with the rel-like domain of p50. Comparison with other ankyrin repeat-containing proteins suggests that a subclass of these proteins acts as regulators of rel-like transcription factors.

Cloning of the DNA-binding subunit of human nuclear factor kappa B: the level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor alpha.

The DNA binding subunit of nuclear factor kappa B (NF-kappa B), a B-cell protein that interacts with the immunoglobulin kappa light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor alpha (TNF alpha), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-kappa B protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-kappa B binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNF alpha or phorbol ester. Thus, both factors not only activate NF-kappa B protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-kappa B.

Traducao "in vitro" e transcricao reversa do mRNA de procolageno. / Translation \"in vitro\" and reverse transcription of procollagen mRNA

Uma fracao de RNA mensageiro (mRNA) foi isolada a partir de RNA total extraido de polissomos de embrioes de galinha e purificada por dois ciclos de cromatografia em oligo (dt) - celulose. A analise deste material por eletroforese em condicoes denaturantes revelou a presenca de um pico homogeneo de moleculas, com um coeficiente de sedimentacao em torno de 28S. A adicao deste mRNA a um sistema de sintese proteica derivado de reticulocitos de coelho induziu a formacao de polipeptideos sensiveis a colagenase bacteriana purificada, com mobilidade eletroforetica igual a das cadeias do procolageno padrao. DNA complementar (cDNA) a mRNA de procolageno foi obtido pelo uso de transcriptase reserva. A analise deste cDNA em gel de agarose alcalino, revelou um tamanho medio correspondente a 565000 daltons. Atraves de experimentos de hibridizacao dos cDNAs de procolageno e globina com seus respectivos mRNAs, em que se assumiu a pureza da preparacao do mRNA de globina como 100%, pode-se estimar, por comparacao, que o mRNA de procolageno apresenta-se 78% puro