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A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment.

ABSTRACT: Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. Given the deleterious effects to the host of TNF-alpha, it has been postulated that TNF-alpha gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-alpha gene transcription in humans remains obscure, although NF-kappaB has been suggested. Our previous studies pertaining to macrophage response to LPS identified a novel DNA-binding domain located from -550 to -487 in the human TNF-alpha promoter that contains transcriptional activity, but lacks any known NF-kappaB-binding sites. We have used this DNA fragment to isolate and purify a 60-kDa protein binding to this fragment and obtained its amino-terminal sequence, which was used to design degenerate probes to screen a cDNA library from THP-1 cells. A novel cDNA clone (1.8 kb) was isolated and fully sequenced. Characterization of this cDNA clone revealed that its induction was dependent on LPS activation of THP-1 cells; hence, the name LPS-induced TNF-alpha factor (LITAF). Inhibition of LITAF mRNA expression in THP-1 cells resulted in a reduction of TNF-alpha transcripts. In addition, high level of expression of LITAF mRNA was observed predominantly in the placenta, peripheral blood leukocytes, lymph nodes, and the spleen. Finally, chromosomal localization using fluorescence in situ hybridization revealed that LITAF mapped to chromosome 16p12-16p13.3. Together, these findings suggest that LITAF plays an important role in the activation of the human TNF-alpha gene and proposes a new mechanism to control TNF-alpha gene expression.

SUBMITTER: Myokai F

PROVIDER: S-EPMC16364 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment.

Myokai F F Takashiba S S Lebo R R Amar S S

Proceedings of the National Academy of Sciences of the United States of America 19990401 8

Lipopolysaccharide (LPS) is a potent stimulator of monocytes and macrophages, causing secretion of tumor necrosis factor alpha (TNF-alpha) and other inflammatory mediators. Given the deleterious effects to the host of TNF-alpha, it has been postulated that TNF-alpha gene expression must be tightly regulated. The nature of the nuclear factor(s) that control TNF-alpha gene transcription in humans remains obscure, although NF-kappaB has been suggested. Our previous studies pertaining to macrophage ...[more]

PMID: 10200294

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Project description:IntroductionThe present study assessed the potential functions of interleukin (IL)-32? on inflammatory arthritis and endotoxin shock models using IL-32? transgenic (Tg) mice. The potential signaling pathway for the IL-32-tumor necrosis factor (TNF)? axis was analyzed in vitro.MethodsIL-32? Tg mice were generated under control of a ubiquitous promoter. Two disease models were used to examine in vivo effects of overexpressed IL-32?: Toll-like receptor (TLR) ligand-induced arthritis developed using a single injection of lipopolysaccharide (LPS) or zymosan into the knee joints; and endotoxin shock induced with intraperitoneal injection of LPS and D-galactosamine. TNF? antagonist etanercept was administered simultaneously with LPS in some mice. Using RAW264.7 cells, in vitro effects of exogenous IL-32? on TNF?, IL-6 or macrophage inflammatory protein 2 (MIP-2) production were assessed with or without inhibitors for nuclear factor kappa B (NF?B) or mitogen-activated protein kinase (MAPK).ResultsSingle injection of LPS, but not zymosan, resulted in development of severe synovitis with substantial articular cartilage degradation in knees of the Tg mice. The expression of TNF? mRNA in inflamed synovia was highly upregulated in the LPS-injected Tg mice. Moreover, the Tg mice were more susceptive to endotoxin-induced lethality than the wild-type control mice 48 hours after LPS challenge; but blockade of TNF? by etanercept protected from endotoxin lethality. In cultured bone marrow cells derived from the Tg mice, overexpressed IL-32? accelerated production of TNF? upon stimulation with LPS. Of note, exogenously added IL-32? alone stimulated RAW264.7 cells to express TNF?, IL-6, and MIP-2 mRNAs. Particularly, IL-32? -induced TNF?, but not IL-6 or MIP-2, was inhibited by dehydroxymethylepoxyquinomicin (DHMEQ) and U0126, which are specific inhibitors of nuclear factor kappa B (NF?B) and extracellular signal regulated kinase1/2 (ERK1/2), respectively.ConclusionsThese results show that IL-32? contributed to the development of inflammatory arthritis and endotoxin lethality. Stimulation of TLR signaling with LPS appeared indispensable for activating the IL-32?-TNF? axis in vivo. However, IL-32? alone induced TNF? production in RAW264.7 cells through phosphorylation of inhibitor kappa B (I?B) and ERK1/2 MAPK. Further studies on the potential involvement of IL-32?-TNF? axis will be beneficial in better understanding the pathology of autoimmune-related arthritis and infectious immunity.

Project description:UNLABELLED:Inflammation may be maladaptive to the control of viral infection when it impairs interferon (IFN) responses, enhancing viral replication and spread. Dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis B virus and hepatitis C virus (HCV). Previous studies from our laboratory have shown that expression of an IFN-stimulated gene (ISG), ubiquitin-like protease (USP)18 is upregulated in chronic HCV infection, leading to impaired hepatocyte responses to IFN-?. We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-?), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-? response. Human hepatoma cells and primary murine hepatocytes were treated with TNF-?/LPS/IL-6/IL-10 and USP18, phosphorylated (p)-STAT1 and myxovirus (influenza virus) resistance 1 (Mx1) expression was determined. Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-?, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-? refractory state, which was reversed by USP18 knockdown. Liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. Hepatic ischemia/reperfusion injury led to an induction of USP18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. These data demonstrate that certain inflammatory stimuli (TNF-? and LPS) but not others (IL-6 and IL-10) target USP18 expression and thus inhibit IFN signaling. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with USP18 representing a potential target for intervention in various inflammatory states. IMPORTANCE:Inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. Blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. Previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic HCV infection, leading to impaired hepatocyte responses. In this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via USP18 induction. These findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of USP18 representing a potential target for intervention in various inflammatory states.

Dataset Information

A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment.

Publications

A novel lipopolysaccharide-induced transcription factor regulating tumor necrosis factor alpha gene expression: molecular cloning, sequencing, characterization, and chromosomal assignment.

Similar Datasets

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