NF-kappaB mediates FGF signal regulation of msx-1 expression.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors is involved in proliferation, differentiation, and apoptosis in a stage- and cell-dependent manner. Recent evidence has shown that NF-kappaB activity is necessary for both chicken and mouse limb development. We report here that the NF-kappaB family member c-rel and the homeodomain gene msx-1 have partially overlapping expression patterns in the developing chick limb. In addition, inhibition of NF-kappaB activity resulted in a decrease in msx-1 mRNA expression. Sequence analysis of the msx-1 promoter revealed three potential kappaB-binding sites similar to the interferon-gamma (IFN-gamma) kappaB-binding site. These sites bound to c-Rel, as shown by electrophoretic mobility shift assay (EMSA). Furthermore, inhibition of NF-kappaB activity significantly reduced transactivation of the msx-1 promoter in response to FGF-2/-4, known stimulators of msx-1 expression. These results suggest that NF-kappaB mediates the FGF-2/-4 signal regulation of msx-1 gene expression.

Nuclear factor-kappaB (NF-kappaB) regulates proliferation and branching in mouse mammary epithelium.

The nuclear factor-kappaB (NF-kappaB) family of transcription factors has been shown to regulate proliferation in several cell types. Although recent studies have demonstrated aberrant expression or activity of NF-kappaB in human breast cancer cell lines and tumors, little is known regarding the precise role of NF-kappaB in normal proliferation and development of the mammary epithelium. We investigated the function of NF-kappaB during murine early postnatal mammary gland development by observing the consequences of increased NF-kappaB activity in mouse mammary epithelium lacking the gene encoding IkappaBalpha, a major inhibitor of NF-kappaB. Mammary tissue containing epithelium from inhibitor kappaBalpha (IkappaBalpha)-deficient female donors was transplanted into the gland-free mammary stroma of wild-type mice, resulting in an increase in lateral ductal branching and pervasive intraductal hyperplasia. A two- to threefold increase in epithelial cell number was observed in IkappaBalpha-deficient epithelium compared with controls. Epithelial cell proliferation was strikingly increased in IkappaBalpha-deficient epithelium, and no alteration in apoptosis was detected. The extracellular matrix adjacent to IkappaBalpha-deficient epithelium was reduced. Consistent with in vivo data, a fourfold increase in epithelial branching was also observed in purified IkappaBalpha-deficient primary epithelial cells in three-dimensional culture. These data demonstrate that NF-kappaB positively regulates mammary epithelial proliferation, branching, and functions in maintenance of normal epithelial architecture during early postnatal development.

RAG2-/-, I kappa B-alpha-/- chimeras display a psoriasiform skin disease.

Nuclear factor-kappa B, a ubiquitous transcription factor involved in inflammatory and immune responses, is inappropriately activated in several immuno-related diseases, such as allograft rejection, or bronchial asthma. As nuclear factor-kappa B activity is regulated by inhibitor of kappa B (I kappa B), the gene encoding I kappa B-alpha was disrupted in mice to observe the in vivo effects of hyperactivation of nuclear factor-kappa B. I kappa B-alpha-/- mice have constitutive nuclear factor-kappa B activity, severe skin disease, and neonatal lethality. To determine the role of I kappa B-alpha deficient immunocytes in the pathogenesis of the skin disease in adult mice, we utilized the RAG2-deficient blastocyst complementation system to generate RAG2-/-, I kappa B-alpha-/- chimeras. These animals display a psoriasiform dermatitis characterized by hyperplastic epidermal keratinocytes and dermal infiltration of immunocytes, including lymphocytes. Skin grafts transferred from diseased chimeras to recipient nude mice produce hyperproliferative psoriasiform epidermal keratinocytes in response to stimulation. Furthermore, adoptive transfer of lymph node cells from diseased chimeras to RAG2-/- recipient mice recapitulates the disease. Taken together, these characterizations provide evidence to suggest that constitutive activation of nuclear factor-kappa B, due to deficiency in I kappa B-alpha, can invoke severe psoriasiform dermatitis in adult mice. J Invest Dermatol 115:1124-1133 2000

Lymphocytes lacking I kappa B-alpha develop normally, but have selective defects in proliferation and function.

NF-kappaB has been implicated in the development, activation, and function of B and T lymphocytes. We have evaluated the in vivo effects of deletion of IkappaB-alpha, a major inhibitor of NF-kappaB, on lymphocyte development, proliferation, and function. To elucidate the long term role of IkappaB-alpha in lymphocytes, fetal liver cells of 14.5-day-old IkappaB-alpha(-/-) or wild-type embryos were transplanted into irradiated recombinase-activating gene-2-deficient mice. Within 4 wk, the IkappaB-alpha(-/-) fetal liver cells reconstitute mature B and T cell populations in the recipients comparable to those produced by wild-type fetal liver cells. However, the proliferative responses of IkappaB-alpha(-/-) B cells are enhanced, whereas those of IkappaB-alpha(-/-) T cells are reduced. The levels of IgG1, IgG2a, IgA, and IgE produced by IkappaB-alpha(-/-) B cells are elevated relative to those produced by IkappaB-alpha(+/+) or IkappaB-alpha(+/-). Moreover, the specific immune responses to OVA and the generation of germinal centers are impaired in recipients of IkappaB-alpha(-/-) fetal liver cells. These results indicate that IkappaB-alpha plays a vital role in signal transduction pathways regulating lymphocyte proliferation and also in the production of specific Ig isotypes.

Dynamic expression and activity of NF-kappaB during post-natal mammary gland morphogenesis.

The Rel/NF-kappaB family of transcription factors has been implicated in such diverse cellular processes as proliferation, differentiation, and apoptosis. As each of these processes occurs during post-natal mammary gland morphogenesis, the expression and activity of NF-kappaB factors in the murine mammary gland were examined. Immunohistochemical and immunoblot analyses revealed expression of the p105/p50 and RelA subunits of NF-kappaB, as well as the major inhibitor, IkappaBalpha, in the mammary epithelium during pregnancy, lactation, and involution. Electrophoretic mobility shift assay (EMSA) demonstrated that DNA-binding complexes containing p50 and RelA were abundant during pregnancy and involution, but not during lactation. Activity of an NF-kappaB-dependent luciferase reporter in transgenic mice was highest during pregnancy, decreased to near undetectable levels during lactation, and was elevated during involution. This highly regulated pattern of activity was consistent with the modulated expression of p105/p50, RelA, and IkappaBalpha.

Mesenchymal expression of nuclear factor-kappaB inhibits epithelial growth and branching in the embryonic chick lung.

It is becoming increasingly recognized that the ubiquitous, inducible transcription factor nuclear factor-kappaB (NF-kappaB) is involved in developmental processes. For example, NF-kappaB acts as a mediator of epithelial-mesenchymal interactions in the developing chick limb. We investigated the role of NF-kappaB in directing the branching morphogenesis of the developing chick lung, a process which relies on epithelial-mesenchymal communication. High level expression of relA was found in the mesenchyme surrounding the nonbranching structures of the lung but was not detected either in the mesenchyme surrounding the branching structures of the distal lung or in the developing lung epithelium. Specific inhibition of mesenchymal NF-kappaB in lung cultures resulted in increased epithelial budding. Conversely, expression of a trans-dominant activator of NF-kappaB in the lung mesenchyme repressed budding. Ectopic expression of RelA was sufficient to inhibit the ability of the distal mesenchyme to induce epithelial bud formation. Cellular proliferation in the mesenchyme was inhibited by hyperactivation of NF-kappaB in the mesenchyme of lung cultures. Interestingly, increased NF-kappaB activity in the mesenchyme also decreased the proliferation of the associated epithelium, while inhibition of NF-kappaB activity increased cellular proliferation in lung cultures. Expression patterns of several genes which are known to influence lung branching morphogenesis were altered in response to changes in mesenchymal NF-kappaB activity, including fgf10, bmp-4, and tgf-beta1. Thus NF-kappaB represents the first transcription factor reported to function within the lung mesenchyme to limit growth and branching of the adjacent epithelium.

Multiorgan nuclear factor kappa B activation in a transgenic mouse model of systemic inflammation.

We utilized a line of transgenic mice expressing Photinus luciferase complementary DNA (cDNA) under the control of a nuclear factor kappa B (NF-kappaB)-dependent promoter (from the 5' human immunodeficiency virus-1 [HIV-1] long terminal repeat) to examine the role of NF-kappaB activation in the pathogenesis of systemic inflammation induced by bacterial endotoxin (lipopolysaccharide [LPS]). After intraperitoneal injection of E. coli LPS, these mice displayed a time- and dose-dependent, organ-specific pattern of luciferase expression, showing that NF-kappaB-dependent gene transcription is transiently activated in multiple organs by systemic LPS administration. Luciferase expression in liver could be specifically blocked by intravenous administration of replication-deficient adenoviral vectors expressing a dominant inhibitor of NF-kappaB (IkappaB-alphaDN), confirming that luciferase gene expression is a surrogate marker for NF-kappaB activation in this line of mice. After treatment with intraperitoneal LPS, the mice were found to have increased lung tissue messenger RNA (mRNA) expression of a variety of cytokines that are thought to be NF-kappaB-dependent, as well as elevated serum concentrations of presumed NF-kappaB-dependent cytokines. In lung tissue homogenates, a close correlation was identified between luciferase activity and KC levels. These studies show that systemic treatment with LPS orchestrates a multiorgan NF-kappaB-dependent response that likely regulates the pathobiology of systemic inflammation.

Nuclear factor-kappaB/Rel is apoptogenic in cytokine withdrawal-induced programmed cell death.

In the complex microenvironment where they evolve, developing cells undergo rapid programmed cell death (PCD) when cytokines that support them become limiting. The transcriptional mechanisms of cytokine-withdrawal apoptosis are poorly understood. In this report, we used early B-lymphocyte tissue culture and transgenic cells to demonstrate that nuclear factor-kappaB (NF-kappaB) promotes apoptosis during cytokine withdrawal-induced PCD. In the progenitor B lymphocyte model FL5.12, whereas NF-kappaB has an antiapoptotic function in response to tumor necrosis factor-alpha, cytokine withdrawal causes nuclear translocation of NF-kappaB/cRel, where it is apoptogenic. Inhibition of NF-kappaB activation delays cytokine withdrawal-induced PCD in both FL5.12 and transgenic early B cells. Additionally, reconstituting a bone marrow microenvironment ex vivo abrogates the differential apoptotic pattern between control and transgenic early B cells.

Reovirus-induced apoptosis requires activation of transcription factor NF-kappaB.

Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate this response, we investigated whether reovirus infection alters the activation state of the transcription factor nuclear factor kappa B (NF-kappaB). As determined in electrophoretic mobility shift assays, reovirus infection of HeLa cells leads to nuclear translocation of NF-kappaB complexes containing Rel family members p50 and p65. Reovirus-induced activation of NF-kappaB DNA-binding activity correlated with the onset of NF-kappaB-directed transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form of NF-kappaB is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a proteasome inhibitor prevents NF-kappaB activation following infection and substantially diminishes reovirus-induced apoptosis. Second, transient expression of a dominant-negative form of IkappaB that constitutively represses NF-kappaB activation significantly reduces levels of apoptosis triggered by reovirus infection. Third, mutant cell lines deficient for either the p50 or p65 subunits of NF-kappaB are resistant to reovirus-induced apoptosis compared with cells expressing an intact NF-kappaB signaling pathway. These findings indicate that NF-kappaB plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.

Isolated abdominal vasculitis as an atypical presentation of Wegener's granulomatosis.

Although current classifications characterize vasculitic syndromes based upon the size of the vessels involved, the histopathology, and the presence or absence of antineutrophil cytoplasmatic antibodies ANCA (1-3), those occasional patients with vasculitis whose features are not typical may evade diagnosis and effective treatment. We report one such patient who presented with bilateral refractory uveitis and abdominal angina who had a positive C-ANCA. Because of his atypical presentation, this patient's disease progressed over 8 yr despite an extensive gastrointestinal evaluation, before a diagnosis of vasculitis was established angiographically, and immunosuppressive therapy was begun.

Dramatic improvement of left ventricular function after cytotoxic therapy in lupus patients with acute cardiomyopathy: report of 6 cases.

Although lupus cardiomyopathy is thought to be clinically uncommon, we encountered 6 patients with systemic lupus erythematosus (SLE) over a 10 year period who had severe left ventricular dysfunction and showed remarkable improvement in their cardiac function after cytotoxic therapy. All patients met the American College of Rheumatology criteria for classification of SLE and presented with signs of severe biventricular failure relatively early in their disease. Concurrent manifestations of SLE at the time of cardiomyopathy included rash, arthritis, myalgias, pleuritis, pericarditis, and nephritis. Four of the 6 patients were taking prednisone 20 mg/day at the time heart failure developed. In all cases the CPK were normal. Evaluation of cardiac function by echocardiogram and/or radionuclide gated blood pool scintigraphy revealed a severe depression of ventricular function with initial left ventricular ejection fraction (LVEF) ranging from 11 to 34% (mean 19%). Within 6 months of initiation of cytotoxic treatment all patients showed a dramatic response: the post-treatment LVEF ranged from 25 to 55%. This series of patients suggests that cardiomyopathy may be a more common complication of SLE than previously reported. Cardiomyopathy occurs relatively early in the course of SLE, may lead to severe cardiac dysfunction despite corticosteroid therapy, and appears to be responsive to cytotoxic therapy.

Differential serine phosphorylation regulates IkappaB-alpha inactivation.

NF-kappaB is a ubiquitous transcription factor involved in the signal transduction mechanisms of the immune response, acute phase reactions, and viral infections. NF-kappaB proteins are retained in the cytoplasm by association with an inhibitor, termed IkappaB. Studies on the regulation of mammalian IkappaB-alpha have revealed that two amino-terminal conserved phosphoserines are the target sites of incoming signals. We report that the corresponding amino-terminal phosphoserines of avian IkappaB-alpha are phosphorylation targets leading to inactivation of IkappaB-alpha upon stimulation. In addition, we show differential roles for these two serines. Mutation of serine 40 to alanine blocks all stimuli tested (TNF-alpha, phorbol ester, and anti-CD3 and anti-CD28), leading to NF-kappaB activation, while mutation of serine 36 to alanine attenuates only certain transduced signals (PMA, TNF-alpha). These novel findings support the hypothesis that the amino-terminal phosphoserine residues of avian IkappaB-alpha differentially mediate NF-kappaB signal transduction pathways and activation by distinct signals, thereby resulting in the activation NF-kappaB.

Arthritis of the forefoot. A review from a rheumatologic and medical perspective.

Pain in the forefoot indicative of metatarsophalangeal synovitis often heralds the presence of significant inflammatory disease, and may be the initial presenting symptom of various systemic disorders. A review of these disorders and their rheumatologic, nonsurgical management is described.

Inhibition of NF-kappaB activity results in disruption of the apical ectodermal ridge and aberrant limb morphogenesis.

In Drosophila, the Dorsal protein establishes the embryonic dorso-ventral axis during development. Here we show that the vertebrate homologue of Dorsal, nuclear factor-kappa B (NF-kappaB), is vital for the formation of the proximo-distal organizer of the developing limb bud, the apical ectodermal ridge (AER). Transcription of the NF-kappaB proto-oncogene c-rel is regulated, in part, during morphogenesis of the limb bud by AER-derived signals such as fibroblast growth factors. Interruption of NF-kappaB activity using viral-mediated delivery of an inhibitor results in a highly dysmorphic AER, reduction in overall limb size, loss of distal elements and reversal in the direction of limb outgrowth. Furthermore, inhibition of NF-kappaB activity in limb mesenchyme leads to a reduction in expression of Sonic hedgehog and Twist but derepresses expression of the bone morphogenetic protein-4 gene. These results are the first evidence that vertebrate NF-kappaB proteins act to transmit growth factor signals between the ectoderm and the underlying mesenchyme during embryonic limb formation.

Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kappa B in gastric epithelial cells.

In vivo, gastric infection with Helicobacter pylori leads to substantial production of the inflammatory cytokines IL-1, IL-6, TNF-alpha, and IL-8. H. pylori strains that contain the cag pathogenicity island (cag+) and are associated with ulceration and gastric carcinoma induce greater cytokine production than cag- strains. Expression of these cytokines is often regulated by the transcription factor complex, nuclear factor-kappa B (NF-kappa B) through kappa B-binding elements in the enhancer/promoter regions of their genes. We report that more virulent cag+ H. pylori strains induce increased NF-kappa B-DNA binding activity, which elevates IL-8 expression in AGS gastric epithelial cells. The cag+ H. pylori strains induce significant stimulation of IL-8 promoter-driven reporter activity, while cag- strains do not. Furthermore, mutation of specific genes within the cag island (picA1 and picB) ablates enhanced NF-kappa B activation and IL-8 transcription. Increased IL-8 expression is inhibited by mutation in either the NF-kappa B or NF-IL-6 binding element. The cag+ strains, compared with the cag- strains, induce enhanced nuclear localization of a RelA-containing NF-kappa B binding complex, but no increase in NF-IL-6 binding activity. These studies demonstrate that the ability of different types of H. pylori strains to activate NF-kappa B correlates with their ability to induce IL-8 transcription and indicate a mechanism for the heightened inflammatory response seen in subjects infected with cag+ H. pylori strains.

TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes.

Signaling through IL-2R, IL-2 induces tyrosine phosphorylation and activation of Jak-1 and Jak-3 kinases and Stat 3 and Stat 5 transcription factors leading to cell cycle progression of activated T cells from G1 to S phase. TGF-beta is an immunosuppressive cytokine, which inhibits T cell proliferation at G1 to S phase transition. We examined the effect of TGF-beta on IL-2R signal transduction pathway in activated T cells. We show here that treatment of activated T cells with TGF-beta inhibited IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 but not Jak-3 and Stat 3. TGF-beta also inhibited IL-2-induced expression of alpha- and beta-chains of IL-2R and induced apoptotic cell death in T cells. These results suggest that TGF-beta-induced growth arrest and apoptosis are associated with the modulation of IL-2-induced activation of Jak-Stat pathway in T cells.

Expression of glutathione and gamma-glutamylcysteine synthetase mRNA is Jun dependent.

The gene GLCLC encodes the catalytic subunit of gamma-glutamylcysteine synthetase (glutamate-cysteine ligase E.C. 6.3.2.2), the rate limiting enzyme for glutathione synthesis. When HepG2 cells were exposed to the serine/threonine phosphatase inhibitor okadaic acid (OA), increased expression of GLCLC was observed, as was the development of resistance to xenobiotic induced GSH depletion. Okadaic acid is known to activate both NF-kappaB and AP-1 activity. Inhibition of NF-kappaB activity by overexpression of an IkappaB alpha transdominant inhibitor or exposure to the protease inhibitor TLCK did not inhibit the OA mediated increase in GLCLC transcripts. Fibroblasts derived from a mouse containing a c-Jun null mutation exhibited diminished AP-1 binding activity, reduced levels of GLCLC message, and a correspondingly low GSH concentration compared to wild type cells. When the null cells, which express Jun B and Jun D, were exposed to OA, AP-1 binding activity increased, as did expression of GLCLC message. These results indicate that AP-1 transcription factors participate in the regulation of glutathione metabolism.