ABSTRACT
NF-kappaB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-kappaB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-kappaB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-kappaB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-kappaB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-kappaB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.
Subject(s)
Acute-Phase Proteins/metabolism , Gene Expression Regulation, Neoplastic , Lipocalins/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins/metabolism , Thyroid Neoplasms/enzymology , Thyroid Neoplasms/pathology , Acute-Phase Proteins/genetics , Cell Line, Tumor , Cell Survival , Health , Humans , I-kappa B Kinase/metabolism , Immunohistochemistry , Lipocalin-2 , Lipocalins/genetics , Proteomics , Proto-Oncogene Proteins/genetics , RNA, Small Interfering/genetics , Thyroid Neoplasms/geneticsABSTRACT
The conformational stability of the rat thyroid transcription factor 1 homeodomain, TTF-1HD, has been investigated by means of circular dichroism (CD) and differential scanning calorimetry (DSC) measurements at pH 5.0 as a function of KCl concentration. Thermal unfolding of TTF-1HD is a reversible two-state transition. The protein is not stable against temperature, showing a denaturation temperature of 32 degrees C in the absence of salt and 50 degrees C at 75 mM KCl. The binding energetics of TTF-1HD to its target DNA sequence has been characterized by means of isothermal titration calorimetry (ITC) measurements, complemented with CD data. At 25 degrees C, pH 5.0 and 75 mM KCl, the binding constant amounts to 1.5 x 10(8)M(-1) and the binding enthalpy change amounts to -41 kJ mol(-1). The process is enthalpy driven, but also the entropy change is favorable to complex formation. To gain a molecular level understanding of the interactions determining the association of TTF-1HD to the target DNA sequence structural information would be requested, but it is not yet available. Therefore, structural models of two complexes, TTF-1HD with the target DNA sequence and TTF-1HD with a modified DNA sequence, have been constructed by using as a template the NMR structure of the complex between NK-2 HD and its target DNA, and by performing molecular dynamics simulations 3.5 ns long. Analysis of these models allows one to shed light on the origin of the DNA binding specificity characteristic of TTF-1HD.
Subject(s)
DNA/chemistry , Homeodomain Proteins/chemistry , Nuclear Proteins/chemistry , Thermodynamics , Transcription Factors/chemistry , Animals , Binding Sites , Calorimetry , Circular Dichroism , DNA/metabolism , Homeodomain Proteins/metabolism , Models, Molecular , Nuclear Proteins/metabolism , Protein Conformation , Rats , Thyroid Nuclear Factor 1 , Transcription Factors/metabolismABSTRACT
Nuclear factor kappaB (NF-kappaB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-kappaB is dependent on the phosphorylation of the inhibitory subunit IkappaBalpha that is mediated by a multimeric, high molecular weight complex, called IkappaB kinase (IKK) complex. This is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, NEMO/IKKgamma. The latter protein is essential for the activation of IKKs and NF-kappaB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-kappaB) as a NEMO/IKKgamma-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-kappaB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-kappaB at the level of the IKK complex and that A20 inhibits activation of NF-kappaB by de-ubiquitination of NEMO/IKKgamma. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKgamma and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-kappaB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKgamma and facilitates A20-mediated de-ubiquitination of NEMO/IKKgamma, thus resulting in inhibition of NF-kappaB.
Subject(s)
DNA-Binding Proteins/metabolism , I-kappa B Kinase/metabolism , NF-kappa B/metabolism , Proteins/metabolism , Animals , Binding Sites , Catalytic Domain/genetics , Cell Line , Cysteine Endopeptidases , DNA-Binding Proteins/genetics , Humans , I-kappa B Kinase/genetics , Intracellular Signaling Peptides and Proteins , Mice , Mutation , NF-kappa B/antagonists & inhibitors , Nuclear Proteins , Protein Binding , Protein Interaction Mapping , Protein Structure, Tertiary/genetics , Proteins/genetics , Rabbits , Tumor Necrosis Factor alpha-Induced Protein 3 , UbiquitinABSTRACT
Gastric mucosa responds to Helicobacter pylori-induced cell damage by increasing the expression of COX-2 and EGF-related peptides. We sought to investigate the bacterial virulence factor/s and the host cellular pathways involved in the upregulation of COX-2, HB-EGF and amphiregulin in MKN 28 and AGS gastric mucosal cells. H. pylori strain CCUG 17874 was grown in Brucella broth supplemented with 0.2% (2,6-dimethyl)-beta-cyclodextrins. The soluble proteins released in the culture medium by the bacterium were fractionated by exclusion size and anion exchange chromatography. A single peak retaining the ability to upregulate COX-2 and HB-EGF mRNA and protein expression was obtained. SDS-PAGE analysis of the peak showed two peptides with an apparent molecular weight of 38 and 22 kDa, which were identified by automated Edman degradation analysis as the N-terminal and C-terminal peptides of H. pylori gamma-glutamyltranspeptidase respectively. Acivicin, a selective gamma-glutamyltranspeptidase inhibitor, counteracted H. pylori-induced upregulation of COX-2 and EGF-related peptide mRNA expression. An H. pylori isogenic mutant gamma-glutamyltranspeptidase-deficient strain did not exert any effect on COX-2, HB-EGF and amphiregulin mRNA expression. Blockade of phosphatidylinositol-3 kinase and p38 kinase, but not MAP kinase kinase, inhibited H. pylori gamma-glutamyltranspeptidase-induced upregulation of COX-2 and EGF-related peptide mRNA expression.