ABSTRACT
T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular Ca(2+) to activate the key transcription factors nuclear factor of activated T lymphocytes (NFAT) and NF-κB. The mechanism of NFAT activation by Ca(2+) has been determined. However, the role of Ca(2+) in controlling NF-κB signaling is poorly understood, and the source of Ca(2+) required for NF-κB activation is unknown. We demonstrate that TCR- but not TNF-induced NF-κB signaling upstream of IκB kinase activation absolutely requires the influx of extracellular Ca(2+) via STIM1-dependent Ca(2+) release-activated Ca(2+)/Orai channels. We further show that Ca(2+) influx controls phosphorylation of the NF-κB protein p65 on Ser-536 and that this posttranslational modification controls its nuclear localization and transcriptional activation. Notably, our data reveal that this role for Ca(2+) is entirely separate from its upstream control of IκBα degradation, thereby identifying a novel Ca(2+)-dependent distal step in TCR-induced NF-κB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca(2+)-dependent PKCα-mediated phosphorylation of p65. Thus, we establish the source of Ca(2+) required for TCR-induced NF-κB activation and define a new distal Ca(2+)-dependent checkpoint in TCR-induced NF-κB signaling that has broad implications for the control of immune cell development and T cell functional specificity.
Subject(s)
Calcium Channels/biosynthesis , Calcium Signaling/physiology , Calcium/metabolism , Membrane Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes/metabolism , Transcription Factor RelA/metabolism , Transcriptional Activation/physiology , Calcium Channels/genetics , Humans , Jurkat Cells , Membrane Proteins/genetics , Neoplasm Proteins/genetics , ORAI1 Protein , Phosphorylation/physiology , Receptors, Antigen, T-Cell/genetics , Stromal Interaction Molecule 1 , Transcription Factor RelA/geneticsABSTRACT
Lung epithelial cells are considered important sources of inflammatory molecules and extracellular matrix proteins that contribute to diseases such as asthma. Understanding the factors that stimulate epithelial cells may lead to new insights into controlling lung inflammation. This study sought to investigate the responsiveness of human lung epithelial cells to the TNF family molecules LIGHT and lymphotoxin αß (LTαß). Bronchial and alveolar epithelial cell lines, and primary human bronchial epithelial cells, were stimulated with LIGHT and LTαß, and expression of inflammatory cytokines and chemokines and markers of epithelial-mesenchymal transition and fibrosis/remodeling was measured. LTß receptor, the receptor shared by LIGHT and LTαß, was constitutively expressed on all epithelial cells. Correspondingly, LIGHT and LTαß strongly induced a limited but highly distinct set of inflammatory genes in all epithelial cells tested, namely the adhesion molecules ICAM-1 and VCAM-1; the chemokines CCL5, CCL20, CXCL1, CXCL3, CXCL5, and CXCL11; the cytokines IL-6, activin A and GM-CSF; and metalloproteinases matrix metalloproteinase-9 and a disintegrin and metalloproteinase domain-8. Importantly, induction of the majority of these inflammatory molecules was insensitive to the suppressive effects of the corticosteroid budesonide. LIGHT and LTαß also moderately downregulated E-cadherin, a protein associated with maintaining epithelial integrity, but did not significantly drive production of extracellular matrix proteins or α-smooth muscle actin. Thus, LIGHT and LTαß induce a distinct steroid-resistant inflammatory signature in airway epithelial cells via constitutively expressed LTß receptor. These findings support our prior murine studies that suggested the receptors for LIGHT and LTαß contribute to development of lung inflammation characteristic of asthma and idiopathic pulmonary fibrosis.
Subject(s)
Epithelial Cells/drug effects , Glucocorticoids/pharmacology , Inflammation Mediators/metabolism , Lymphotoxin alpha1, beta2 Heterotrimer/pharmacology , Tumor Necrosis Factor Ligand Superfamily Member 14/pharmacology , Bronchi/cytology , Budesonide/pharmacology , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Line , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cells, Cultured , Chemokines/genetics , Chemokines/metabolism , Cytokines/genetics , Cytokines/metabolism , Drug Resistance , Epithelial Cells/metabolism , Flow Cytometry , Gene Expression/drug effects , Humans , Immunoblotting , Lymphocytes/drug effects , Lymphocytes/metabolism , Monocytes/drug effects , Monocytes/metabolism , Receptors, Leukotriene B4/genetics , Receptors, Leukotriene B4/metabolism , Receptors, Tumor Necrosis Factor, Member 14/genetics , Receptors, Tumor Necrosis Factor, Member 14/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The signaling and adaptor protein Homer3 plays a role in controlling immune homeostasis and self-reactivity. Homer3 is recruited to the immune synapse (IS) following TCR ligation, although the mechanisms regulating this subcellular localization are unknown. We show that Homer3 specifically associates with a novel ubiquitin-like domain in the IkappaB kinase (IKK) beta subunit of the IKK complex. Homer3 associates with IKKbeta in T cells and colocalizes with the IKK complex at the IS. However, Homer3 is not required for IKK activation, as NF-kappaB signaling is intact in Homer3-deficient T cells. Instead, the IKK complex recruits Homer3 to the IS following TCR engagement, and we present evidence that this association regulates actin dynamics in T cells. These findings identify a novel interaction between two major signaling proteins and reveal an unexpected NF-kappaB-independent function for the IKK complex in regulating the subcellular localization of Homer3.
Subject(s)
Carrier Proteins/metabolism , I-kappa B Proteins/metabolism , Carrier Proteins/physiology , Cell Line , HeLa Cells , Homer Scaffolding Proteins , Humans , I-kappa B Proteins/physiology , Immunological Synapses/enzymology , Immunological Synapses/immunology , Jurkat Cells , Protein Structure, Tertiary , Receptors, Antigen, T-Cell/physiology , Signal Transduction/immunology , Subcellular Fractions/immunology , Subcellular Fractions/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolismABSTRACT
Ligation of the lymphotoxin-ß receptor (LTßR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)ß (IKKß(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKß(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.
Subject(s)
Cell Nucleus/metabolism , Chemokine CXCL12/biosynthesis , Endothelial Cells/metabolism , Signal Transduction/physiology , Transcription Factor RelB/metabolism , Umbilical Veins/metabolism , Up-Regulation/physiology , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Cell Line , Cell Nucleus/genetics , Chemokine CXCL12/genetics , Endothelial Cells/cytology , Humans , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Mutation, Missense , Signal Transduction/drug effects , Transcription Factor RelB/genetics , Tumor Necrosis Factor Ligand Superfamily Member 14/genetics , Tumor Necrosis Factor Ligand Superfamily Member 14/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Umbilical Veins/cytology , Up-Regulation/drug effectsABSTRACT
Proinflammatory NF-kappaB activation requires the IkappaB (inhibitor of NF-kappaB) kinase (IKK) complex that contains two catalytic subunits named IKKalpha and IKKbeta and a regulatory subunit named NF-kappaB essential modulator (NEMO). NEMO and IKKbeta are essential for tumor necrosis factor (TNF)-induced NF-kappaB activation, and we recently demonstrated that NEMO and IKKalpha are sufficient for interleukin (IL)-1-induced signaling. IKKalpha and IKKbeta both contain a functional NEMO-binding domain (NBD); however, the role of NEMO association with each kinase in NF-kappaB signaling and IKK complex formation remains unclear. To address this question, we stably reconstituted IKKalpha(-/-) and IKKbeta(-/-) murine embryonic fibroblasts (MEFs) with wild-type (WT) or NBD-deficient (DeltaNBD) versions of IKKalpha and IKKbeta, respectively. TNF-induced classical NF-kappaB activation in IKKbeta(-/-) MEFs was rescued by IKKbeta(WT) but not IKKbeta(DeltaNBD), whereas neither IKKbeta(WT) nor IKKbeta(DeltaNBD) affected IL-1-induced NF-kappaB signaling. As previously described, classical NF-kappaB transcriptional activity was absent in IKKalpha(-/-) cells. Reconstitution with either IKKalpha(WT) or IKKalpha(DeltaNBD) rescued both IL-1 and TNF-induced transcription, demonstrating that NEMO association is not required for IKKalpha-dependent regulation of NF-kappaB-dependent transcription. Stably expressed IKKalpha(WT) or IKKbeta(WT) associated with endogenous IKKs and NEMO in IKKalpha(-/-) or IKKbeta(-/-) MEFs, respectively, resulting in formation of the heterotrimeric IKKalpha-IKKbeta-NEMO complex. In contrast, although the IKKalpha(DeltaNBD) and IKKbeta(DeltaNBD) mutants associated with endogenous IKKs containing an NBD, these dimeric endogenous IKK-IKK(DeltaNBD) complexes did not associate with NEMO. These findings therefore demonstrate that formation of the heterotrimeric IKKalpha-IKKbeta-NEMO holocomplex absolutely requires two intact NEMO-binding domains.
Subject(s)
I-kappa B Kinase/chemistry , I-kappa B Kinase/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , NF-kappa B/metabolism , Animals , Cell Line , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , I-kappa B Kinase/genetics , Interleukin-1/pharmacology , Mice , Protein Structure, Tertiary , Sequence Deletion , Signal Transduction , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
To identify Janus kinase (JAK) inhibitors that selectively target gastrointestinal tissues with limited systemic exposures, a class of imidazopyrrolopyridines with a range of physical properties was prepared and evaluated. We identified compounds with low intrinsic permeability and determined a correlation between permeability and physicochemical properties, clogP and tPSA, for a subset of compounds. This low intrinsic permeability translated into compounds displaying high colonic exposure and low systemic exposure after oral dosing at 25 mg/kg in mouse. In a mouse PK/PD model, oral dosing of lead compound 2 demonstrated dose-dependent inhibition of pSTAT phosphorylation in colonic explants post-oral dose but low systemic exposure and no measurable systemic pharmacodynamic activity. We thus demonstrate the utility of JAK inhibitors with low intrinsic permeability as a feasible approach to develop gut-restricted, pharmacologically active molecules with a potential advantage over systemically available compounds that are limited by systemic on-target adverse events.
Subject(s)
Inflammatory Bowel Diseases/drug therapy , Janus Kinase Inhibitors/pharmacology , Janus Kinase Inhibitors/pharmacokinetics , Pyridines/pharmacology , Pyridines/pharmacokinetics , Administration, Oral , Animals , Dogs , Drug Discovery , Female , Humans , Inflammatory Bowel Diseases/metabolism , Janus Kinase Inhibitors/administration & dosage , Janus Kinase Inhibitors/chemistry , Janus Kinases/antagonists & inhibitors , Janus Kinases/metabolism , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred C57BL , Permeability , Phosphorylation/drug effects , Pyridines/administration & dosage , Pyridines/chemistryABSTRACT
Nuclear factor kappa B (NF-kappaB) is an inducible transcription factor that regulates the expression of many genes involved in normal immune and inflammatory responses. NF-kappaB activation is normally a rapid and transient response to pro-inflammatory stimuli however dysregulated constitutively active NF-kappaB signaling leads to chronic inflammation and provides a cell survival signal in many types of cancer. NF-kappaB signaling is therefore an important target for the development of novel anti-inflammatory or anti-cancer drugs. We previously identified and characterized a cell-permeable peptide that blocks NF-kappaB signaling by disrupting the critical upstream IkappaB kinase (IKK) complex. We describe in this chapter three separate methods to determine the effects of this NEMO-binding domain (NBD) peptide on pro-inflammatory NF-kappaB signaling in response to tumor necrosis factor (TNF).
Subject(s)
I-kappa B Kinase/pharmacology , NF-kappa B/antagonists & inhibitors , Signal Transduction/drug effects , Cells, Cultured , Electrophoretic Mobility Shift Assay , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , I-kappa B Kinase/metabolism , Immunoblotting , Luciferases/metabolism , Protein Structure, Tertiary , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND: Human hypomorphic nuclear factor-kappaB essential modulator (NEMO) mutations cause diverse clinical and immunologic phenotypes, but understanding of their scope and mechanistic links to immune function and genotype is incomplete. OBJECTIVE: We created and analyzed a database of hypomorphic NEMO mutations to determine the spectrum of phenotypes and their associated genotypes and sought to establish a standardized NEMO reconstitution system to obtain mechanistic insights. METHODS: Phenotypes of 72 individuals with NEMO mutations were compiled. NEMO L153R and C417R were investigated further in a reconstitution system. TNF-alpha or Toll-like receptor (TLR)-5 signals were evaluated for nuclear factor-kappaB activation, programmed cell death, and A20 gene expression. RESULTS: Thirty-two different mutations were identified; 53% affect the zinc finger domain. Seventy-seven percent were associated with ectodermal dysplasia, 86% with serious pyogenic infection, 39% with mycobacterial infection, 19% with serious viral infection, and 23% with inflammatory diseases. Thirty-six percent of individuals died at a mean age of 6.4 years. CD40, IL-1, TNF-alpha, TLR, and T-cell receptor signals were impaired in 15 of 16 (94%), 6 of 7 (86%), 9 of 11 (82%), 9 of 14 (64%), and 7 of 18 (39%), respectively. Hypomorphism-reconstituted NEMO-deficient cells demonstrated partial restoration of NEMO functions. Although both L153R and C417R impaired TLR and TNF-alpha-induced NF-kappaB activation, L153R also increased TNF-alpha-induced programmed cell death with decreased A20 expression. CONCLUSION: Distinct NEMO hypomorphs define specific disease and genetic characteristics. A reconstitution system can identify attributes of hypomorphisms independent of an individual's genetic background. Apoptosis susceptibility in L153R reconstituted cells defines a specific phenotype of this mutation that likely contributes to the excessive inflammation with which it is clinically associated.
Subject(s)
Databases, Genetic , I-kappa B Kinase/genetics , I-kappa B Kinase/metabolism , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/metabolism , Mutation, Missense , Amino Acid Substitution , Apoptosis/genetics , CD40 Antigens/metabolism , DNA-Binding Proteins , Gene Expression Regulation/genetics , Genetic Complementation Test/methods , Genotype , Humans , Intracellular Signaling Peptides and Proteins , Jurkat Cells , NF-kappa B/metabolism , Nuclear Proteins/biosynthesis , Phenotype , Protein Structure, Tertiary/genetics , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/genetics , Toll-Like Receptor 5/metabolism , Tumor Necrosis Factor alpha-Induced Protein 3 , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The cytokine TNFSF14 [homologous to Lymphotoxin, exhibits Inducible expression and competes with HSV Glycoprotein D for binding to HVEM, a receptor expressed on T lymphocytes (LIGHT)] has been shown in mouse models to be important for development of lung tissue remodeling that is characteristic of asthma, idiopathic pulmonary fibrosis (IPF), and systemic sclerosis (SSc). However, its cellular targets are not fully delineated. In the present report, we show that LTßR and HVEM, the receptors for LIGHT, are constitutively expressed in primary human lung fibroblasts (HLFs). We asked whether LIGHT could promote inflammatory and remodeling-relevant activity in HLFs and how this was similar to, or distinct from, IL-13 or TGF-ß, two cytokines strongly implicated in the pathogenesis of asthma, IPF, and SSc. Accumulation of myofibroblasts expressing alpha smooth muscle actin is a feature of lung inflammatory diseases. LIGHT promoted cell cycle progression and proliferation of HLFs, but not alpha smooth muscle actin expression. In contrast, TGF-ß upregulated alpha smooth muscle actin but did not drive their proliferation. LIGHT also increased the gene or protein expression of a number of proinflammatory mediators, including ICAM-1 and VCAM-1, IL-6 and GM-CSF, the chemokines CCL5 and 20, and CXCL5, 11, and 12, and lung remodeling-associated proteinases MMP-9 and ADAM8. These were dependent on LTßR but not HVEM. LIGHT displayed overlapping and synergistic activities with IL-13 for a number of the activities, but LIGHT additionally enhanced the gene expression of several molecules, including the innate cytokines IL-33 and TSLP, which were not upregulated by IL-13. Our results highlight the varied and pleiotropic effects of LIGHT in HLFs. LIGHT might then be a therapeutic target for modulation of inflammation and remodeling associated with asthma and other similar diseases of the lung that involve fibroblasts.
Subject(s)
Fibroblasts/metabolism , Inflammation Mediators/metabolism , Interleukin-13/metabolism , Lung/metabolism , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor Ligand Superfamily Member 14/metabolism , Biomarkers , Cell Line , Cell Proliferation/drug effects , Cells, Cultured , Cytokines/metabolism , Fibroblasts/drug effects , Gene Expression , Humans , Inflammation Mediators/pharmacology , Interleukin-13/genetics , Lung/pathology , Transforming Growth Factor beta/genetics , Tumor Necrosis Factor Ligand Superfamily Member 14/genetics , Tumor Necrosis Factor Ligand Superfamily Member 14/pharmacologyABSTRACT
Activation of the classical and noncanonical NF-kappaB pathways by ligation of the lymphotoxin (LT)-beta receptor (LTbetaR) plays a crucial role in lymphoid organogenesis and in the generation of ectopic lymphoid tissue at sites of chronic inflammation. Within these microenvironments, LTbetaR signaling regulates the phenotype of the specialized high endothelial cells. However, the direct effects of LTbetaR ligation on endothelial cells remain unclear. We therefore questioned whether LTbetaR ligation could directly activate endothelial cells and regulate classical and noncanonical NF-kappaB-dependent gene expression. We demonstrate that the LTbetaR ligands LIGHT and LTalpha1beta2 activate both NF-kappaB pathways in HUVECs and human dermal microvascular endothelial cells (HDMEC). Classical pathway activation was less robust than TNF-induced signaling; however, only LIGHT and LTalpha1beta2 and not TNF activated the noncanonical pathway. LIGHT and LTalpha1beta2 induced the expression of classical NF-kappaB-dependent genes in HUVEC, including those encoding the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Consistent with this stimulation, LTbetaR ligation up-regulated T cell adhesion to HUVEC. Furthermore, the homeostatic chemokine CXCL12 was up-regulated by LIGHT and LTalpha1beta2 but not TNF in both HUVEC and HDMEC. Using HUVEC retrovirally transduced with dominant negative IkappaB kinase alpha, we demonstrate that CXCL12 expression is regulated by the noncanonical pathway in endothelial cells. Our findings therefore demonstrate that LTbetaR ligation regulates gene expression in endothelial cells via both NF-kappaB pathways and we identify CXCL12 as a bona fide noncanonical NF-kappaB-regulated gene in these cells.
Subject(s)
Endothelium, Vascular/immunology , Endothelium, Vascular/pathology , Gene Expression Regulation/immunology , Inflammation Mediators/physiology , Lymphotoxin alpha1, beta2 Heterotrimer/physiology , NF-kappa B/physiology , Signal Transduction/immunology , Tumor Necrosis Factor Ligand Superfamily Member 14/physiology , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Adhesion Molecules/biosynthesis , Cell Adhesion Molecules/genetics , Cell Line , Endothelium, Vascular/metabolism , Humans , Ligands , Lymphotoxin alpha1, beta2 Heterotrimer/metabolism , Lymphotoxin beta Receptor/biosynthesis , Lymphotoxin beta Receptor/metabolism , Lymphotoxin beta Receptor/physiology , Lymphotoxin-alpha/metabolism , Lymphotoxin-alpha/physiology , Lymphotoxin-beta/metabolism , Lymphotoxin-beta/physiology , NF-kappa B/metabolism , Signal Transduction/genetics , Skin/blood supply , Skin/cytology , Skin/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocyte Subsets/pathology , Tumor Necrosis Factor Ligand Superfamily Member 14/metabolism , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
Cells lacking functional NF-kappaB die after ligation of some tumor necrosis factor (TNF) receptor family members through failure to express NF-kappaB-dependent anti-apoptotic genes. NF-kappaB activation requires the IkappaB kinase (IKK) complex containing two catalytic subunits named IKKalpha and IKKbeta that regulate distinct NF-kappaB pathways. IKKbeta is critical for classical signaling that induces pro-inflammatory and anti-apoptotic gene profiles, whereas IKKalpha regulates the non-canonical pathway involved in lymphoid organogenesis and B-cell development. To determine whether IKKalpha and IKKbeta differentially function in rescuing cells from death induced by activators of the classical and non-canonical pathways, we analyzed death after ligation of the TNF and lymphotoxin-beta receptors, respectively. Using murine embryonic fibroblasts (MEFs) lacking each of the IKKs, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, and dominant negative Fas-associated death domain protein, we found that deletion of these kinases sensitized MEFs to distinct cell death pathways. MEFs lacking IKKalpha were sensitized to death in response to both cytokines that was entirely caspase-dependent, demonstrating that IKKalpha functions in this process. Surprisingly, death of IKKbeta-/- MEFs was not blocked by caspase inhibition, demonstrating that IKKbeta negatively regulates caspase-independent cell death (CICD). CICD was strongly activated by both TNF and lymphotoxin-beta receptor ligation in IKKbeta-/- MEFs and was accompanied by loss of mitochondrial membrane potential and the generation of reactive oxygen species. CICD was inhibited by the anti-oxidant butylated hydroxyanosole and overexpression of Bcl-2, neither of which blocked caspase-dependent apoptosis. Our findings, therefore, demonstrate that both IKKalpha and IKKbeta regulate cytokine-induced apoptosis, and IKKbeta additionally represses reactive oxygen species- and mitochondrial-dependent CICD.
Subject(s)
Apoptosis/genetics , Caspases/metabolism , Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Protein Serine-Threonine Kinases/deficiency , Reactive Oxygen Species/metabolism , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Antioxidants/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Caspase Inhibitors , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Embryo, Mammalian/cytology , Fibroblasts/cytology , I-kappa B Kinase/deficiency , I-kappa B Kinase/metabolism , Lymphotoxin beta Receptor/agonists , Lymphotoxin beta Receptor/metabolism , Mice , Mice, Knockout , Mitochondria/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Organogenesis/drug effects , Organogenesis/genetics , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Receptor Aggregation/drug effects , Receptor Aggregation/genetics , Tumor Necrosis Factor-alpha/pharmacology , NF-kappaB-Inducing KinaseABSTRACT
Studies of patients with congenital immunodeficiency due to mutation of the NF-kappaB essential modulator (NEMO) gene have demonstrated that NEMO integrity is required for NK cell cytotoxicity. Thus, we have studied the physiology of NF-kappaB activation in NK cells during the cytolytic program. In resting ex vivo human NK cells or cell lines, IkappaB was degraded after 10 min exposure to PMA and ionomycin, or TNF and was maximally degraded by 30 min. Ligation of several NK cell activation receptors including NKp30 induced a similar response and was blocked by pretreatment with the proteosome inhibitor MG132. There was no short-term effect on p100 processing, the signature of noncanonical NF-kappaB activation. NK cell IkappaB degradation corresponded to increases in nuclear NF-kappaB as detected by EMSA. Supershift of stimulated NK cells and fluorescence microscopy of individual NK cells in cytolytic conjugates demonstrated that the p65/p50 heterodimer was the primary NF-kappaB used. NF-kappaB function was evaluated in NK92 cells transduced with a kappaB GFP reporter, and their conjugation with K562 cells or ligation of NKp30 ligation resulted in rapid GFP accumulation. The latter was prevented by the Syk inhibitor piceatannol. Thus, NK cell activation signaling specifically induces transcriptional activation and synthesis of new NF-kappaB dependent proteins during the initiation of cytotoxicity.
Subject(s)
Killer Cells, Natural/immunology , NF-kappa B/metabolism , Receptors, Immunologic/immunology , Cell Line , Green Fluorescent Proteins/immunology , Humans , Ionomycin/pharmacology , K562 Cells , Leupeptins/pharmacology , NF-kappa B/antagonists & inhibitors , Natural Cytotoxicity Triggering Receptor 3 , Receptors, Immunologic/antagonists & inhibitors , Signal Transduction/immunology , Stilbenes/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.
Subject(s)
Gene Expression Regulation , I-kappa B Kinase/metabolism , Interleukin-1/physiology , Intracellular Signaling Peptides and Proteins/physiology , NF-kappa B/metabolism , Animals , Catalysis , Cell Line , Enzyme Activation , Humans , I-kappa B Kinase/physiology , Mice , Peptides/chemistry , Protein Binding , Recombinant Proteins/chemistry , Signal TransductionABSTRACT
Activation of NF-kappaB requires two highly related kinases named IKKalpha and IKKbeta that share identity in the nature and positioning of their structural domains. Despite their similarity, the kinases are functionally divergent, and we therefore sought to identify any structural features specific for IKKalpha or IKKbeta. We performed bioinformatics analysis, and we identified a region resembling a ubiquitin-like domain (UBL) that exists only in IKKbeta and that we named the UBL-like domain (ULD). Deletion of the ULD rendered IKKbeta catalytically inactive and unable to induce NF-kappaB activity, and overexpression of only the ULD dose-dependently inhibited tumor necrosis factor-alpha-induced NF-kappaB activity. The ULD could not be functionally replaced within IKKbeta by ubiquitin or the corresponding region of IKKalpha, whereas deletion of the equivalent section of IKKalpha did not affect its catalytic activity against IkappaBalpha or its activation by NF-kappaB-inducing kinase. We identified five residues conserved among the larger family of UBL-containing proteins and IKKbeta, and alanine scanning revealed that the leucine at position 353 (Leu(353)) is absolutely critical for IKKbeta-induced NF-kappaB activation. Most intriguingly, the L353A mutant was catalytically active but, unlike wild-type IKKbeta, formed a stable complex with the NF-kappaB p65 subunit. Our findings therefore establish the ULD as a critical functional domain specific for IKKbeta that might play a role in dissociating IKKbeta from p65.
Subject(s)
Protein Serine-Threonine Kinases/chemistry , Ubiquitin/physiology , Amino Acid Sequence , Animals , COS Cells , Catalysis , HeLa Cells , Humans , I-kappa B Kinase , Molecular Sequence Data , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/physiology , Transcription Factor RelAABSTRACT
In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.
Subject(s)
Apoptosis/physiology , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Interleukin-1/pharmacology , Intracellular Signaling Peptides and Proteins , Phosphoinositide-3 Kinase Inhibitors , Protein Serine-Threonine Kinases , Amino Acid Chloromethyl Ketones/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein , Carrier Proteins/genetics , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolism , Cathepsin B/metabolism , Cell Nucleus , Cells, Cultured , Chromones/pharmacology , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/genetics , Retroviridae/genetics , Transduction, Genetic , Tumor Necrosis Factor-alpha/pharmacology , Umbilical Veins/cytologyABSTRACT
We have examined the effects of interferon (IFN)-gamma on expression and function of CD95 (APO-1/Fas) and associated proteins in cultured human umbilical vein and dermal microvascular endothelial cells (HUVEC and HDMEC, respectively). Unstimulated cells express only low levels of CD95; IFN-gamma produces a time- and concentration-dependent increase of CD95 in both cell types at the mRNA and cell surface protein levels. IFN-gamma also produces an increase in expression of pro-caspase-8 (FLICE/MACH) but does not significantly change expression of either Fas-associated death domain (FADD) protein or cellular FLICE inhibitory protein (cFLIP), other proteins associated with the CD95 death-inducing signaling complex (DISC). Neither resting nor IFN-gamma-treated EC express detectable CD95L mRNA or protein. Untreated HUVEC and HDMEC show minimal apoptosis when transduced to express CD95L. Treatment of CD95L-transduced cells with IFN-gamma causes apoptosis within 24 to 36 hours that can be blocked by antagonistic anti-CD95 antibody or by the caspase-inhibitory peptide zVAD-FMK. The extent of apoptosis is increased by co-treatment with either the protein synthesis inhibitor cycloheximide or the phosphatidylinositol 3-kinase inhibitor LY294002. Untransduced HUVEC treated with IFN-gamma also undergo CD95-initiated apoptosis when mixed with CD95L-transduced HUVEC or when incubated with pharmacologically activated cytolytic T lymphocytes. Overexpression of CD95 in HUVEC confers sensitivity to CD95L in the absence of IFN-gamma-treatment. We conclude that IFN-gamma induces sensitivity of endothelium to CD95L-mediated apoptosis, and that this response may result from increased expression of CD95 and/or pro-caspase-8.
Subject(s)
Apoptosis , Caspases/genetics , Endothelium, Vascular/physiology , Enzyme Precursors/genetics , Interferon-gamma/pharmacology , fas Receptor/genetics , Amino Acid Chloromethyl Ketones/pharmacology , Antigens, CD/genetics , Apoptosis/drug effects , Caspase 8 , Caspase 9 , Cell Death/drug effects , Cells, Cultured , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Enzyme Inhibitors/pharmacology , Fas Ligand Protein , Gene Expression Regulation/drug effects , Genetic Vectors , Humans , Membrane Glycoproteins/genetics , Microcirculation , Retroviridae/genetics , Reverse Transcriptase Polymerase Chain Reaction , Skin/blood supply , Transfection , Umbilical Veins , fas Receptor/physiologyABSTRACT
Tumor necrosis factor (TNF) receptor 1-associated death domain protein (TRADD) is an adaptor protein known to be involved in the TNF signaling pathway as well as signaling of other members of the TNF receptor superfamily, including DR3, DR6, p75(NTR), and the Epstein-Barr virus latent membrane protein 1. Current knowledge of the function of the adaptor protein has been derived from studies examining its over-expression in either wild-type or mutated forms. In this study, we analyzed the consequences of antisense oligonucleotide (ASO)-mediated depletion of endogenous TRADD on TNF induction of inflammation-related gene products, such as intercellular adhesion molecule-1, and associated kinase signaling pathways in human umbilical vein endothelial cells. A broader perspective of TRADD's role in TNF signaling was indicated by microarray gene expression analysis, where 20 of 24 genes that showed a 5-fold or greater increase in TNF-induced mRNA expression levels displayed a reduction in TNF-induced expression as a consequence of ASO-mediated knockdown of TRADD. Reduced activation of the nuclear factor-kappaB and c-Jun NH(2)-terminal kinase pathways, as measured by IkappaB-alpha protein levels and the extent of c-Jun phosphorylation, was also observed. These results indicate usage of antisense inhibitors of TRADD expression for modulating diseases associated with TRADD-dependent signal transduction pathways.