Tumor Necrosis Factor Biosynthesis Research Articles

Therapeutic approaches for tumor necrosis factor inhibition

Tumor necrosis factor (TNF) consists of an inflammatory cytokine essential for homeostasis and organism defense. Despite its physiological relevance, both increased biosynthesis and release of TNF lead to the exacerbation of inflammatory and oxidative responses, which are related to the pathogenesis of a host of diseases of an inflammatory, autoimmune and/or infectious nature. In this context, effective therapeutic approaches for the modulation of TNF have been the focus of research efforts. Approximately one million individuals worldwide have been treated with biotechnological inhibitors of this cytokine, the so-called anti-TNF biopharmaceuticals. However, given the high risk of infection and the limitations related to cost and administration routes, new therapeutic approaches aimed at biological targets that directly or indirectly modulate the production and/or activation of TNF appear promising alternatives for the discovery of new anti-inflammatory and immunomodulatory orally active drugs and are therefore discussed in this paper.

The Biosynthesis Characteristics of TTP and TNF Can Be Regulated through a Posttranscriptional Molecular Loop

The abundant expression of tumor necrosis factor (TNF) is a hallmark of chronic inflammation of the gastrointestinal tract. Prolonged inflammation can lead to inflammatory bowel disease. TNF biosynthesis is regulated both at transcription and posttranscriptional levels. However, the stimulation-induced increase in translation rate is much larger. This might indicate the possibility of a posttranscriptional regulatory mechanism. How, during basal conditions, is the free concentration of TNF tightly regulated at low levels? The stability and translational efficiency of TNF transcript are regulated by an AU-rich element (ARE) in the 3'-UTR of messenger RNA. A transacting protein, TTP, binds to ARE and enhances the mRNA turnover. Here, we examine a proposal that TNF homeostasis is regulated by a TTP-TNF interaction loop at the posttranscriptional level. We propose a computational framework of this regulatory loop by modeling the role of AREs in mediating the messenger RNA stability and translation. This posttranscriptional regulatory loop between TTP and TNF is composed of two feedback loops (i.e. positive and negative). The mutual interaction of these feedback loops regulates the biosynthesis response of TNF during basal and inflammatory conditions. Here, we also propose an explanation for why the p38 inhibitors become insensitive for TTP knock-out mice.

MAPKAPK-2 Signaling Is Critical for Cutaneous Wound Healing

Cutaneous wound healing is a complex process, which is heavily dependent on successful inflammatory action. Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2 or MK2), a major substrate of p38 MAPK, has been shown to be a major player in multiple inflammatory diseases, but its role in cutaneous wound healing has not yet been explored. In this study, by comparing excisional wounds made on the backs of MK2 knockout (KO) and MK2 wild-type (WT) mice, we found that the kinetics of wound healing are significantly affected by the absence of MK2 (P=0.010 to P<0.001). Histological examination showed a higher level of acanthosis of the migrating wound keratinocyte layer as well as a higher level of collagen deposition in the granulation tissue of the wounds from MK2 WT mice compared with those from MK2 KO mice. Interestingly, although MK2 did not influence macrophage and neutrophil infiltration of the wounds, the expression of many cytokines and chemokines was significantly affected at different days post wounding. Furthermore, the delayed healing rate of wounds in MK2 KO mice can be significantly improved by passive transfer of macrophages with intact MK2. Overall, these results show a critical role for MK2 gene expression in macrophages participating in the process of cutaneous wound healing.

Interleukin‐1β and tetradecanoylphorbol acetate‐induced biosynthesis of tumor necrosis factor α in human hepatoma cells involves the transcription factors ATF2 and c‐Jun and stress‐activated protein kinases

The proinflammatory cytokine tumor necrosis factor (TNF) alpha is mainly produced in cells from the monocyte/macrophage lineage. TNFalpha is also a key signaling molecule in the liver functioning as an important physiological and pathogenic mediator. In hepatocytes or human hepatoma cells TNFalpha is expressed at extremely low levels but TNFalpha biosynthesis can be induced by interleukin (IL)-1beta or 12-O-tetradecanoylphorbol-13-acetate (TPA). Here, we show that IL-1beta and TPA stimulated TNFalpha gene transcription in hepatoma cells mediated by a composite TPA-responsive element/cAMP response element. Both IL-1beta and TPA triggered phosphorylation and activation of the basic region leucine zipper transcription factors c-Jun and ATF2 and expression of dominant-negative mutants of c-Jun and ATF2-reduced TNFalpha promoter activity and secretion of TNFalpha. Expression of the nuclear dual-specific MAP kinase phosphatase-1 (MKP-1) blocked TNFalpha promoter activity and TNFalpha secretion following IL-1beta or TPA stimulation, indicating that MKP-1 functions as a nuclear shut-of-device of IL-1beta and TPA-induced TNFalpha expression.

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Regulates Tumor Necrosis Factor mRNA Stability and Translation Mainly by Altering Tristetraprolin Expression, Stability, and Binding to Adenine/Uridine-Rich Element

The mitogen-activated protein kinase (MAPK) p38/MAPK-activated protein kinase 2 (MK2) signaling pathway plays an important role in the posttranscriptional regulation of tumor necrosis factor (TNF), which is dependent on the adenine/uridine-rich element (ARE) in the 3' untranslated region of TNF mRNA. After lipopolysaccharide (LPS) stimulation, MK2-deficient macrophages show a 90% reduction in TNF production compared to the wild type. Tristetraprolin (TTP), a protein induced by LPS, binds ARE and destabilizes TNF mRNA. Accordingly, macrophages lacking TTP produce large amounts of TNF. Here, we generated MK2/TTP double knockout mice and show that, after LPS stimulation, bone marrow-derived macrophages produce TNF mRNA and protein levels comparable to those of TTP knockout cells, indicating that in the regulation of TNF biosynthesis TTP is genetically downstream of MK2. In addition, we show that MK2 is essential for the stabilization of TTP mRNA, and phosphorylation by MK2 leads to increased TTP protein stability but reduced ARE affinity. These data suggest that MK2 inhibits the mRNA destabilizing activity of TTP and, in parallel, codegradation of TTP together, with the target mRNA resulting in increased cellular levels of TTP.

Post-transcriptional regulation of tumor necrosis factor alpha expression

The success of tumor necrosis factor (TNF) antagonists in the therapy of inflammatory arthritides has established the central role of this cytokine in the pathogenesis of these disorders. TNF is notable in that it is predominantly controlled at the post-transcriptional level in macrophages. TNF biosynthesis is highly regulated by the AU-rich element (AURE) found in its 3' untranslated region. This AURE regulates both the stability and rate of translation of TNF mRNA. In the macrophage, the ERK, JNK, and p38 SAPK signaling pathways converge on the AURE in regulating the nuclear export, stability and translation of TNF mRNA. Second, the C3H zinc finger protein, tristetraprolin (TTP), appears to play a major role in the post-transcriptional regulation of TNF by binding the AURE. Recent studies have identified that TTP is a target of the p38 SAPK/MAPKAP K2 kinase (MK2) pathway. Phosphorylation of TTP by MK2 has been reported in vitro and in vivo; data suggest that MK2 activation inactivates the function of TTP as a destabilizing protein. Intriguingly, TTP binding to the AURE does not appear to be regulated by this phosphorylation. Rather, the function of TTP seems to be modulated through interactions with specific proteins that alter its subcellular localization. This creates a model where TTP provides specificity in binding cytokine-type AURE but the consequences of this interaction are determined by protein–protein interactions. Second, we have identified that TTP does not bind all AURE, but rather exhibits specificity for nUAUUUAUn sequences. Third, we have identified that TTP regulates its own mRNA stability. Fourth, we have demonstrated that TTP localizes to the polysomes in the context of macrophage activation by lipopolysaccharide. Fifth, we have demonstrated that TTP is expressed in many different hematopoietic cells and appears to function as an AURE binding protein. Thus, although the role of TTP in TNF biology is best understood in the macrophage, it appears that some, if not all, of these concepts may be relevant to other cells involved in the immune and inflammatory response.

Regulation and localization of endogenous human tristetraprolin

Tumor necrosis factor (TNF) has been implicated in the development and pathogenicity of infectious diseases and autoimmune disorders, such as septic shock and arthritis. The zinc-finger protein tristetraprolin (TTP) has been identified as a major regulator of TNF biosynthesis. To define its intracellular location and examine its regulation of TNF, a quantitive intracellular staining assay specific for TTP was developed. We establish for the first time that in peripheral blood leukocytes, expression of endogenous TTP is confined to the cytoplasm. Baseline expression of TTP was higher in monocytes than in lymphocytes or neutrophils. After in vitro incubation with lipopolysaccharide (LPS), leukocyte TTP levels increased rapidly, peaking after approximately 2 hours. Monocytes showed the greatest response to LPS stimulation and lymphocytes the least. TTP levels were also studied in leukocytes isolated from healthy volunteers infused with a bolus dose of LPS. TTP expression and initial upregulation in response to LPS infusion were consistent with the in vitro data. Neutrophil TTP levels responded first, reaching an initial peak within 1 hour, monocyte levels peaked next at 2 hours, followed by lymphocytes at 4 hours. This response paralleled plasma TNF levels, which peaked 2 hours after infusion and were no longer detectable after 12 hours. A second rise in intracellular TTP levels, which did not parallel plasma TNF levels, was observed in all leukocyte populations, starting 12 hours after infusion. These data establish the cytoplasmic location of TTP, supporting a major role for this protein in regulating TNF production, and suggest that TTP levels are not regulated solely by TNF.

Preclinical and clinical assessment of the safety and potential efficacy of thalidomide in heart failure

Background: Inflammatory mediators, especially tumor necrosis factor (TNF), have been implicated in heart failure (HF). Thalidomide has anti-inflammatory properties and selectively inhibits TNF. Thus far, thalidomide or thalidomide analogues have not been evaluated in patients with heart failure. Methods: Thalidomide was assessed in preclinical and clinical studies. First, isolated cardiac myocytes were pretreated with thalidomide or thalidomide analogues, and TNF production was assessed after lipopolysaccharide (LPS) provocation. Second, to determine the safety and potential efficacy of thalidomide, an open-label dose escalation safety study was conducted in seven patients with advanced heart failure. Results: Thalidomide and thalidomide analogues inhibited LPS-induced TNF biosynthesis in cardiac myocytes in a dose-dependent manner. Thalidomide analogues had a greater inhibitory effect on TNF production than did thalidomide. In patients with advanced HF, thalidomide was safe and potentially effective when used at lower doses. However, dose-limiting toxicity was observed in two patients. There was a significant increase in the 6-minute walk distance and a trend toward improvement in left ventricular ejection fraction and quality of life after 12 weeks of maintenance therapy with thalidomide. Conclusions: Taken together these results suggest that thalidomide or its derivatives may be useful in selected patients with HF. This potential needs to be studied in larger clinical trials.

Distinct cellular functions of MK2.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK alpha and -beta, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.

Angiotensin II induces tumor necrosis factor biosynthesis in the adult mammalian heart through a protein kinase C-dependent pathway.

Previous studies suggest that angiotensin II (Ang II) upregulates the expression of tumor necrosis factor (TNF) in nonmyocyte cell types; however, the effect of Ang II on TNF expression in the adult mammalian heart is not known. To determine whether Ang II was sufficient to provoke TNF biosynthesis in the adult heart, we examined the effects of Ang II in isolated buffer-perfused Langendorff feline hearts. Ang II (10(-7) mol/L) treatment resulted in a time- and dose-dependent increase in myocardial TNF mRNA and protein biosynthesis in the heart as well as in cultured adult cardiac myocytes. The effects of Ang II on myocardial TNF mRNA and protein synthesis were mediated through the angiotensin type 1 receptor (AT1R), insofar as an AT1R antagonist (AT1a) blocked the effects of Ang II, whereas an angiotensin type 2 receptor (AT2R) antagonist (AT2a) had no effect. Stimulation with Ang II led to the activation of nuclear factor-kappaB and activator protein-1 (AP-1), two transcription factors that are important for TNF gene expression. Nuclear factor-kappaB activation was accompanied by phosphorylation of IkappaBalpha on serine 32 as well as degradation of IkappaBalpha, suggesting that the effects of Ang II were mediated through an IkappaBalpha-dependent pathway. The important role of protein kinase C (PKC) was suggested by studies in which a phorbol ester triggered TNF biosynthesis, and a PKC inhibitor abrogated Ang II-induced TNF biosynthesis. These studies suggest that Ang II provokes TNF biosynthesis in the adult mammalian heart through a PKC-dependent pathway.

MK2 Targets AU-rich Elements and Regulates Biosynthesis of Tumor Necrosis Factor and Interleukin-6 Independently at Different Post-transcriptional Levels

We demonstrate that lipopolysaccharide-induced tumor necrosis factor (TNF) biosynthesis becomes independent of MAPKAP kinase 2 (MK2) when the AU-rich element (ARE) of the TNF gene is deleted. In spleen cells and macrophages where TNF biosynthesis is restored as a result of this deletion, interleukin (IL)-6 biosynthesis is still dependent on MK2. In MK2-deficient macrophages the half-life of IL-6 mRNA is reduced more than 10-fold, whereas the half-life of TNF mRNA is only weakly decreased. It is shown that the stability of a reporter mRNA carrying the AU-rich 3'-untranslated region (3'-UTR) of IL-6 is increased by MK2. The data provide in vivo evidence that the AU-rich 3'-UTRs of TNF and IL-6 are downstream to MK2 signaling and make MK2 an essential component of mechanisms that regulate biosynthesis of IL-6 at the levels of mRNA stability, and of TNF mainly through TNF-ARE-dependent translational control.

Nuclear Factor-κB Blockade Attenuates but Does Not Abrogate Lipopolysaccharide-Dependent Tumor Necrosis Factor-α Biosynthesis in Alveolar Epithelial Cells

We have investigated the role that the nuclear factor (NF)-κB plays in regulating the biosynthesis of tumor necrosis factor (TNF)-α, an inflammatory cytokine. Irreversible inhibition of the proteasome complex by carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; 1–50 μM) had no inhibitory effect on LPS-mediated TNF-α biosynthesis. Furthermore, selective inhibition of NF-κB by the action of caffeic acid phenylethyl ester (CAPE; 1–100 μM) and sulfasalazine (SSA; 0.1–10 mM), a potent and irreversible inhibitor of NF-κB, partially attenuated, but did not abolish, LPS-dependent TNF-α secretion. Incorporation of a selectively permeant inhibitor of NF-κB, SN-50 (1–20 μM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-κB subunit, and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, attenuated in a dose-dependent manner LPS-mediated release of TNF-α. It is concluded that the NF-κB pathway is partially implicated and that its blockade attenuates, but does not abrogate, LPS-dependent TNF-α biosynthesis in alveolar epithelial cells.

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.

A reporter transgene indicates renal-specific induction of tumor necrosis factor (TNF) by shiga-like toxin. Possible involvement of TNF in hemolytic uremic syndrome.

We have examined the hypothesis that TNF may play a pathogenetically important role in the hemolytic uremic syndrome. Specifically, we considered the possibility that shigatoxin, which eventuates this syndrome, might induce TNF biosynthesis, and/or that TNF and shigatoxin might sensitize animals, each to the toxic effects of the other agent. Shigatoxin was found to sensitize mice to the lethal effect of LPS and to the lethal effect of TNF. On the other hand, pretreatment of animals with either TNF or LPS did not noticeably sensitize mice to the lethal effect of shigatoxin. Intraperitoneal injections of shigatoxin did not induce the production of detectable quantities of TNF in the plasma of mice. When shigatoxin was injected into transgenic mice bearing a chloramphenicol acetyltransferase (CAT) reporter gene that indicates TNF synthesis, CAT activity was induced within the kidney, but not in other tissues. We therefore conclude that shigatoxin acts to induce TNF synthesis within the kidney, and at the same time increases renal sensitivity to the toxic effects of TNF. While this mouse model does not reproduce the hemolytic uremic syndrome as it occurs in humans, it does suggest that local synthesis of TNF within the kidney may contribute to renal injury induced by shigatoxin.

Thalidomide blocks virus that causes AIDS

Thalidomide, the tranquilizer associated with birth defects in the 1960s, blocks activation of HIV-1—the virus that causes AIDS—in certain immune system cells, find researchers at Rockefeller University in New York City. The drug could be useful in delaying the progression of an HIV-1 infection and onset of AIDS symptoms, the researchers suggest. They now plan clinical trials to test this idea. Thalidomide was never approved for use in the U.S. It was withdrawn from the market in Europe in 1961 after the discovery that it causes limb deformities in children of women who took it. Since then, researchers have found that thalidomide is useful in treating inflammation associated with severe cases of leprosy. There is evidence that the drug could also be useful in treating other inflammatory processes. While investigating the mechanism of thalidomide's anti-inflammatory activity, Gilla Kaplan, an associate professor at Rockefeller, and coworkers showed that the drug blocks biosynthesis of tumor necrosis factor...

Constitutive synthesis of tumor necrosis factor in the thymus.

Although tumor necrosis factor (TNF) is a major mediator of endotoxic shock, the normal function of TNF that has preserved this protein throughout mammalian evolution remains unknown. If the protein serves a role in normal development or homeostasis, it must be produced under physiologic conditions. To determine whether TNF secretion occurs in normal animals, and to define the tissue sources of the protein, we prepared a reporter construct in which the TNF coding sequence and introns are replaced by the chloramphenicol acetyltransferase (CAT) coding sequence. This construct was inserted into the murine genome, yielding 13 transgenic founders. Macrophages harvested from 4 of the transgenic lines expressed CAT activity after stimulation with Escherichia coli lipopolysaccharide in vitro. Each of these 4 transgenic lines also constitutively expressed CAT activity in the thymus but in no other tissue examined. Cultured thymocytes secrete TNF, as demonstrated both by cytotoxicity assays and by immunoprecipitation of radiolabeled thymic culture medium. CAT activity was associated with the thymic lymphocyte population and not with thymic macrophages or dendritic cells. CAT activity was present in thymic lymphocytes irrespective of CD4 or CD8 expression; T cells from the spleen, however, had no detectable CAT activity. The biosynthesis of TNF in the thymus of normal animals implies a role for this protein in the development or regulation of the immune response.

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.

Optimal induction of tumor necrosis factor production in human monocytes requires complete S-form lipopolysaccharide.

Optimal activation of human monocytes in vitro for the biosynthesis of tumor necrosis factor was achieved only with complete S-form lipopolysaccharide. Endotoxin preparations with shorter carbohydrate chains or the lipid A component of lipopolysaccharide were not able to induce release of comparable amounts of tumor necrosis factor by monocytes under the conditions described. The same differences in the level of tumor necrosis factor mRNA were observed. Moreover, addition of these agents to appropriate monocyte-activating substances inhibited the production of tumor necrosis factor. The regulatory implications of this phenomenon are discussed.