Tumor Necrosis Factor Gene Transcription Research Articles

Transcriptional regulation of TNF/LT locus in immune cells

Tumor necrosis factor (TNF) is one of the most important proinflammatory cytokines. It demonstrates a complex pattern of tissue-specific expression and behaves as a product of immediate early transcriptional response in macrophages. These properties have made the regulation of TNF gene, as well as regulation of tightly linked related lymphotoxin alpha (LTalpha) and beta (LTbeta) genes the object of thorough investigation for more than two decades. Some aspects of TNF/LT locus regulation, such as the role of distal TNF-promoter and of NF-kappaB factors in TNF gene transcription, still remain the object of discussion. Moreover, several recent studies uncovering the molecular mechanisms of immediate early gene activation and directly related to TNF gene regulation have not been reflected in published reviews yet. Here we briefly overview the modern concepts of transcriptional regulation of the TNF/LT locus, with an accent on new data and unanswered questions.

Post-induction, Stimulus-specific Regulation of Tumor Necrosis Factor mRNA Expression

The tumor necrosis factor (TNF) gene is activated by multiple extracellular signals in a stimulus- and cell type-specific fashion. Based on the presence of kappaB-like DNA motifs in the region upstream of the TNF gene, some have proposed a direct role for NF-kappaB in lipopolysaccharide (LPS)-induced TNF gene transcription in cells of the monocyte/macrophage lineage. However, we have previously demonstrated a general and critical role for a minimal TNF promoter region bearing only one of the kappaB-like motifs, kappa3, which is bound by nuclear factor of activated T cell proteins in lymphocytes and fibroblasts in response to multiple stimuli and Ets proteins in LPS-stimulated macrophages. Here, in an effort to resolve these contrasting findings, we used a combination of site-directed mutagenesis of the TNF promoter, quantitative DNase I footprinting, and analysis of endogenous TNF mRNA production in response to multiple stimuli under conditions that inhibit NF-kappaB activation (using the proteasome inhibitor lactacystin and using cells lacking either functional NF-kappaB essential modulator, which is the IkappaB kinase regulatory subunit, or the Nemo gene itself). We find that TNF mRNA production in response to ionophore is NF-kappaB-independent, but inhibition of NF-kappaB activation attenuates virus- and LPS-induced TNF mRNA levels after initial induction. We conclude that induction of TNF gene transcription by virus or LPS does not depend upon NF-kappaB binding to the proximal promoter; rather, a stimulus-specific post-induction mechanism involving NF-kappaB, yet to be characterized, is involved in the maintenance of maximal TNF mRNA levels.

NFAT5 binds to the TNF promoter distinctly from NFATp, c, 3 and 4, and activates TNF transcription during hypertonic stress alone

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays an important role in a variety of infectious and autoimmune disorders. Its transcription is regulated in a stimulus- and cell-type-specific manner via the recruitment of distinct DNA/activator complexes forming secondary structures or enhanceosomes. NFATp, a member of the nuclear factor of activated T cells (NFAT) family of transcription factors, plays a critical role in TNF gene regulation under a variety of conditions. In this study, we show that NFAT5, the most recently described NFAT family member, binds to the TNF promoter in a manner distinct from other NFAT proteins and is a key mediator in the activation of TNF gene transcription during hypertonic stress alone.

Transcriptional Down-regulation of Tumor Necrosis Factor-α Gene by Early Given Pentoxifylline: A Quantitative Demonstration in Rats with Peritoneal Sepsis

Effects of pentoxifylline (PTX; 20 mg/kg/day) on mortality, blood pressure, acidosis, and inflammatory mediators were studied in rats subjected to cecal ligation and puncture (CLP). PTX was intravenously given once 30 minafter CLP. Compared with controls with CLP but no drug, the PTX group showed improved survival ever 10 days of observation. PTX treatment significantly attenuated the decrease in blood pressure and increase in serum lactate concentrations in comparison to controls administrated with normal saline at 24 h after operation. Tumor necrosis factor (TNF)-a concentrations were reduced significantly by PTX in serum measured by radioimmunoassays. According to the semi-quantitative and quantitative (TaqMan) reverse-transcription polymerase chain reactions, PTX had significantly decreased TNF-a but not IL-1β gene expression in lung and liver (both p < 0.05) at 24 h postoperatively. Thus, PTX attenuated mortality, TNF-a, and lactate in this sepsis model, in association with down-regulation of TNF gene transcription. Moreover, PTX may have other various benefitical actions on sepsis. Clinical trials of PTX given early in sepsis should be considered.

Advances in medical therapy for Crohn's disease.

Management of Crohn's disease has changed considerably in recent years. The discovery of tumor necrosis factor (TNF) as a pivotal cytokine in the inflammatory cascade has led to the development of several neutralizing antibodies, soluble receptors, and small molecules, interfering with TNF gene transcription and expression. Infliximab is the only monoclonal antibody that is commercially available. This potent molecule is effective for both active and fistulizing disease in the acute and maintenance phases of treatment. In addition to anti-TNF agents, weekly methotrexate injection and the classic "antimetabolites" azathioprine and 6-mercaptopurine remain highly valuable as maintenance drugs. "Tailored" antimetabolite therapy has now become possible with metabolite measurements and determination of the TPMT gene. The active metabolite thioguanine itself could be a promising alternative in patients who are intolerant of 6-mercaptopurine. In fistulizing disease, infliximab is becoming the treatment of choice, although fistula tracks do not disappear permanently and many patients still need surgical intervention.

The macrophage response to endotoxin requires platelet activating factor.

Platelet activating factor (PAF) is a key proinflammatory mediator of septic shock and is metabolized by PAF-acetylhydrolase (PAF-AH). Low circulating levels of PAF-AH have been associated with the development of autodestructive excessive inflammatory responses such as post-injury multiple organ failure, and recombinant PAF-AH is being studied for the prevention of acute respiratory distress syndrome (ARDS). However, the potential role of PAF as an autocrine mediator of macrophage activation is unclear. We wanted to examine the role of PAF in the endotoxin- (LPS) induced macrophage response using PAF-AH. Rabbit alveolar macrophages were stimulated with LPS (10 ng/mL) with or without PAF-AH (0.1-10 microg/mL). Supernatants were collected to measure the production of tumor necrosis factor (TNF), interleukin 8 (Il-8), and prostaglandin E2 (PGE2). Cell monolayers were assessed for procoagulant activity (PCA). TNF mRNA production was determined by Northern blot and RNA stability was assessed. Evaluation of intracellular signaling pathways for LPS included western blots for phosphorylated p38 and ERK kinases and gel shift for nuclear factor-kappaB. There was a dose-response inhibition of TNF, PCA, Il-8, and PGE2 production following pretreatment with PAF-AH. Time course studies revealed effective inhibition of TNF production with administration of PAF-AH up to 2 h after LPS challenge. TNF mRNA production was inhibited, while mRNA stability was not affected. There was no effect on the phosphorylation of p38 or ERK 1 kinases; however, the nuclear translocation of NF-kappaB was inhibited. Macrophage cytokine production in response to endotoxin is PAF dependent. This effect involves the inhibition of TNF gene transcription and concomitant inhibition of NF-kappaB.

Genetic polymorphism at position-308 in the promoter region of the tumor necrosis factor (TNF): implications of its allelic distribution on susceptibility or resistance to diseases in the Chilean population.

Several single-nucleotide polymorphisms have been identified in the human TNF gene promoter. The polymorphism at position-308 (TNF-308), which involves substituting G for A and designing the TNF2 allele, leads to a higher rate of TNF gene transcription than the wild-type TNF1 allele in in vitro expression studies. It has also been linked to increased susceptibility to a variety of illnesses. Using PCR-RFLP analysis we detected significant differences in the TNF-308 genotypes of Chilean and other populations. We conclude that there is a gradient in the distribution of the TNF2 allele according to ethnicity; we have also hypothesized that populations bearing a higher proportion of the TNF2 allele may have an increased predisposition toward or incidence of several chronic metabolic, degenerative, inflammatory and autoimmune diseases.

Fish Oil Decreases Macrophage Tumor Necrosis Factor Gene Transcription by Altering the NFκB Activity

Background.Fish oil-supplemented diets have anti-inflammatory and immunomodulating effects, though the exact mechanism(s) are unknown. This study investigated the effects of eicosapentanenoic acid (EPA), a major component of fish oil, on transcriptional regulation of tumor necrosis factor (TNF) gene in lipopolysaccharide (LPS)-stimulated macrophages (MØ).Methods.RAW 264.7 cells, a mouse MØ cell line, were grown in EPA-rich media for 24–48 h. MØ were washed and exposed toEscherichia coliLPS (1 μg/ml) for 2 h. TNF mRNA expression was measured by Northern blot assays. Total nuclear extracts were harvested for the measurement of NFκB with electrophoretic mobility shift assays. Supershift assays were performed with anti-P50 or anti-P65 antibodies to show components of NFκB dimers. TNF production was determined by L929 bioassays.Results.LPS stimulated RAW cell TNF mRNA expression and NFκB activity. In contrast, RAW cells grown in EPA-rich media had less TNF mRNA expression and an altered composition of the NFκB subunits (P65/P50 dimers) in the presence of LPS. TNF production by LPS-stimulated MØ was reduced by EPA.Conclusions.The inhibitory effect of EPA on LPS-stimulated MØ TNF gene transcription and protein elaboration is, in part, mediated through altering NFκB activation by reducing the P65/P50 dimers.

Genetic Polymorphisms in the Tumor Necrosis Factor Locus Influence Non-Hodgkin's Lymphoma Outcome

Systemic release of tumor necrosis factor (TNF) and lymphotoxin-α (LTα) has been found to contribute to the severity of non-Hodgkin's lymphoma (NHL). We investigated whether genetic polymorphisms in the TNF locus, previously shown to influence TNF and LTα genes expression, might contribute to these cytokines production and to the clinical course of NHL. Genomic DNA from 273 lymphoma patients was typed for TNF (−308) polymorphism using an allele-specific polymerase chain reaction (PCR) and for LTα (+252) polymorphism with a PCR-based restriction fragment length polymorphism. The presence of the TNF allele involved in increased TNF gene transcription was associated with higher plasma levels of this cytokine at the time of lymphoma diagnosis (χ2 test, P = .013). An extended haplotype analysis showed that the presence of at least two TNF or LTα high-producer alleles constituted a risk factor for first-line treatment failure (χ2 test, P = .021), shorter progression-free survival (log-rank test, P = .0007), and overall survival (log-rank test, P = .012). In the subgroup of 126 patients with diffuse large-cell lymphoma, the presence of two or more TNF/LTα high producing alleles contributed significantly to a higher rate of relapse and progression (log-rank test, P = .045 and P = .027). In multivariate Cox regression models including the variables of the International Prognostic Index, the TNF/LTα haplotype status was found to be an independent risk factor for progression-free survival (relative risk 2.33, 95% confidence interval [1.17 to 4.64], P = .0053) and overall survival (relative risk 1.92, 95% confidence interval [0.63 to 5.80],P = .081) of large-cell lymphoma patients. These results indicate that genetic polymorphism leading to increased TNF production influences the outcome of NHL and suggest a pathophysiological role for the genetic control of the immune response in lymphoid malignancies.

Genetic Polymorphisms in the Tumor Necrosis Factor Locus Influence Non-Hodgkin's Lymphoma Outcome

AbstractSystemic release of tumor necrosis factor (TNF) and lymphotoxin-α (LTα) has been found to contribute to the severity of non-Hodgkin's lymphoma (NHL). We investigated whether genetic polymorphisms in the TNF locus, previously shown to influence TNF and LTα genes expression, might contribute to these cytokines production and to the clinical course of NHL. Genomic DNA from 273 lymphoma patients was typed for TNF (−308) polymorphism using an allele-specific polymerase chain reaction (PCR) and for LTα (+252) polymorphism with a PCR-based restriction fragment length polymorphism. The presence of the TNF allele involved in increased TNF gene transcription was associated with higher plasma levels of this cytokine at the time of lymphoma diagnosis (χ2 test, P = .013). An extended haplotype analysis showed that the presence of at least two TNF or LTα high-producer alleles constituted a risk factor for first-line treatment failure (χ2 test, P = .021), shorter progression-free survival (log-rank test, P = .0007), and overall survival (log-rank test, P = .012). In the subgroup of 126 patients with diffuse large-cell lymphoma, the presence of two or more TNF/LTα high producing alleles contributed significantly to a higher rate of relapse and progression (log-rank test, P = .045 and P = .027). In multivariate Cox regression models including the variables of the International Prognostic Index, the TNF/LTα haplotype status was found to be an independent risk factor for progression-free survival (relative risk 2.33, 95% confidence interval [1.17 to 4.64], P = .0053) and overall survival (relative risk 1.92, 95% confidence interval [0.63 to 5.80],P = .081) of large-cell lymphoma patients. These results indicate that genetic polymorphism leading to increased TNF production influences the outcome of NHL and suggest a pathophysiological role for the genetic control of the immune response in lymphoid malignancies.

Regulation of the Human TNF Promoter by the Transcription Factor Ets

Tumor necrosis factor (TNF) affects the growth, differentiation, and function of a multitude of cell types and is viewed as a potent mediator of inflammation and cellular immune responses. In order to delineate functional domains that control TNF gene transcription, we have analyzed a 5' flanking region of the human TNF promoter spanning base pairs -115 to -98. This region contains a PEA3/Ets-1 binding motif 5' GAGGA 3' in direct juxtaposition to an AP-1/ATF-like palindromic sequence motif 5' TGAGCTCA 3'. Specific binding of Ets and Jun to their respective elements is demonstrated by competition analysis as well as by supershift assays. As shown by promoter deletion analysis, these two binding sites were essential for both basal promoter activity and responsiveness to the phorbol ester phorbol 12-myristate 13-acetate. Co-transfection of c-ets or c-jun expression plasmids along with TNF promoter-CAT reporter constructs revealed the participation of both transcription factors in the regulation of TNF gene transcription. Correspondingly, site-specific mutation of either Ets or Jun sites led to a complete loss of responsiveness to the respective transcription factor. These data suggest an essential role of Ets for the activation of TNF gene transcription.

Lipopolysaccharide Signals Activation of Tumor Necrosis Factor Biosynthesis Through the Ras/Raf-1/MEK/MAPK Pathway

Lipopolysaccharide (LPS) is known to activate macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One of the most important and best studied of these cytokines is tumor necrosis factor (TNF). Details of the signaling pathway leading to TNF biosynthesis remain unclear. The pathway is branched in the sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP kinase homologs become phosphorylated in LPS-stimulated cells, suggesting their possible involvement in signal transduction. We sought to test this hypothesis. Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensitive cells. Transfection studies, in which dominant inhibitors of ras and raf-1 were used to block signaling to the level of MAP kinase, were carried out in order to judge whether the TNF gene transcription and TNF mRNA translation are modulated through this pathway. In RAW 264.7 mouse macrophages, both ERK2 and MEK1 activity are induced by LPS treatment. In the same cell line, dominant negative inhibitors of ras and raf-1 block LPS-induced activation of the TNF promoter, as well as derepression of the translational blockade normally imposed by the TNF 3'-untranslated region. A constitutively active form of raf-1 (raf-BXB) was found to augment, but not replace, the LPS signal. In LPS-insensitive cells (RAW 264.7 x NIH 3T3 fusion hybrid cells and primary macrophages derived from C3H/HeJ mice), ERK2 activity was found to be refractory to induction by LPS. The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to the level of the TNF gene and mRNA. raf and raf-1 lie upstream from (or actually represent) the physical branchpoints of the transcriptional and translation activation signals generated by LPS. The lesions that prevent LPS signaling in macrophages from C3H/HeJ mice, or in RAW 264.7 x NIH 3T3 fusion hybrid cells, occupy a proximal position in the signaling pathway.

Silica increases tumor necrosis factor (TNF) production, in part, by upregulating the TNF promoter.

Silica causes release of tumor necrosis factor (TNF) from mononuclear phagocytes. One hypothesis is that silica increases TNF production, in part, by upregulating the TNF gene. To evaluate this hypothesis, THP-1 cells (a myelomonocytic cell line) were exposed to various amounts of silica and then the TNF gene transcription was evaluated. In this study silica caused a dose-dependent increase in TNF mRNA and the peak response occurred at 3 h following stimulation. A transient transfection assay also showed that silica upregulated expression of a TNF CAT construct in THP-1 cells. Furthermore, a nuclear run-on assay demonstrated that silica particles induce increased TNF gene transcription in exposed cells. THP-1 cells cultured for various periods of time in the presence of silica released TNF into the cell supernatants. These studies show that silica can upregulate the TNF gene, which results in the release of TNF protein from the cells.

Differential regulation of human B-lymphocyte tumor necrosis factor- alpha (TNF-alpha) and lymphotoxin (TNF-beta) production by protein phosphatase 1 and 2A inhibitor

Tumor necrosis factor (TNF) and lymphotoxin (LT; TNF-beta) are major cytokines produced by B lymphocytes. Stimulation by okadaic acid, a phosphatase 1 and 2A inhibitor, markedly increased TNF mRNA accumulation and cytokine production. On the other hand, the accumulation of LT mRNA was not affected by okadaic acid despite structural and functional similarities between TNF and LT. The increase in TNF mRNA accumulation was due to the stimulation of gene transcription and a marked stabilization of this mRNA. The binding activities of the transcription factors AP-1 and AP-2 and NF kappa B, which regulates TNF gene transcription, were also stimulated by okadaic acid. In addition, okadaic acid was shown to increase TNF production at the protein level. These results show the importance of protein phosphatases in the regulation of cytokine production in B cells, and further identifies differences in the regulation of TNF-alpha and LT production.

Transcriptional regulation of cytokine expression by diethyldithiocarbamate in human HL-60 promyelocytic leukemia cells

Diethyldithiocarbamate (DDTC) is an investigational agent used to ameliorate chemotherapy-or radiotherapy-induced myelosuppression. We studied the effects of DDTC on the regulation of hematopoietic cytokine production in human myeloid cells. The results demonstrated that DDTC decreases proliferation of human HL-60 promyelocytic leukemia cells in a concentration-dependent manner. DDTC treatment also increased interleukin-α- (IL-1α), IL-1β, and tumor necrosis factor (TNF) expression in these cells. Similar findings were obtained in normal human peripheral blood monocytes. Peak induction of these cytokines occurred 6–12 hr after exposure to DDTC; levels returned to those in control cells by 24–48 hr in HL-60 cells. This effect was specific for IL-1 and TNF in that there was no detectable increase in IL-3, macrophage colony-stimulating factor or granulocyte/macrophage colony-stimulating factor RNA expression. Transcriptional run-on analysis demonstrated that exposure to DDTC increased the rate of TNF gene transcription in HL-60 cells. These data suggest that the myeloprotective effects of DDTC may be mediated, at least in part, by the induction of TNF, IL-1α, and IL-1β.

Interactive effects of the tumor necrosis factor promoter and 3'-untranslated regions.

The tumor necrosis factor (TNF) promoter and 3'-untranslated region (3'-UTR) each contain sequence elements that mediate a response to bacterial endotoxin. Although the promoter contains sequences that permit augmented TNF gene transcription in response to LPS, the 3'-UTR contains sequences that normally confer translational repression, but which allow "derepression" to occur after cell contact with endotoxin. We now show that both genetic elements act in concert during activation of TNF gene expression in macrophages. In order to do so, we have made use of chloramphenicol acetyltransferase reporter constructs in which the TNF promoter and 3'-UTR are represented either independently or in combination with one another. Suppression of chloramphenicol acetyltransferase and TNF mRNA synthesis, observed after treatment of the macrophages with dexamethasone, 2-aminopurine, pentoxifylline, or dibutyryl cAMP, has also been studied in detail. Each class of inhibitor suppresses TNF biosynthesis through a separate mechanism. Interestingly, suppression by pentoxifylline is manifested partly (but not entirely) at the level of transcription, and depends upon the presence of both the TNF promoter and 3'-UTR. The data suggest that other sequences within the TNF gene could also be required for the full effect of pentoxifylline, which may act to prevent processing of the primary transcript. The suppressive effect of dexamethasone is manifested both at the level of transcription and at the level of translation, and is mediated both by sequences present in the TNF promoter and by sequences present in the 3'-UTR. Suppression by 2-aminopurine is solely dependent upon promoter sequences.