Classical and alternative NF-κB signaling cooperate in regulating adipocyte differentiation and function.

BACKGROUND AND OBJECTIVE: Inflammation of adipose tissue (AT) is a central mediator of insulin resistance. However, the molecular mechanisms triggered by inflammatory cells are not fully understood. The aim of this study was to analyze the metabolic functions of lymphotoxin-ß-receptor (LTßR)-mediated alternative NF-κB signaling in adipocytes and to reveal its effects on body weight and insulin sensitivity in vivo. METHODS: RelB(FatKO) mice and littermate controls were treated with LTßR agonistic antibody (α-LTßR) or a LTßR antagonist (LTßR:Ig fusion protein) after feeding a high-fat diet or standard diet. Mice were analyzed by insulin tolerance and glucose tolerance tests prior to analysis by necropsy and qRT-PCR of abdominal white adipose tissue. 3T3-L1 preadipocytes and mouse embryonic fibroblasts were used for differentiation and expression analysis after treatment with α-LTßR and differentiation to adipocytes. The molecular mechanism was elucidated by chromatin immunoprecipitation and combinatorial treatment with α-LTßR and tumor necrosis factor (TNF). RESULTS: RelB(FatKO) mice showed improved insulin sensitivity despite increased adiposity and adipocyte hypertrophy. LTßR-induced activation of p52-RelB in 3T3-L1 cells attenuated adipogenesis and modulated adipocyte functions via transcriptional downregulation of peroxisome proliferator-activated receptor γ (PPARγ). This LTßR-mediated pathway was synergistically regulated via a TNF-induced increase in p100 and RelB expression and nuclear translocation. CONCLUSIONS: Our data describe an anti-adipogenic action of LTßR signaling and a novel synergism of alternative and classical NF-κB signaling in the regulation of adipocytes. In conclusion, this strong synergism between the two NF-κB pathways shows a method to inhibit adipocyte differentiation and to improve insulin sensitivity and can be a potential target to treat metabolic disorders more efficiently than with other known drugs.

Cross talk between stimulated NF-kappaB and the tumor suppressor p53.

Nuclear factor-kappaB (NF-kappaB) and p53 critically determine cancer development and progression. Defining the cross talk between these transcription factors can expand our knowledge on molecular mechanisms of tumorigenesis. Here, we show that induction of replicational stress activates NF-kappaB p65 and triggers its interaction with p53 in the nucleus. Experiments with knockout cells show that p65 and p53 are both required for enhanced NF-kappaB activity during S-phase checkpoint activation involving ataxia-telangiectasia mutated and checkpoint kinase-1. Accordingly, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) also triggers formation of a transcriptionally active complex containing nuclear p65 and p53 on kappaB response elements. Gene expression analyses revealed that, independent of NF-kappaB activation in the cytosol, TNF-induced NF-kappaB-directed gene expression relies on p53. Hence, p53 is unexpectedly necessary for NF-kappaB-mediated gene expression induced by atypical and classical stimuli. Remarkably, data from gain- and loss-of function approaches argue that anti-apoptotic NF-kappaB p65 activity is constitutively evoked by a p53 hot-spot mutant frequently found in tumors. Our observations suggest explanations for the outstanding question why p53 mutations rather than p53 deletions arise in tumors of various origins.

Repression of inflammatory responses in the absence of DNA binding by the glucocorticoid receptor.

The glucocorticoid receptor (GR) acts both as a transcription factor itself on genes carrying GR response elements (GREs) and as a modulator of other transcription factors. Using mice with a mutation in the GR, which cannot activate GRE promoters, we examine whether the important anti-inflammatory and immune suppressive functions of glucocorticoids (GCs) can be established in this in vivo animal model. We find that most actions are indeed exerted in the absence of the DNA-binding ability of the GR: inhibition of the inflammatory response of locally irritated skin and of the systemic response to lipopolysaccharides. GCs repress the expression and release of numerous cytokines both in vivo and in isolated primary macrophages, thymocytes and CD4(+) splenocytes. A transgenic reporter gene controlled by NF-kappa B exclusively is also repressed, suggesting that protein- protein interaction with other transcription factors such as NF-kappa B forms the basis of the anti-inflammatory activity of GR. The only defect of immune suppression detected so far concerns the induced apoptosis of thymocytes and T lymphocytes.

Inhibition of NF-kappaB in T cells blocks lymphoproliferation and partially rescues autoimmune disease in gld/gld mice.

The Fas ligand (FasL)/Fas pathway is crucial for the maintenance of homeostasis of the peripheral immune system. Its importance is illustrated by the spontaneous mouse mutants gld andlpr which lack functional FasL and Fas receptor, respectively. These animals develop lymphadenopathy, splenomegaly, increased serum Ig and autoantibodies, leading to an autoimmune syndromeand premature death. The Rel/NF-kappaB family of transcription factors plays an important role in peripheral lymphocyte proliferation and survival. In this report, we studied the consequences of T cell-specific inhibition of NF-kappaB on the development of the gld phenotype. Transgenic gld/gld mice expressing a non-degradable form of IkappaBalpha under the control of T cell-specific regulatory elements show dramatically reduced lymphadenopathy, splenomegaly, and an almost complete elimination of Thy-1(+)B220(+)CD4(-)CD8(-) abnormal T cells, correlating with reduced proliferative responses and increased apoptosis of peripheral T cells upon TCR triggering. Interestingly, the B cell abnormalities that are characteristic of gld/gld mice, such as the production of autoantibodies, high levels of serum Ig, and the development of glomerulonephritis, are partially corrected. These results suggest that the T cell-specific inhibition of NF-kappaB opens apoptotic pathways distinct from FasL/Fas which, along with a diminished proliferative response, blocks splenomegaly and lymphadenopathy and partially rescues autoimmune disease in gld/gld mice.

Essential role of RelB in germinal center and marginal zone formation and proper expression of homing chemokines.

High levels of the Rel/NF-kappaB family member RelB are restricted to specific regions of thymus, lymph nodes, and Peyer's patches. In spleen, RelB is expressed in periarteriolar lymphatic sheaths, germinal centers (GCs), and the marginal zone (MZ). In this study, we report that RelB-deficient (relB(-/-)) mice, in contrast to nfkb1(-/-), but similar to nfkb2(-/-) mice, are unable to form GCs and follicular dendritic cell networks upon Ag challenge in the spleen. RelB is also required for normal organization of the MZ and its population by macrophages and B cells. Reciprocal bone marrow transfers demonstrate that RelB expression in radiation-resistant stromal cells, but not in bone marrow-derived hemopoietic cells, is required for proper formation of GCs, follicular dendritic cell networks, and MZ structures. However, the generation of MZ B cells requires RelB in hemopoietic cells. Expression of TNF ligand/receptor family members is only moderately altered in relB(-/-) splenocytes. In contrast, expression of homing chemokines is strongly reduced in relB(-/-) spleen with particularly low mRNA levels of the chemokine B lymphocyte chemoattractant. Our data indicate that activation of p52-RelB heterodimers in stromal cells downstream of TNF/lymphotoxin is required for normal expression of homing chemokines and proper development of spleen microarchitecture.

Nuclear factor-kappaB activation is not involved in a MPTP model of Parkinson's disease.

In the present study the involvement of hydroxyl free radicals and nuclear factor-kappaB (NF-kappaB) activation was investigated in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease. MPTP (30 mg/kg, s.c.) produced a significant 2-fold increase in hydroxyl free radicals in the striatum of C57BL/6 mice determined by microdialysis in combination with the salicylate hydroxylation assay. Electrophoretic mobility shift assays did not detect NF-kappaB activation after MPTP treatment. Furthermore, p50-deficient mice showed only minor differences in striatal dopamine and metabolite levels as well as tyrosine hydroxylase immunoreactivity after MPTP administration in comparison to wildtype mice. We postulate that, although hydroxyl radical production was enhanced, NF-kappaB plays only a minor role in the MPTP model because neither neurochemical nor immunocytochemical parameters were altered in p50-deficient mice in comparison to controls.

MHC class II and CD40 play opposing roles in dendritic cell survival.

In contrast to very immature dendritic cells (DC), mature DC are largely resistant to death by CD95 (CD95/APO-1) ligation. Investigation of other potential death-inducing ligands showed that mature DC were instead highly susceptible to apoptosis induced by cross-linking of MHC class II. Thus, increasing DC maturity correlates with increased resistance to CD95 killing, but an increased susceptibility to class II-mediated killing. Anti-I-A/I-E monoclonal antibodies (mAb) induced rapid (<2 h) apoptotic cell death in mature epidermal, spleen and bone marrow-derived DC, as determined by annexin/propidium iodide staining, morphological changes, decreased diploidy and loss in mitochondrial membrane potential. Although full class II-mediated killing required DC cytoskeletal motion, divalent cations and phosphatase activity, neither caspase activation, respiration, RNA or protein synthesis, NO production, nor CD95:CD95L interactions were required. Strikingly, DC pretreated by CD40 mAb cross-linking, but not by lipopolysaccharide or TNF-alpha, were completely resistant to class II-mediated killing. CD40-mediated protection was reduced in the presence of the SB202190 inhibitor of the mitogen-activated protein kinase p38 pathway, but appeared to be independent of p42/44 extracellular signal-related kinase or NF-KB activation. Our findings show that in addition to its role as an activator of antigen-presenting cell function, CD40 provides an important counter-signal against class II-induced apoptosis. Thus, these data point to an important role of the T cell in regulating DC survival.

Mice lacking the transcription factor RelB develop T cell-dependent skin lesions similar to human atopic dermatitis.

Mice with a targeted disruption of the Rel / NF-kappaB family member RelB develop a complex inflammatory phenotype and hematopoietic abnormalities. RelB-deficient (relB(- / -)) mice were clinically normal until 4 - 10 weeks after birth when thickening of the skin and hair loss developed. Histological and immunohistochemical evaluation of relB(- / -) skin lesions revealed hyperkeratosis and marked epidermal hyperplasia. Many CD4(+) T cells and eosinophils mixed with lesser numbers of CD8(+) T cells and neutrophils were present in the dermis. There was a moderate increase of MHC class II-positive dermal dendritic cells and dermal mast cells. Increased expression of Th2 cytokines correlated with increased mRNA levels of eotaxin and CCR3 in relB(- / -) skin. The dermatitis did not develop in the offspring of relB(- / -) mice crossed with transgenic mice that lack peripheral T cells, demonstrating that the skin lesions were T cell dependent. The dermatitis observed in RelB-deficient mice had many similarities with atopic dermatitis in human patients including infiltrating CD4(+) T cells and eosinophils in the skin, increased number of eosinophils in the blood and increased serum IgE. Thus, the relB(- / -) mouse should be a useful model to study the pathogenesis of this common allergic human disease.

NF-kappaB is activated and promotes cell death in focal cerebral ischemia.

The transcription factor NF-kappaB is a regulator of cell death or survival. To investigate the role of NF-kappaB in neuronal cell death, we studied its activation in a rodent model of stroke. In the ischemic hemisphere, NF-kappaB was activated, as determined by increased expression of an NF-kappaB-driven reporter transgene, nuclear translocation of NF-kappaB in neurons and enhanced DNA binding of NF-kappaB subunits RelA and p50. In p50 knockout mice, ischemic damage was significantly reduced. This indicates a cell death-promoting role of NF-kappaB in focal ischemia. NF-kappaB may provide a new pharmacological target in neurologic disease.

Multifocal defects in immune responses in RelB-deficient mice.

Mice with a targeted disruption of the Rel/nuclear factor-kappaB family member RelB develop a complex inflammatory phenotype, myeloid hyperplasia, and splenomegaly due to extramedullary hemopoiesis. In this work, we report that RelB-deficient mice, in addition to the pathologic changes, were highly susceptible to infection by the facultative intracellular bacterium Listeria monocytogenes. RelB binds transcriptionally active kappaB motifs in the TNF-alpha promoter in normal cells, and in vitro studies with macrophages isolated from RelB-deficient animals revealed impaired production of TNF-alpha in response to LPS and IFN-gamma. RelB-deficient mice also were unable to mount a protective immune response against lymphocytic choriomeningitis virus. These results indicate a defective T cell-macrophage interaction and cytotoxic T cell response, respectively, in mice lacking RelB. Analysis of resting and specific Ab production demonstrated that while RelB is not required for the secretion of Ig isotypes that result from heavy chain class switching, it is necessary for normal production of Ag-specific IgG in response to T cell-dependent and -independent stimuli. Thus, RelB is not only essential for a normal hemopoietic system in the unchallenged animal, but also involved in various specific and nonspecific immune responses.

p50-NF-kappaB complexes partially compensate for the absence of RelB: severely increased pathology in p50(-/-)relB(-/-) double-knockout mice.

RelB-deficient mice (relB(-/-)) have a complex phenotype including multiorgan inflammation and hematopoietic abnormalities. To examine whether other NF-kappaB/Rel family members are required for the development of this phenotype or have a compensatory role, we have initiated a program to generate double-mutant mice that are deficient in more than one family member. Here we report the phenotypic changes in relB(-/-) mice that also lack the p50 subunit of NF-kappaB (p50(-/-)). The inflammatory phenotype of p50(-/-)relB(-/-) double-mutant mice was markedly increased in both severity and extent of organ involvement, leading to premature death within three to four weeks after birth. Double-knockout mice also had strongly increased myeloid hyperplasia and thymic atrophy. Moreover, B cell development was impaired and, in contrast to relB(-/-) single knockouts, B cells were absent from inflammatory infiltrates. Both p50(-/-) and heterozygous relB(-/+) animals are disease-free. In the absence of the p50, however, relB(-/+) mice (p50(-/-)relB(-/+)) had a mild inflammatory phenotype and moderate myeloid hyperplasia. Neither elevated mRNA levels of other family members, nor increased kappaB-binding activities of NF-kappaB/Rel complexes could be detected in single- or double-mutant mice compared to control animals. These results indicate that the lack of RelB is, in part, compensated by other p50-containing complexes and that the "classical" p50-RelA-NF-kappaB activity is not required for the development of the inflammatory phenotype.

Genetic approaches to study Rel/NF-kappa B/I kappa B function in mice.

The generation of animal models in which individual members of a gene family are genetically altered is a particularly attractive way to elucidate their function-Members of the Rel/NF-kappa B/I kappa B family constitute an important network of transcription factors and regulatory proteins that control the expression of numerous cellular and viral genes crucial for a variety of processes. A few examples are developmental pattern formation and immune response in Drosophila, viral replication, and immune, inflammatory, acute phase and stress responses in vertebrates. The findings from knockout and transgenic mice developed to study Rel/NF-kappa B/I kappa B function in vivo are reviewed here. In general, these studies point to the essential role of these factors in the development and function of the vertebrate immune system.

Both multiorgan inflammation and myeloid hyperplasia in RelB-deficient mice are T cell dependent.

Mice with a targeted disruption of RelB, a member of the Rel/NF-kappaB family of transcription factors, have multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, and splenomegaly due to extramedullary hemopoiesis. To elucidate the cellular requirements for this complex phenotype, we have bred RelB-deficient (RelB(kappaO)) animals to two strains of immunodeficient mice, recombinase-activating gene-1-deficient (RAG-1(kappaO), lacking B and T cells), and Nur77/N10-transgenic mice (Nur77/N10(TG), lacking only T cells). We also generated mutant mice deficient in both RelB and the p50 subunit of NF-kappaB (p50(kappaO), multiple defects in B cell function). RelB(kappaO)RAG-1(kappaO) and RelB(kappaO)Nur77/N10(TG) mice are disease-free, while RelB(kappaO)p50(kappaO) double-mutant animals develop an even more severe phenotype despite the absence of B cells in the inflammatory infiltrates. Thus, both multiorgan inflammation and myeloid hyperplasia in RelB-deficient mice are T cell dependent, whereas B cells are not crucially involved.

B cells lacking RelB are defective in proliferative responses, but undergo normal B cell maturation to Ig secretion and Ig class switching.

A number of distinct functional abnormalities have been observed in B cells derived from p50/ NF-kappa B or c-rel knockout mice. RelB, another member of the NF-kappa B/Rel family of transcription factors, is expressed during the latter stages of B cell maturation and can bind to regulatory sites within the Ig heavy chain locus. Therefore, we tested the ability of B cells from relB knockout mice (relB-/-) to proliferate, undergo maturation to IgM secretion, and switch to the expression of downstream Ig isotypes in response to distinct activators including LPS, anti-CD40 mAb or CD40 ligand, and/or dextran anti-IgD antibodies in combination with various cytokines, including IL-4, IL-5, IFN-gamma, and TGF-beta. B cells lacking RelB showed up to 4-fold reductions in DNA synthesis in response to LPS, CD40, and membrane Ig-dependent activation relative to controls. However, relB-/- B cells were comparable to control B cells in their ability to undergo maturation to IgM secretion and switch to the expression of IgG3, IgG1, IgG2b, IgG2a, IgE, and/or IgA under all activation conditions tested. Thus, RelB, like c-Rel and p50/NF-kappa B, plays a role in B cell proliferation. However, in contrast to c-Rel and p50/ NF-kappa B, it is not critically involved in maturation to Ig secretion or expression of Ig isotypes.

RelB, a member of the Rel/NF-kappa B family of transcription factors.

RelB, originally identified as an immediate early gene product, is a member of the Rel/NF-kappa B family of transcription factors important for the regulation of genes involved in immune and inflammatory processes. RelB by itself is inactive due to its inability to homodimerize and to bind to kappa B sequences. However, in the presence of the Rel/NF-kappa B proteins p50 or p52, RelB is a potent transactivator. Transcriptional activation domains were identified in the NH2 and COOH termini of RelB separated by the approximately 300 amino acids spanning the Rel homogy domain (RHD). The last 120 amino acids of this domain are necessary for the dimerization of RelB and were analyzed in detail by in vitro mutagenesis. RelB forms complexes with p50 and p52 but not with RelA and c-Rel. In contrast to RelA-containing complexes, RelB-containing complexes are only weakly inhibited in their activity by I kappa B alpha. Furthermore, in lymphoid tissues RelB is not associated with I kappa B alpha. In contrast to other members of the Rel/NF-kappa B family, high expression of RelB is limited to interdigitating dendritic cells. Mice with a targeted disrupted relB locus show phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells.

Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway.

The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell populations due to an early onset of apoptosis. CD4-CD8- double-negative and CD25+ precursor cells, however, are unaffected. Moreover, nur77/N10-transgenic thymocytes show increased expression of Fas ligand (FasL), while the levels of the Fas receptor (Fas) are not increased. The mouse spontaneous mutant gld (generalized lymphoproliferative disease) carries a point mutation in the extracellular domain of the FasL gene that abolishes the ability of FasL to bind to Fas. Thymuses from nur77/N10-transgenic mice on a gld/gld background have increased cellularity and an almost normal profile of thymocyte subpopulations. Our results demonstrate that one pathway of apoptosis triggered by Nur77/N10 in double-positive thymocytes occurs through the upregulation of FasL expression resulting in increased signaling through Fas.

Overexpression of RelB in transgenic mice does not affect I kappa B alpha levels: differential regulation of RelA and RelB by the inhibitor protein.

In mouse lymphoid tissues, RelB heterodimers represent the constitutive kappa B-binding activity, whereas RelA and c-Rel complexes most likely are involved in inducible kappa B-binding and gene activation. Our laboratory has previously shown that the potential excess of NF-kappa B activity in transgenic mice overexpressing RelA is counteracted by a dramatic increase in I kappa B alpha, mainly due to its increased stability through association with RelA. As an attempt to elucidate the in vivo mechanisms that lead to the constitutive DNA-binding activity of RelB heterodimers, we have generated mouse lines overexpressing a relB transgene in a position-independent and copy number-dependent manner. Expression of RelB in these transgenic animals is very high in immature thymocytes and restricted to T cell areas in secondary lymphoid tissues. In contrast to the results obtained with RelA-transgenic thymocytes, we demonstrate here that overexpression of RelB results in a dramatic increase in overall kappa B-binding activity. Interestingly, I kappa B alpha protein levels are not altered in the RelB-transgenic animals, indicating that within the same cell type RelA and RelB complexes are differentially regulated by I kappa B alpha.

Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-kappa B/Rel family.

RelB, a member of the NF-kappa B/Rel family of transcription factors, has been implicated in the constitutive expression of kappa B-regulated genes in lymphoid tissues. We have generated mice carrying a germline mutation of the relB gene, resulting in the absence of RelB protein and a dramatic reduction of constitutive kappa B-binding activity in thymus and spleen. Mice homozygous for the disrupted relB locus had phenotypic abnormalities including multifocal, mixed inflammatory cell infiltration in several organs, myeloid hyperplasia, splenomegaly due to extramedullary hematopoiesis, and a reduced population of thymic dendritic cells. RelB-deficient animals also had an impaired cellular immunity, as observed in contact sensitivity experiments. Thus, RelB plays a decisive role in the hematopoietic system, and its absence cannot be functionally compensated by any other member of the NF-kappa B/Rel family.

Constitutive and inducible Rel/NF-kappa B activities in mouse thymus and spleen.

We have studied the expression of members of the rel family of transcription factors and ikba in mouse thymus and spleen by in situ hybridization. Our results show that the rel genes have different temporal and spatial patterns of expression suggesting distinct roles in these lymphoid tissues. The Rel/NF-kappa B proteins and I kappa B alpha in thymus and spleen were also analysed by Western blotting and electrophoretic mobility shift assays. Although RelB protein is present at significantly lower levels in thymus and spleen extracts when compared to RelA, in both tissues the predominant kappa B-binding activity consists of p50/RelB and p52/RelB heterodimers and only very little binding of RelA-containing complexes to kappa B sites was detected. Significant binding of c-Rel complexes was only found in spleen extracts. Treatment of thymus and spleen extracts with deoxycholate (DOC), however, results in a strong increase in binding to kappa B sites of both RelA and c-Rel complexes. In contrast, binding of RelB complexes is not induced after DOC treatment. Our results suggest a differential role of Rel/NF-kappa B complexes in mouse thymus and spleen with RelB heterodimers representing the constitutive kappa B-binding activity, whereas RelA and c-Rel complexes most likely are involved in inducible kappa B-binding and gene activation.

Developmental expression of the mouse c-rel proto-oncogene in hematopoietic organs.

We have studied the expression of the c-rel proto-oncogene during mouse embryonic development and adult animals using in situ hybridization and immunocytochemical analysis. c-rel transcripts were detected late in development with an expression pattern that parallels the emergence and diversification of hematopoietic cells. In the embryo, c-rel is expressed first in the mesoderm-derived hematopoietic cells of the liver and later also in other hematopoietic tissues such as thymus and spleen. This correlation between c-rel expression and places of hematopoietic infiltration is conserved in the postnatal period, with expression of c-rel mRNA in the medullary region of the thymus and in splenic B cell areas, including the marginal zone and the outer region of the periarterial sheath. High levels of c-rel transcripts were also detected in the splenic germinal centers, lymph nodes and Peyer's patches. Using double immunofluorescence and cell preparations from different embryonic and adult hematopoietic organs, we have defined the pattern and cell types of c-rel expression in different hematopoietic cell lineages and in the stromal cell content of the thymus. By using electrophoretic mobility shift assays, we have also correlated c-Rel expression in spleen with kappa B-binding activity in the form of c-Rel/p50 and c-Rel/p52 heterodimers. The timing and pattern of expression of the c-rel proto-oncogene in the different cell lineages suggest that temporally regulated changes in c-Rel expression may be required for vertebrate hematopoiesis.