Polymorphism of the Tumor Necrosis Factor α Gene in Patients with Infiltrative Tuberculosis from the Bashkortostan Populations

The polymorphism at position –308 of the TNF- gene promoter was analyzed in three ethnic groups and in patients with infiltrative pulmonary tuberculosis from Bashkortostan. No interethnic difference in allele or genotype frequency distribution was observed. The frequency of allele TNF2 in tuberculosis patients was significantly higher than in controls (² = 11.69, p = 0.001), suggesting association of this allele with higher risk of pulmonary tuberculosis or with markedly disturbed immune response.     

The IVVY Motif and Tumor Necrosis Factor Receptor-associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor κB (RANK) Cooperate to Induce Osteoclastogenesis

Receptor activator of NF-κB (RANK) activation by RANK ligand (RANKL) mediates osteoclastogenesis by recruiting TNF receptor-associated factors (TRAFs) via three cytoplasmic motifs (motif 1, PFQEP^369–373; motif 2, PVQEET^559–564; and motif 3, PVQEQG^604–609) to activate the NF-κB and MAPK signaling pathways. RANK also has a TRAF-independent motif (IVVY^535–538), which is dispensable for the activation of TRAF-induced signaling pathways but essential for osteoclast lineage commitment by inducing the expression of nuclear factor of activated T-cells c1 (NFATc1) to regulate osteoclast gene expression. Notably, TNF/IL-1-mediated osteoclastogenesis requires RANK ligand assistance, and the IVVY motif is also critical for TNF/IL-1-mediated osteoclastogenesis by rendering osteoclast genes responsive to these two cytokines. Here we show that the two types of RANK cytoplasmic motifs have to be on the same RANK molecule to mediate osteoclastogenesis, suggesting a functional cooperation between them. Subsequent osteoclastogenesis assays with TNF or IL-1 revealed that, although all three TRAF motifs play roles in TNF/IL-1-mediated osteoclastogenesis, motifs 2 and 3 are more potent than motif 1. Accordingly, inactivation of motifs 2 and 3 blocksTNF/IL-1-mediated osteoclastogenesis. Mechanistically, double mutation of motifs 2 and 3, similar to inactivation of the IVVY motif, abrogates the expression of nuclear factor of activated T-cells c1 and osteoclast genes in assays reflecting RANK-initiated and TNF/IL-1-mediated osteoclastogenesis. In contrast, double inactivation of motifs 2 and 3 did not affect the ability of RANK to activate the NF-κB and MAPK signaling pathways. Collectively, these results indicate that the RANK IVVY motif cooperates with the TRAF-binding motifs to promote osteoclastogenesis, which provides novel insights into the molecular mechanism of RANK signaling in osteoclastogenesis.     

Engineering Tumor Cells with Tumor Necrosis Factor α (TNF-α) or CD40 Ligand (CD40L) Genes Induce Anti-tumor Immune Responses

International Journal of Peptide Research and Therapeutics - One of the main problems in tumor therapy is immunosuppressive microenvironment of the tumor. To overcome this, we assessed targeted...     

Molecular Genetic Analysis of the Interleukin 6 and Tumor Necrosis Factor α Gene Polymorphisms in Multiple Myeloma

The –174G C polymorphism of the interleukin 6 (IL-6) gene promoter and the –308G A polymorphism of the tumor necrosis factor (TNF) gene promoter were tested for association with multiple myeloma (MM) varying in severity. Of 69 patients, 19 had aggressive MM, 26 had benign MM, and 24 had unidentified MM. The control group (N = 102) matched the test group in age and sex composition. The two groups did not significantly differ in allele and genotype frequencies of the IL-6 and TNF genes. Genotype CC, which determines low-level expression of IL-6, occurred at a frequency of 0.35 in patients with low-progressing MM and was absent from patients with aggressive MM. The TNF gene showed no association with predisposition to MM or clinical variant of the disease. As for MM progression, genotype CC of the IL-6 gene was associated with milder clinical signs in patients from Bashkortostan.     

Tumor Necrosis Factor �� Represses Bone Morphogenetic Protein (BMP) Signaling by Interfering with the DNA Binding of Smads through the Activation of NF-��B

Bone morphogenetic proteins (BMPs) induce not only bone formation in vivo but also osteoblast differentiation of mesenchymal cells in vitro. Tumor necrosis factor α (TNFα) inhibits both osteoblast differentiation and bone formation induced by BMPs. However, the molecular mechanisms of these inhibitions remain unknown. In this study, we found that TNFα inhibited the alkaline phosphatase activity and markedly reduced BMP2- and Smad-induced reporter activity in MC3T3-E1 cells. TNFα had no effect on the phosphorylation of Smad1, Smad5, and Smad8 or on the nuclear translocation of the Smad1-Smad4 complex. In p65-deficient mouse embryonic fibroblasts, overexpression of p65, a subunit of NF-κB, inhibited BMP2- and Smad-induced reporter activity in a dose-dependent manner. Furthermore, this p65-mediated inhibition of BMP2- and Smad-responsive promoter activity was restored after inhibition of NF-κB by the overexpression of the dominant negative IκBα. Although TNFα failed to affect receptor-dependent formation of the Smad1-Smad4 complex, p65 associated with the complex. Chromatin immunoprecipitation and electrophoresis mobility shift assays revealed that TNFα suppressed the DNA binding of Smad proteins to the target gene. Importantly, the specific NF-κB inhibitor, BAY11-7082, abolished these phenomena. These results suggest that TNFα inhibits BMP signaling by interfering with the DNA binding of Smads through the activation of NF-κB.     

Kinetic and Equilibrium Studies of (-)125Iodocyanopindolol Binding to β-Adrenoceptors on Human Lymphocytes: Evidence for the Existence of two Classes of Binding Sites

Abstract

Saturation experiments were performed on intact human peripheral mononuclear leucocytes (MNL) and MNL membranes with (-)¹²⁵Iodocyanopindolol (¹²⁵ICYP) over a large concentration range (1.5-600pmol/l). The corresponding Scatchard plots were curvilinear suggesting two saturable classes of binding sites: A high affinity binding site (B_max1=1000±400 sites/cell, K_d1= 2.1±0.9 pmol/l for intact MNL and B_max1=550±190 sites/cell, K_d1=4.1±0.9 pmol/l for MNL membranes)and a low affinity binding site (B_max2=9150±3590 binding sites/cell, K_d2=440±50 pmol/l for intact MNL and B_max2=11560±4690 sites/cell, K_d2=410±70 pmol/l for MNL membranes). Dissociation of (-)¹²⁵ICYP from MNL was biphasic consisting of a slow dissociating component (dissociation rate constant k_-1=(0.5±0.2)x10^?3 min^?1 for intact MNL and k_-1=(1.0±0.1)x10^?3min^?1 for MNL membranes) and a fast dissociating component (k_-2=(80±20)x10^?3min^?1 for intact MNL and k_-2=(60±10)x10^?3min^?1 for MNL membranes). In dissociation experiments started after equilibration with various (-)¹²⁵ICYP concentrations k_-1 and k_-2 were independent of the equilibrium concentration, whereas the percentual occupancy of the slow and the fast dissociating component varied and was similar to the estimated fractional occupancy of either binding site at the same (-)¹²⁵ICYP concentrations in saturation experiments. The association rate constant was in the same order of magnitude for both binding sites. These results suggest two independent classes of binding sites for (-)¹²⁵ICYP on MNL.     

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.     

β-Transducin Repeat-containing Protein 1 (β-TrCP1)-mediated Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Protein Degradation Promotes Tumor Necrosis Factor α (TNFα)-induced Inflammatory Gene Expression

Cytokine modulation of the endothelium is considered an important contributor to the inflammation response. TNFα is an early response gene during the initiation of inflammation. However, the detailed mechanism by which TNFα induces proinflammatory gene expression is not completely understood. In this report, we demonstrate that silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) represses the expression of a subset of TNFα target genes in human umbilical vein endothelial cells. Upon TNFα stimulation, we observed an increase in the E3 ubiquitin ligase β-TrCP1 and a decrease in SMRT protein levels. We show that β-TrCP1 interacts with SMRT in a phosphorylation-independent manner and cooperates with the E2 ubiquitin-conjugating enzyme E2D2 to promote ubiquitination-dependent SMRT degradation. Knockdown of β-TrCP1 increases SMRT protein accumulation, increases SMRT association with its targeted promoters, and decreases SMRT target gene expression. Taken together, our results support a model in which TNFα-induced β-TrCP1 accumulation promotes SMRT degradation and the subsequent induction of proinflammatory gene expression.     

Influence of GABA(A) Receptor α Subunit Isoforms on the Benzodiazepine Binding Site

Classical benzodiazepines, such as diazepam, interact with α_xβ₂γ₂ GABA_A receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and with receptors containing the homologous mutations in α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂. The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α₁S204Cβ₂γ₂, α₁S205Cβ₂γ₂ and α₁T206Cβ₂γ₂ and homologous positions in α₂, α₃, α₅ and α₆ with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA_A receptor isoform α₁β₂γ₂, α₂β₂γ₂, α₃β₂γ₂, α₅β_1/2γ₂ and α₆β₂γ₂.     

The Suppressive Effect of β-Glucan on the Production of Tumor Necrosis Factor-Alpha in BV2 Microglial Cells

β-Glucan was recently shown to have the ability to enhance and stimulate the immune system in humans, but little is known about its the anti-inflammatory effects. We investigated the effect of β-glucan on the production of tumor necrosis factor-alpha (TNF-α), a major pro-inflammatory mediator, in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. β-Glucan decreased the production and expression of TNF-α. In addition, it blocked LPS-stimulated activation of nuclear factor kappa B (NF-κB). Hence β-glucan might suppress LPS-stimulated TNF-α production by inhibiting NF-κB in BV2 microglial cells.     

Characterization of Single-Nucleotide Polymorphisms in the Tumor Necrosis Factor α Promoter Region and in Lymphotoxin α in Squamous Intraepithelial Lesions, Precursors of Cervical Cancer

Development of cervical cancer is a long process of abnormal cancerous cell growth in the cervix and is primarily the result of infection with specific high-risk types of human papillomavirus (HPV). The cytokines tumor necrosis factor α (TNFα) and lymphotoxin α (LTA) have an important role in all stages of cervical cancer and have the ability to induce the regression or promote the development of human tumors. Biologically important single-nucleotide polymorphisms (SNPs) occur within the TNFα and LTA genes. Therefore, the purpose of this study was to investigate the SNPs in the TNFα promoter region (-163, -238, -244, -308, -376, -857, -863, and -1031) and in the first intron of LTA (+252) in women with precursor lesions of cervical cancer. Overall, we studied 396 women from Mexico City. A total of 191 patients with HPV infection and precursor cervical lesions were subdivided in two groups: those with low-grade squamous intraepithelial lesions (n = 132) and those with high-grade squamous intraepithelial lesions (n = 59). Women (n = 205) negative for HPV and without cervical lesions were also included in the study. DNA was extracted from peripheral white blood cells and from cervical samples, and detection of biallelic polymorphisms of TNFα and LTA was performed using the polymerase chain reaction-sequence-specific oligonucleotide probe and restriction fragment length polymorphism techniques, respectively. We demonstrated that risk is associated with the genotype G/A (odds ratio = 2.48) and that protection is associated with the genotype G/G of SNP TNFα -376 (odds ratio = 0.37).     

γ-Secretase Activity Is Required for Regulated Intramembrane Proteolysis of Tumor Necrosis Factor (TNF) Receptor 1 and TNF-mediated Pro-apoptotic Signaling

The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.