Tumor necrosis factor (TNF) interferes with insulin signaling through the p55 TNF receptor death domain

Tumor necrosis factor (TNF) contributes to insulin resistance by binding to the 55kDa TNF receptor (TNF-R55), resulting in serine phosphorylation of proteins such as insulin receptor (IR) substrate (IRS)-1, followed by reduced tyrosine phosphorylation of IRS-1 through the IR and, thereby, diminished IR signal transduction. Through independent receptor domains, TNF-R55 activates a neutral (N-SMase) and an acid sphingomyelinase (A-SMase), that both generate the sphingolipid ceramide. Multiple candidate kinases have been identified that serine-phosphorylate IRS-1 in response to TNF or ceramide. However, due to the fact that the receptor domain of TNF-R55 mediating inhibition of the IR has not been mapped, it is currently unknown whether TNF exerts these effects with participation of N-SMase or A-SMase. Here, we identify the death domain of TNF-R55 as responsible for the inhibitory effects of TNF on tyrosine phosphorylation of IRS-1, implicating ceramide generated by A-SMase as a downstream mediator of inhibition of IR signaling.     

Chromosome assignments of the human TNF p55 and p75 receptor genes.

At least two different receptor molecules have been described that are capable of binding tumor necrosis factor alpha, a cytokine that plays an important role in inflammation and antitumor activity. Comparative analyses at the nucleotide sequence level suggest that these receptors are members of a newly defined protein family that also includes human and rat nerve growth factor receptors. In this study, we determine the chromosome assignments of the human TNF alpha receptor genes, one of which may have evolved as part of a conserved Hox locus-containing chromosome segment.     

Expression in Escherichia coli of the death domain of the human p55 tumor necrosis factor receptor.

The p55 tumor necrosis factor receptor (TNF-RI) is the main receptor by which TNF exerts its effects. The signaling capacity largely depends on the presence of an intact C-terminal protein-protein interaction domain, a so-called death domain (DD). Here we report the expression and purification of the human TNF-RI DD as a fusion with the Escherichia coli thioredoxin A (TRX) protein. When expressed under control of the bacteriophage T7 promoter, TRX-DD accumulates as a soluble protein in the cytoplasm of E. coli. The TRX-DD protein was released from the cells into the periplasmic fraction after osmotic shock. Due to self-association of the DD, a large part of the material appeared as multimers; it could be removed by selective precipitation and a combination of ion-exchange and size-exclusion chromatography. This purification protocol yielded 30 mg of purified, monomeric protein from 1 liter of shake-flask culture. The purified TRX-DD was found to be functional as it still bound to the TNF-RI-associated DD protein and the intracellular part of TNF-RI. We conclude that TRX-DD is correctly folded and can be used for further structure/function analysis.     

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with > 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.     

Inhibition of receptor internalization by monodansylcadaverine selectively blocks p55 tumor necrosis factor receptor death domain signaling

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the "death domain" of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.     

Tumor necrosis factor (TNF)-alpha induces leptin production through the p55 TNF receptor

Tumor necrosis factor (TNF)-alpha acts directly on adipocytes to increase production of the lipostatic factor, leptin. However, which TNF receptor (TNFR) mediates this response is not known. To answer this question, leptin was measured in plasma of wild-type (WT), p55, and p75 TNFR knockout (KO) mice injected intraperitoneally with murine TNF-alpha and in supernatants from cultured WT, p55, and p75 TNFR KO adipocytes incubated with TNF-alpha. Leptin also was measured in supernatants from C3H/HeOuJ mouse adipocytes cultured with blocking antibodies to each TNFR and TNF-alpha as well as in supernatants from adipocytes incubated with either human or murine TNF-alpha, which activate either one or both TNFR, respectively. The results using all four strategies show that the induction of leptin production by TNF-alpha requires activation of the p55 TNFR and that although activation of the p75 TNFR alone cannot cause leptin production, its presence affects the capability of TNF-alpha to induce leptin production through the p55 TNFR. These results provide new information on the interplay between cells of the immune system and adipocytes.     

NMR structure of the death domain of the p75 neurotrophin receptor.

The intracellular domain of the p75 neurotrophin receptor (p75ICD) lacks catalytic activity but contains a motif similar to death domains found in the cytoplasmic regions of members of the tumor necrosis factor receptor family and their downstream targets. Although some aspects of the signaling pathways downstream of p75 have been elucidated recently, mechanisms of receptor activation and proximal signaling events are unknown. Here we report the nuclear magnetic resonance (NMR) structure of the 145 residue long p75ICD. The death domain of p75ICD consists of two perpendicular sets of three helices packed into a globular structure. The polypeptide segment connecting the transmembrane and death domains as well as the serine/threonine-rich C-terminal end are highly flexible in p75ICD. Unlike the death domains involved in signaling by the TNF receptor and Fas, p75ICD does not self-associate in solution. A surface area devoid of charged residues in the p75ICD death domain may indicate a potential site of interaction with downstream targets.     

TNF-induced haptoglobin release from human neutrophils: pivotal role of the TNF p55 receptor

Haptoglobin (Hp), TNF-alpha, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-alpha on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF-alpha induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF-alpha was not due to nonspecific lysis of the cells. TNF-alpha is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF-alpha mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF-alpha for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF-alpha-induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF-alpha may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.     

Expression of TNF and TNF receptors (p55 and p75) in the rat brain after focal cerebral ischemia.

Cerebral ischemia induces a rapid and dramatic up-regulation of tumor necrosis factor (TNF) protein and mRNA, but the cellular sources of TNF in the ischemic brain have not been defined. The diverse activities of TNF are mediated via ligand interaction with two distinct receptors, p55 and p75, which activate separate intracellular signal transduction pathways, leading to distinct biological effects. Since the effects of cerebral ischemia on TNF receptor (TNFR) expression are unknown, we examined the cellular localization and protein expression of TNF and its two receptors in the rat cerebral cortex in response to permanent middle cerebral artery (MCA) occlusion. The results indicate that focal. cerebral ischemia up-regulates expression of TNF and both TNFRs within the ischemic cortex. The most abundant type of TNF immunoreactivity (IR) was a punctate and filamentous pattern of transected cellular processes; however, cell bodies of neurons, astrocytes, and microglia, as well as infiltrating polymorphonuclear (PMN) leukocytes also showed TNF IR. Brain vasculature displayed TNF IR not only within endothelial cells but also in the perivascular space. MCA occlusion induced significant up-regulation of TNF receptors, with p55 IR appearing within 6 hr, significantly before the appearance of p75 IR at 24 hr after the onset of ischemia. Since p55 has been implicated in transducing cytotoxic signalling of TNF, these results support the proposed injurious role of excessive TNF produced during the acute response to cerebral ischemia.     

TNF receptor p55 is required for elimination of inflammatory cells following control of intracellular pathogens.

The elimination of lymphocytes within inflammatory lesions is a critical component in the resolution of disease once pathogens have been cleared. We report here that signaling through the TNF receptor p55 (TNFRp55) is required to eliminate lymphocytes from lesions associated with intracellular pathogens. Thus, TNFRp55-/- mice, but not Fas-deficient mice, maintained inflammatory lesions associated with either Leishmania major or Rhodococcus equi infection, although they developed a Th1 response and controlled the pathogens. Inflammatory cells from either L. major- or R. equi-infected C57BL/6 mice were sensitive to TNF-induced apoptosis, and conversely the number of apoptotic cells in the lesions from TNFRp55-/- mice was dramatically reduced compared with wild-type mice. Furthermore, in vivo depletion of TNF in wild-type mice blocked lesion regression following R. equi infection. Taken together, our results suggest that signaling through the TNFRp55, but not Fas, is required to induce apoptosis of T cells within inflammatory lesions once pathogens are eliminated, and that in its absence lesions fail to regress.     

A low level of TNF-alpha mediates hemorrhage-induced acute lung injury via p55 TNF receptor

Acute lung injury after hemorrhagic shock (HS) is associated with the expression of tumor necrosis factor (TNF)-alpha in the lung. However, the role of TNF-alpha and its receptors in this pulmonary disorder remains obscure. This study examined the temporal relationship of pulmonary TNF-alpha production to neutrophil accumulation during HS and determined the role of TNF-alpha in neutrophil accumulation and lung leak. HS was induced in mice by removal of 30% of total blood volume. Lung TNF-alpha was measured by ELISA. Neutrophil accumulation was detected by immunofluorescent staining, and microvascular permeability was assessed using Evans blue dye. Although HS induced a slight and transient increase in lung TNF-alpha, neutrophil accumulation preceded the increase in TNF-alpha. However, lung neutrophil accumulation and lung leak were abrogated in TNF-alpha knockout mice, and both were restored by administration of recombinant TNF-alpha to TNF-alpha knockout mice before HS. Neutrophil accumulation and lung leak were abrogated in mice lacking the p55 TNF-alpha receptor, but neither was influenced by p75 TNF-alpha receptor knockout. This study demonstrates that a low level of pulmonary TNF-alpha is sufficient to mediate HS-induced acute lung injury during HS and that the p55 TNF-alpha receptor plays a dominant role in regulating the pulmonary inflammatory response to HS.     

Structure/Function analysis of p55 tumor necrosis factor receptor and fas-associated death domain. Effect on necrosis in L929sA cells

Tumor necrosis factor (TNF) induces a typical apoptotic cell death program in various cell lines by interacting with the p55 tumor necrosis factor receptor (TNF-R55). In contrast, triggering of the fibrosarcoma cell line L929sA gives rise to characteristic cellular changes resulting in necrosis. The intracellular domain of TNF-R55 can be subdivided into two parts: a membrane-proximal domain (amino acids 202-325) and a C-terminal death domain (DD) (amino acids 326-413), which has been shown to be necessary and sufficient for apoptosis. Structure/function analysis of TNF-R55-mediated necrosis in L929sA cells demonstrated that initiation of necrotic cell death, as defined by swelling of the cells, rapid membrane permeabilization, absence of nuclear condensation, absence of DNA hypoploidy, and generation of mitochondrial reactive oxygen intermediates, is also confined to the DD. The striking synergistic effect of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone on TNF-induced necrosis was also observed with receptors solely containing the DD. TNF-R55-mediated necrosis is not affected by the dominant negative deletion mutant of the Fas-associated death domain (FADD-(80-205)) that lacks the N-terminal death effector domain. Moreover, overexpression of FADD-(80-205) in L929sA is cytotoxic and insensitive to CrmA, while the cytotoxicity due to overexpression of the deletion mutant FADD-(1-111) lacking the DD is prevented by CrmA. These results demonstrate that the death domain of FADD can elicit an active necrotic cell death pathway.     

Recombinant 55-kDa tumor necrosis factor (TNF) receptor. Stoichiometry of binding to TNF alpha and TNF beta and inhibition of TNF activity.

The extracellular domain of the 55-kDa TNF receptor (rsTNFR beta) has been expressed as a secreted protein in baculovirus-infected insect cells and Chinese hamster ovary (CHO)/dhfr- cells. A chimeric fusion protein (rsTNFR beta-h gamma 3) constructed by inserting the extracellular part of the receptor in front of the hinge region of the human IgG C gamma 3 chain has been expressed in mouse myeloma cells. The recombinant receptor proteins were purified from transfected cell culture supernatants by TNF alpha- or protein G affinity chromatography and gel filtration. In a solid phase binding assay rsTNFR beta was found to bind TNF alpha with high affinity comparable with the membrane-bound full-length receptor. The affinity for TNF beta was slightly impaired. However, the bivalent rsTNFR beta-h gamma 3 fusion protein bound both ligands with a significantly higher affinity than monovalent rsTNFR beta reflecting most likely an increased avidity of the bivalent construct. A molecular mass of about 140 kDa for both rsTNFR beta.TNF alpha and rsTNFR beta.TNF beta complexes was determined in analytical ultracentrifugation studies strongly suggesting a stoichiometry of three rsTNFR beta molecules bound to one TNF alpha or TNF beta trimer. Sedimentation velocity and quasielastic light scattering measurements indicated an extended structure for rsTNFR beta and its TNF alpha and TNF beta complexes. Multiple receptor binding sites on TNF alpha trimers could also be demonstrated by a TNF alpha-induced agglutination of Latex beads coated with the rsTNFR beta-h gamma 3 fusion protein. Both rsTNFR beta and rsTNFR beta-h gamma 3 were found to inhibit binding of TNF alpha and TNF beta to native 55- and 75-kDa TNF receptors and to prevent TNF alpha and TNF beta bioactivity in a cellular cytotoxicity assay. Concentrations of rsTNFR beta-h gamma 3 equimolar to TNF alpha were sufficient to neutralize TNF activity almost completely, whereas a 10-100-fold excess of rsTNFR beta was needed for similar inhibitory effects. In view of their potent TNF antagonizing activity, recombinant soluble TNF receptor fragments might be useful as therapeutic agents in TNF-mediated disorders.     

Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor.

The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4 beta-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct.     

Identification of tumor necrosis factor (TNF) amino acids crucial for binding to the murine p75 TNF receptor and construction of receptor-selective mutants.

The bioactivity of tumor necrosis factor (TNF) is mediated by two TNF receptors (TNF-Rs), more particularly TNF-RI and TNF-RII. Although human TNF (hTNF) and murine TNF (mTNF) are very homologous, hTNF binds only to mTNF-RI. By measuring the binding of a panel of mTNF/hTNF chimeras to both mTNF-R, we pinpointed the TNF region that mediates the interaction with mTNF-RII. Using site-specific mutagenesis, we identified amino acids 71-73 and 89 as the main interacting residues. Mutein hTNF-S71D/T72Y/H73 Delta/T89E interacts with both types of mTNF-R and is active in CT6 cell proliferation assays mediated by mTNF-RII. Mutein mTNF-D71S/Y72T/Delta 73H/E89T binds to mTNF-RI only and is no longer active on CT6 cells. However, the L929s cytotoxicity of this mutein (an effect mediated by mTNF-RI triggering) was also 100-fold lower than that of wild-type mTNF due to enhanced dissociation during incubation at subnanomolar concentrations. The additional mutation of amino acid 102, resulting in the mutein mTNF-D71S/Y72T/Delta 73H/E89T/P102Q, restored the trimer stability, which led to an enhanced specific activity on L929s cells. Hence the specific activity of a TNF species is governed not only by its receptor binding characteristics but also by its trimer stability after incubation at subnanomolar concentrations. In conclusion, the mutation of TNF amino acids 71-73, 89, and 102 is sufficient to obtain a mTNF mutein selective for mTNF-RI and a hTNF mutein that, unlike wild-type hTNF, also acts on mTNF-RII.     

Structure-activity relationship of N-methyl-bisindolylmaleimide derivatives as cell death inhibitors

A series of N-methyl-bisindolylmaleimide derivatives was synthesized and evaluated as cell death inhibitors. N-Methyl-2-[1-(3-aminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide (21) was the most potent inhibitor of H2O2-induced necrotic death of human leukemia HL60 cells among them.     

Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants.

p53 is a conformationally flexible sequence-specific DNA binding protein mutated in many human tumors. To understand why the mutant p53 proteins associated with human tumors fail to bind DNA, we mapped the DNA binding domain of wild-type p53 and examined its regulation by changes in the protein conformation. Using site-directed mutagenesis, residues 90-286 of mouse p53 were shown to form the sequence-specific DNA binding domain. Two highly conserved regions within this domain, regions IV and V, were implicated in contacting DNA. Wild-type p53 bound DNA as a tetramer, each subunit recognizing five nucleotides of the 20 nucleotide-long DNA site. Conformational shifts of the oligomerization domain propagated to the tetrameric DNA binding domain, regulating DNA binding activity, but did not affect the subunit stoichiometry of wild-type p53 oligomers. Interestingly, conformational shifts could also be propagated within certain p53 mutants, rescuing DNA binding. One of these mutants was the mouse equivalent of human histidine 273, which is frequently associated with human tumors.