Cardiodepression by tumor necrosis factor-alpha

A 75-year-old man with carcinoma of the prostate presented with a pruritic, erythematous plaque involving the scrotal skin. Histological examination revealed extramammary Paget's disease. The intraepidermal tumour cells expressed prostate-specific antigen in keeping with a prostatic origin.     

Protein kinase C regulates macrophage tumor necrosis factor secretion: direct protein kinase C activation restores tumor necrosis factor production in endotoxin tolerance

BACKGROUND: Macrophages pretreated in vitro with endotoxin (LPSp) secrete less tumor necrosis factor (TNF) in response to a second LPS activating (LPSa) stimulus. Protein kinase C (PKC) is required for TNF secretion in a macrophage stimulated with LPSa. In these experiments we examined the role of PKC in TNF signal transduction in naive and tolerant macrophages. METHODS: Murine macrophages were cultured +/- LPSp for 24 hours. Cultures were washed and treated for 1 hour with PKC inhibitors or phorbol myristate acetate (PMA), a direct PKC activator. Cells were then stimulated with a range of LPSa for 6 hours, and TNF was determined by bioassay. RESULTS: LPSa-stimulated TNF secretion by nontolerant macrophages was inhibited by LPSp in the absence of PMA. PKC inhibitors decreased TNF by naive macrophages and exaggerated inhibition in tolerant cells. Depletion of PKC by 24 hours of PMA decreased TNF production by both naive and tolerant macrophages. PKC activation with PMA 1 hour before LPSa augmented TNF secretion in naive cells and reversed TNF inhibition of tolerant cells. CONCLUSIONS: Direct PKC activation with PMA restored TNF secretion in LPS-tolerant macrophages. Endotoxin tolerance may alter the LPSa signal transduction pathway between the LPS receptor and PKC activation.     

Selective induction of tumor necrosis factor receptor type II gene expression by tumor necrosis factor-alpha in C6 glioma cells

The aim of this study, in rabbit tibia, was an evaluation of the early reactions of the tissues to the insertion of polylactic membranes, used in connection with titanium implants. The specimens were retrieved after 1-4 weeks, and a histological analysis was performed. It was possible to see that, in the early implantation phases, no degradation of the macrostructure of the membrane was present. On the outer portion of the membrane many multinucleated giant cells (MGC) were present and membrane fragments were present inside the cytoplasm of these cells. These cells could explain the inflammatory processes reported, in some reports, with the use of materials made by polylactic and polyglycolic acid. We did not observe detrimental effects in the bone tissue around the membrane, and the membrane appeared to have a mechanical stability for the time necessary for bone regeneration.     

Involvement of double-stranded RNA-activated protein kinase in the synergistic activation of nuclear factor-kappaB by tumor necrosis factor-alpha and gamma-interferon in preneuronal cells

Tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma) cooperate during a variety of biological responses and ultimately synergistically enhance the expression of genes involved in immune and inflammatory responses. Recently, we demonstrated that IFN-gamma can significantly potentiate TNF-alpha-induced nuclear factor (NF)-kappaB nuclear translocation in neuronal derived and endothelial cell lines. The mechanism by which these two cytokines exert their synergistic effect on NF-kappaB involves the de novo degradation of the NF-kappaB inhibitor, IkappaBbeta. The double-stranded RNA-dependent kinase PKR is IFN-inducible and has been implicated in the activation of NF-kappaB; therefore, we examined the possibility that PKR may play a role in the synergistic activation of NF-kappaB during TNF-alpha/IFN-gamma cotreatment. The PKR inhibitor 2-aminopurine (2-AP) inhibited TNF-alpha/IFN-gamma-induced NF-kappaB nuclear translocation in neuronal derived cells but not in endothelial cells. The induced degradation of IkappaBbeta, which is normally observed upon TNF-alpha/IFN-gamma cotreatment, was blocked completely by 2-AP in neuronal derived cells. Also, 2-AP treatment or overexpression of a catalytically inactive PKR inhibited the TNF-alpha/IFN-gamma-induced synergistic activation of kappaB-dependent gene expression. Our results suggest that the signal generated by IFN-gamma during TNF-alpha/IFN-gamma cotreatment may require PKR to elicit enhanced NF-kappaB activity, and this signal may affect the stability of the IkappaBbeta protein.     

Dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha induced by guanidine hydrochloride

The dynamic equilibrium unfolding pathway of human tumor necrosis factor-alpha (TNF-alpha) during denaturation at different guanidine hydrochloride (GdnHCl) concentrations (0-4.2 M) was investigated by steady-state fluorescence spectroscopy, potassium iodide (KI) fluorescence quenching, far-UV circular dichroism (CD), picosecond time-resolved fluorescence lifetime, and anisotropy decay measurements. We utilized the intrinsic fluorescence of Trp-28 and Trp-114 to characterize the conformational changes involved in the equilibrium unfolding pathway. The detailed unfolding pathway under equilibrium conditions was discussed with respect to motional dynamics and partially folded structures. At 0-0.9 M [GdnHCl], the rotational correlation times of 22-25 ns were obtained from fluorescence anisotropy decay measurements and assigned to those of trimeric states by hydrodynamic calculation. In this range, the solvent accessibility of Trp residues increased with increasing [GdnHCl], suggesting the slight expansion of the trimeric structure. At 1.2-2.1 M [GdnHCl], the enhanced solvent accessibility and the rotational degree of freedom of Trp residues were observed, implying the loosening of the internal structure. In this [GdnHCl] region, TNF-alpha was thought to be in soluble aggregates having distinct conformational characteristics from a native (N) or fully unfolded state (U). At 4.2 M [GdnHCl], TNF-alpha unfolded to a U-state. From these results, the equilibrium unfolding pathway of TNF-alpha, trimeric and all beta-sheet protein, could not be viewed from the simple two state model (N-->U).     

Cytokine-specific activation of distinct mitogen-activated protein kinase subtype cascades in human neutrophils stimulated by granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor-alpha

To clarify the differences of the signaling pathways used by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor- (TNF), we investigated activation of mitogen-activated protein kinase (MAPK) subtype cascades in human neutrophils stimulated by these cytokines. G-CSF exclusively tyrosine-phosphorylated extracellular signal-regulated kinase (ERK). GM-CSF tyrosine-phosphorylated ERK strongly and p38 MAPK weakly, whereas TNF tyrosine-phosphorylated p38 MAPK strongly and ERK weakly. Consistent with these findings, MEK, an upstream kinase of ERK, was phosphorylated by G-CSF, GM-CSF, and TNF, whereas MKK3/MKK6, an upstream kinase of p38 MAPK, was phosphorylated by GM-CSF and TNF, but not by G-CSF. The potency of these cytokines to phosphorylate ERK and MEK was GM-CSF > G-CSF > TNF, whereas that to phosphorylate p38 MAPK and MKK3/MKK6 was TNF > GM-CSF. C-Jun amino-terminal kinase (JNK) was not tyrosine-phosphorylated by any cytokine despite the existence of JNK proteins in human neutrophils, whereas it was tyrosine-phosphorylated by TNF in undifferentiated and all-trans retinoic acid-differentiated HL-60 cells. Increased phosphorylation of ERK or p38 MAPK was detected within 1 to 5 minutes after stimulation with each cytokine and was dependent on the concentrations of cytokines used. MEK inhibitor (PD98059) reduced tyrosine phosphorylation of ERK, but not p38 MAPK, induced by G-CSF, GM-CSF, or TNF. GM-CSF- or TNF-induced superoxide (O2-) release was inhibited by p38 MAPK inhibitor (SB203580) in a dose-dependent manner, suggesting the possible involvement of p38 MAPK in GM-CSF- or TNF-induced O2- release. The results indicate that G-CSF, GM-CSF, and TNF activate the overlapping but distinct MAPK subtype cascades in human neutrophils and suggest that the differential activation of ERK and p38 MAPK cascades may explain the differences of the effects of these cytokines on human neutrophil functions.     

Activation of host cell protein kinase C by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) consists of a group of diarrhea-producing E. coli strains, common in developing countries, which do not produce classical toxins and are not truly invasive. EPEC strains adhere to mammalian cells in an intimate fashion, trigger a localized increase in intracellular calcium levels, and elevate inositol phosphate production. We hypothesized that these mediators could activate host cell protein kinase C (PKC) and tested this idea in vitro with two cultured human cell lines, HeLa cells and T84 cells. Using a recently described subculturing protocol to "induce" or accelerate EPEC adherence, we infected the cells with EPEC at a multiplicity of infection of approximately 100:1 for 30 to 60 min. Under these conditions, EPEC E2348 increased membrane-bound PKC activity 1.5- to 2.3-fold in HeLa cells and T84 cells, respectively. The increase in membrane-bound PKC activity was accompanied by a decrease in cytosolic PKC activity in EPEC-infected HeLa cells. Nonadherent laboratory E. coli strains such as HB101 and H.S. failed to trigger any consistent change in PKC production, similar to the nonadherent mutant strains derived from E2348, JPN15 (plasmid cured) and CVD206 (eaeA). In addition, immunoblots performed on extracts of T84 cells with a monoclonal antibody against PKC-alpha showed an increased PKC content in membranes of EPEC-infected cells. Finally, EPEC-infected T84 cells showed a 60% increase in responsiveness to the E. coli heat-stable toxin. We conclude that mediators produced in response to EPEC adherence activate PKC in intestinal and nonintestinal cells.     

Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: acid-induced denaturation

Acid-induced unfolding of proteins often results in an intermediate structure, called the molten globule structure or "A" state, which retains at least partial secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding has been studied extensively for alpha-helical proteins, while few studies have been done on proteins containing only beta-strands. Tumor necrosis factor-alpha (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel beta-sheet, organized into a jellyroll beta-sandwich. We have found previously [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of TNF-alpha results in an aggregate which contains a substantial amount of alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert to a monomeric form, as determined by sedimentation and diffusion constants obtained from sedimentation velocity experiments. The sedimentation coefficient indicated that this monomer was only slightly expanded relative to the native state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary structure. These structural features coincide with the notion that the acid-induced structure of TNF-alpha is a molten globule. What is unique in this protein is that TNF-alpha acquires alpha-helical structure, which is not present in the native structure as determined by both CD and Fourier transform infrared spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very gradual noncooperative change in secondary structure upon heating, which results in an increase in alpha-helical content. At pH 2.2 in the absence of salt, TNF-alpha shows considerable alpha-helix, although heating does not change the spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at ambient temperature, and upon heating, we see the noncooperative increase in alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH induces reassociation but to a range of oligomers rather than a unique trimer structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha may be related to its known interaction with lipid bilayers.     

Tumor necrosis factor-alpha stimulates human Clara cell secretory protein production by human airway epithelial cells

OBJECTIVE: Low serum levels of mannan binding lectin (MBL) are associated with increased risk of recurrent infections. We determined whether there was an association between serum MBL levels and the course and prognosis of rheumatoid arthritis (RA). METHODS: MBL was analyzed in sera from 99 patients with RA who were included in a longterm prospective study. RESULTS: Compared with controls, a high fraction of patients lacked detectable MBL in serum (11 vs 3%; p = 0.025). Comparing patients with MBL serum levels above and below the median revealed that those with levels below the median were younger at onset of RA (p = 0.043) and had higher erythrocyte sedimentation rate (p = 0.006), joint swelling score (p = 0.019), limitation of joint motion score (p = 0.027), and annual increase in radiographic destruction score (p = 0.053). CONCLUSION: MBL insufficiency may be a contributing pathogenetic factor in RA.     

Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin

Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of both acute and chronic inflammatory responses in many diseases. Tristetraprolin (TTP), the prototype of a class of Cys-Cys-Cys-His (CCCH) zinc finger proteins, inhibited TNF-alpha production from macrophages by destabilizing its messenger RNA. This effect appeared to result from direct TTP binding to the AU-rich element of the TNF-alpha messenger RNA. TTP is a cytosolic protein in these cells, and its biosynthesis was induced by the same agents that stimulate TNF-alpha production, including TNF-alpha itself. These findings identify TTP as a component of a negative feedback loop that interferes with TNF-alpha production by destabilizing its messenger RNA. This pathway represents a potential target for anti-TNF-alpha therapies.     

TNF-R55-specific form of human tumor necrosis factor-alpha induces collagenase gene expression by human skin fibroblasts

Tumor necrosis factor-alpha (TNF-alpha) is a potent inhibitor of connective tissue formation. The cellular effects of TNF-alpha are mediated by two distinct cell-surface receptors, TNF-R55 and TNF-R75, both present on various types of cells, including fibroblasts. In this study we wanted to elucidate the role of TNF-R55 as a mediator of the connective tissue effects of TNF-alpha by using a mutant, TNF-R55-specific form of human TNF-alpha. This mutant TNF-alpha markedly induced collagenase and stromelysin-1 gene expression in dermal fibroblasts, the maximal activation (up to 42-fold) being 65%-89% of that noted with wild-type human TNF-alpha. In addition, TNF-R55-specific TNF-alpha suppressed type I collagen mRNA levels as potently as wild-type TNF-alpha (by 60%). The enhancement of collagenase gene expression by TNF-R55-specific TNF-alpha was augmented by simultaneous treatment of normal and scleroderma skin fibroblasts with interferon-gamma, indicating specific enhancement of TNF-R55 signaling pathway by interferon-gamma. These results show that stimulation of the TNF-R55 signaling pathway is sufficient for the inhibitory effects of TNF-alpha on extracellular matrix formation by dermal fibroblasts. It is conceivable that due to reduced systemic toxicity, TNF-R55-specific forms of human TNF-alpha may prove to be feasible in the therapy of fibrotic disorders.     

Activation of protein kinase C delta by the c-Abl tyrosine kinase in response to ionizing radiation

The c-Abl protein tyrosine kinase is activated by ionizing radiation (IR) and certain other DNA-damaging agents. The present studies demonstrate that c-Abl associates constitutively with protein kinase C delta (PKCdelta). The results show that the SH3 domain of c-Abl interacts directly with PKCdelta. c-Abl phosphorylates and activates PKCdelta in vitro. We also show that IR treatment of cells is associated with c-Abl-dependent phosphorylation of PKCdelta and translocation of PKCdelta to the nucleus. These findings support a functional interaction between c-Abl and PKCdelta in the cellular response to genotoxic stress.     

Hemodynamic regulation of tumor necrosis factor-alpha gene and protein expression in adult feline myocardium

During the metastatic cascade, a tumor cell passes through several connective tissue barriers which consist of various adhesive molecules, such as fibronectin, laminin, collagens, and other glucoproteins and proteoglycans. Tumor invasion is a complex process involving cell adhesion, motility (migration), and the degradation of tissue barriers caused by the different proteases secreted by tumor cells. Therefore, understanding the invasion mechanism and the control mechanisms of the invasive properties of tumor cells may help in the development of anti-metastatic and anti-invasive therapies. We here focused our attention on the functional molecules involved in the invasive process as targets to control tumor metastasis, and screened the inhibitors of tumor invasion into basement membranes.     

Migration of retinal pigment epithelium cells in vitro is regulated by protein kinase C

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions, including subretinal neovascularization (SRN) and proliferative vitreoretinopathy (PVR). Therefore, elucidation of the mechanism of RPE migration may be useful in devising effective treatment for these disorders. Since protein kinase C (PKC) has been shown to regulate the migration of other cell types, we studied the effects of PKC agonists and antagonists on RPE migration. We used an in vitro wound healing model in which a small area of a confluent monolayer of bovine RPE cells was denuded with a razor blade. The cultures were subsequently incubated with agents known to stimulate [phorbol 12-myristate 13-acetate (PMA)] or inhibit (calphostin C, staurosporine) PKC. After 20 hr, migration was measured as the number of cells that had entered the denuded area. We also measured the translocation of PKC from the cytosol to the membrane in order to determine the activation or inhibition of PKC by PMA and calphostin C in the cells. The phorbol ester PMA stimulated migration by 41%, and calphostin C and staurosporine inhibited migration by 38% and 31%, respectively, in a medium supplemented with 10% serum. To determine the requirement for serum in this modulation, we also measured the effects of PMA and calphostin C on RPE migration in serum-free medium. Under these conditions, basal migration was greatly decreased, but PMA stimulated migration by 177% and calphostin C inhibited migration by 93%. Since PKC modulation is known to induce the proliferation of cells, we also tested the effects of these agents on growth-inhibited migration by pretreating the cells with the antiproliferative drug mitomycin C. We found that modulation of PKC under these conditions equally affected growth-inhibited and growth-dependent migration. Therefore, based on the increase in RPE migration induced by a PKC agonist, and the decrease in migration caused by PKC antagonists, it is suggested that PKC-mediated signal transduction plays a crucial role in RPE cell migration. This knowledge may be useful in devising effective treatments for SRN and PVR.     

Transient exposure of human myoblasts to tumor necrosis factor-alpha inhibits serum and insulin-like growth factor-I stimulated protein synthesis

Tumor necrosis factor-alpha (TNF-alpha) induces cachexia and is postulated to be responsible for muscle wasting in several pathophysiological conditions. The purpose of the present study was to investigate whether exposure of human myoblasts to TNF-alpha could directly inhibit the ability of serum or insulin-like growth factor I (IGF-I) to stimulate protein synthesis as assessed by the incorporation of [3H]phenylalanine into protein. Serum and IGF-I stimulated protein synthesis dose dependently. Half-maximal stimulation of protein synthesis occurred at 05% serum and 8 ng/ml of IGF-I, respectively. TNF-alpha inhibited IGF-I-stimulated protein synthesis in a dose-dependent manner. Additionally, as little as 2 ng/ml of TNF-alpha impaired the ability of IGF-I to stimulate protein synthesis by 33% and, at a dose of 100 ng/ml, TNF-alpha completely prevented the increase in protein synthesis induced by either serum or a maximally stimulating dose of IGF-I. Inhibition of protein synthesis was independent of whether TNF-alpha and growth factors were added to cells simultaneously or if the cells were pretreated with growth factors. Exposure ofmyoblasts to TNF-alpha for 10 min completely inhibited serum-induced stimulation of protein synthesis. TNF-alpha inhibited protein synthesis up to 48 h after addition of the cytokine. TNF-alpha also inhibited serum-stimulated protein synthesis in human myoblasts that were differentiated into myotubes. In contrast, exposure of myoblasts to TNF-alpha had no effect on IGF-I binding and failed to alter the ability of either IGF-I or serum to stimulate [3H]thymidine uptake. These data indicate that transient exposure of myoblasts or myotubes to TNF-alpha inhibits protein synthesis. Thus, the anabolic actions of IGF-I on muscle protein synthesis may be impaired during catabolic conditions in which TNF-alpha is over expressed.