NF-κB RelA Research Articles

CREB-binding protein in androgen receptor-mediated signaling.

CREB-binding protein (CBP) is a transcriptional coregulator that interacts with different DNA binding proteins and components of the general transcription machinery. CBP enhanced androgen receptor (AR)-dependent transcription under transient transfection conditions in CV-1 cells. The ligand binding domain (LBD) and residues 38-296 of the N-terminal region of AR are not required because the activity of a receptor mutant devoid of these domains was augmented by coexpressed CBP. There is physical interaction between AR and CBP in vivo, as judged by coimmunoprecipitation experiments from cell extracts. Consistent with the role of CBP as a coactivator for AR, the 12S E1A adenoviral protein that inactivates CBP function strongly inhibited AR-dependent transactivation. Exogenous CBP was also capable of overcoming the inhibitory effect of AR on AP-1 activity and diminished the mutual transcriptional repression between AR and NF-kappaB (RelA). Collectively, these data imply that transcriptional interference between AR and AP-1 or NF-kappaB is mediated, at least in part, through competition for intracellular CBP and that this coactivator serves as an integrator between androgen-mediated and other signaling pathways.

Induction of endotoxin tolerance depletes nuclear factor-kappaB and suppresses its activation in rat alveolar macrophages.

To investigate the mechanism of endotoxin tolerance in macrophages, a rat alveolar macrophage cell line (NR8383) was rendered endotoxin tolerant by treatment with endotoxin at 40 ng/mL for 48 h. This treatment induced a state of tolerance such that subsequent exposure to high-dose endotoxin (5 microg/mL) resulted in decreased production of macrophage inflammatory protein-2, tumor necrosis factor alpha, and nitric oxide compared to endotoxin-sensitive cells. Suppressed mediator production by endotoxin-tolerant cells was associated with impaired activation of nuclear factor-kappaB (NF-kappaB) in response to treatment with 5 microg/mL of endotoxin. This impairment of NF-kappaB activation was found to be associated with depletion of latent NF-kappaB (both RelA and p50) in the cytoplasm of endotoxin-tolerant cells. These data suggest that a mechanism of endotoxin tolerance is depletion of RelA/p50, which could limit the amount of NF-kappaB available for activation by subsequent stimuli and thereby inhibit transcription of NF-kappaB-dependent genes. Limiting NF-kappaB-dependent inflammatory gene transcription by inducing endotoxin tolerance is a potential therapeutic strategy for diseases where excessive production of inflammatory mediators leads to tissue injury.

Distinct Domains of the RelA NF-κB Subunit Are Required for Negative Cross-talk and Direct Interaction with the Glucocorticoid Receptor

The RelA subunit of NF-kappaB and the glucocorticoid receptor mutually repress each others transcriptional activity, thus providing a mechanism for immunosuppression. Deletion analysis of the glucocorticoid receptor has shown that the DNA binding domain and the ligand binding domain are essential components for repression. Here, we show by deletions and point mutations that both the Rel homology domain and the transactivation domains of RelA are required for repression of the transcriptional activity of the glucocorticoid receptor in intact cells. However, only the Rel homology domain of RelA was found to associate with the glucocorticoid receptor in vitro. RelA mutants, not able to repress glucocorticoid receptor activity, but still able to dimerize, behaved as transdominant inhibitors of the repressive activity of wild type RelA. Furthermore, we show that the 13 S E1A protein is able to interfere with the transrepressive activity of RelA. We propose that negative cross-talk between the glucocorticoid receptor and RelA is due to direct interaction via the Rel homology domain of RelA and the DNA binding domain of the glucocorticoid receptor in combination with interference by the transactivation domains of RelA with the transcriptional activity of the glucocorticoid receptor.

NF-kappa B RelA-deficient lymphocytes: normal development of T cells and B cells, impaired production of IgA and IgG1 and reduced proliferative responses.

To investigate the function of NF-kappa B RelA (p65), we generated mice deficient in this NF-kappa B family member by homologous recombination. Mice lacking RelA showed liver degeneration and died around embryonic day 14.5. To elucidate the role of RelA in lymphocyte development and function, we transplanted fetal liver cells of 13.5-day embryos from heterozygote matings into irradiated SCID mice. Within 4 weeks, both T and B cells had developed in the SCID mice receiving relA-/- fetal liver transplants, similar to the relA+/+ and +/- cases. T cells were found to mature to Thy-1+/TCR alpha beta +/CD3+/CD4+ or CD8+, while B cells had the ability to differentiate to IgM+/B220+ and to secrete immunoglobulins. However, the secretion of IgG1 and IgA was reduced in RelA-deficient B cells. Furthermore, both T and B cells lacking RelA showed marked reduction in proliferative responses to stimulation with Con A, anti-CD3, anti-CD3 + anti-CD28, LPS, anti-IgM, and PMA + calcium ionophore. The results indicate that RelA plays a critical role in production of specific Ig isotypes and also in signal transduction pathways for lymphocyte proliferation.

Identification of a tumor-associated mutant form of the NF-kappaB RelA gene with reduced DNA-binding and transactivating activities.

Alterations of NF-kappaB family members have been found to be associated with various forms of lymphoid malignancies. In order to determine whether alterations of the RelA gene are involved in lymphomagenesis, we analysed a large and representative panel (200 cases) of such tumors. Southern blot analysis did not reveal any rearrangements or locus amplification, suggesting that structural alterations of the RelA gene may represent rare events in lymphoid neoplasia. By means of PCR-SSCP analysis, we were able to identify a single point mutation leading to amino acid substitution (codon 494, Glu-Asp) in the transactivating (TA) domain in one case of multiple myeloma. The mutated allele was expressed in the pathological bone marrow sample but not in the peripheral blood cells of the patient. We demonstrate that the RelA protein carrying this specific mutation (called RelA494D) has less transactivating ability than the normal RelA protein. Interestingly, the mutated protein has a lower affinity for kappaB binding sites both as a homodimer or in association with the NFKB1/p50 subunit. Transfection experiments using a Gal4-RelA494D fusion protein indicated that the mutation does not alter the intrinsic transactivating ability of the TA domain of RelA. Furthermore, in vitro translated RelA494D is able to dimerize efficiently with other NF-kappaB members, such as p50, cREL and Ikappa Balpha. Our data therefore suggest that this mutation may alter the specific structural conformation needed for the DNA interaction of RelA, and provide insights into the amino acid sequences involved in mediating the biological activities of RelA.

LPS Does Not Directly Induce STAT Activity in Mouse Macrophages

Induction of gene expression in cytokine-treated cells involves the protein tyrosine kinase-dependent activation of members of the STAT family of transcription factors. To determine if lipopolysaccharide (LPS) might activate one or more STAT factors, nuclear extracts from LPS-treated RAW264.7 macrophages were assayed for STAT-like DNA binding activity using oligonucleotides recognized by different members of this protein family. Within 30 min a single LPS-inducible DNA–protein complex was detected using three separate oligonucleotides. This activity was not reactive with anti-STAT antibodies and was subsequently identified as composed of the NFκB components NFκB1 and Rel-A. Thus, LPS does not directly stimulate STAT factors with known sequence-specific DNA binding activity.

Regulation of adhesion and growth of fibrosarcoma cells by NF-kappa B RelA involves transforming growth factor beta.

The NF-kappa B transcription factor is a pleiotropic activator that participates in the induction of a wide variety of cellular genes. Antisense oligomer inhibition of the RelA subunit of NF-kappa B results in a block of cellular adhesion and inhibition of tumor cell growth. Investigation of the molecular basis for these effects showed that in vitro inhibition of the growth of transformed fibroblasts by relA antisense oligonucleotides can be reversed by the parental-cell-conditioned medium. Cytokine profile analysis of these cells treated with relA antisense oligonucleotides revealed inhibition of transforming growth factor beta 1 (TGF-beta 1 to the transformed fibroblasts reversed the inhibitory effects of relA antisense oligomers on soft agar colony formation and cell adhesion to the substratum. Direct inhibition of TGF-beta 1 expression by antisense phosphorothioates to TGF-beta 1 mimicked the in vitro effects of blocking cell adhesion that are elicited by antisense relA oligomers. These results may explain the in vitro effects of relA antisense oligomers on fibrosarcoma cell growth and adhesion.