Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

T Cell Receptor-Induced NF-kB Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-Mediated Calcium Entry

ABSTRACT: T cell activation following antigen binding to the T cell receptor (TCR) involves the mobilization of intracellular calcium (Ca2+) to activate the key transcription factors NFAT and NF-ÎºB. The mechanism of NFAT activation by Ca2+ has been determined; however, the role of Ca2+ in controlling NF-ÎºB signaling is poorly understood and the source of Ca2+ required for NF-ÎºB activation is unknown. We demonstrate that TCR- but not TNF- induced NF-ÎºB signaling upstream of IÎºB kinase (IKK) activation absolutely requires the influx of extracellular Ca2+ via STIM1-dependent CRAC/Orai channels. We further show that Ca2+ influx controls phosphorylation of the NF-ÎºB protein p65 on Ser536 and that this post- translational modification controls its nuclear localization and transcriptional activation. Notably our data reveal that this role for Ca2+ is entirely separate from its upstream control of IÎºBÎ± degradation, thereby identifying a novel Ca2+- dependent distal step in TCR-induced NF-ÎºB activation. Finally, we demonstrate that this control of distal signaling occurs via Ca2+-dependent PKCk-mediated phosphorylation of p65. Thus, we establish the source of Ca2+ required for TCR induced NF-kB activation and we define a new distal Ca2+-dependent checkpoint in TCR-induced NF-kB signaling that has broad implications for the control of immune cell development and T cell functional specificity. 3 treatments were analyzed, with biological replicates for each treatment. In addition, three timepoints (1 hour, 4 hour, and 8 hour) were examined for each treatment, as well as an untreated control. In total 19 samples were analyzed

ORGANISM(S): Homo sapiens

SUBMITTER: daniel beiting

PROVIDER: E-GEOD-76804 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:Breast cancers with HER2 overexpression are sensitive to drugs targeting the receptor or its kinase activity. HER2-targeting drugs are initially effective against HER2- positive breast cancer, but resistance inevitably occurs. We previously found that nuclear factor kappa B is hyper-activated in the subset of HER-2 positive breast cancer cells and tissue specimens. In this study, we report that constitutively active NF-ÎºB rendered HER2-positive cancer cells resistant to anti-HER2 drugs, and cells selected for Lapatinib resistance up-regulated NF-ÎºB. In both circumstances, cells were anti-apoptotic and grew rapidly as xenografts. Lapatinib-resistant cells were refractory to HER2 and NF-ÎºB inhibitors alone but were sensitive to their combination, suggesting a novel therapeutic strategy. A subset of NF-ÎºB-responsive genes was overexpressed in HER2-positive and triple-negative breast cancers, and patients with this NF-ÎºB signature had poor clinical outcome. Anti-HER2 drug resistance may be a consequence of NF-ÎºB activation, and selection for resistance results in NF-ÎºB activation, suggesting this transcription factor is central to oncogenesis and drug resistance. Clinically, the combined targeting of HER2 and NF-ÎºB suggests a potential treatment paradigm for patients who relapse after anti-HER2 therapy. Patients with these cancers may be treated by simultaneously suppressing HER2 signaling and NF-ÎºB activation. We used microarrays to detail the gene expression differences underlying the characterictic survival differences between the SKR6, SKR6-Vector, SKR6CA, and SKR6LR cell lines, which are defined as follows: SKR6: A clonal derivative of SKBR3 cells isolated by fluorescence-activated cell sorting (FACS) to enrich for elevated HER2 levels, SKR6CA: SKR6 cells retrovirally transduced with constitutively active NF-ÎºB relA/p65 (CAp65) and selected with puromycin, SKR6 vector: SKR6 cells transduced with the pQCXIP empty retroviral vector and selected with puromycin, and SKR6LR: SKR6 cells treated with increasing lapatinib concentrations (0.2 to 5 Î¼M) for several months. We sorted SKBR-3 cells by fluorescence-activated cell sorting (FACS) to enriched for cell population with elevated HER2 expression, which we termed SKR6. The following cell lines were then created from SKR6 cells: SKR6CA: SKR6 cells retrovirally transduced with constitutively active NF-ÎºB relA/p65 (CAp65), SKR6 vector: SKR6 cells transduced with the pQCXIP empty retroviral vector and selected with puromycin, and SKR6LR: SKR6 cells treated with increasing lapatinib concentrations (0.2 to 5 Î¼M) for several months.

Project description:Macrophages and dendritic cells (DCs) differently contribute to the generation of coordinated immune system responses against infectious agents. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. By contrast, following antigen recognition, macrophages initiate first the inflammatory process and then switch to an anti-inflammatory phenotype for the restoration of tissue homeostasis. Following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. DC stimulation with lipopolysaccharide (LPS) induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. We asked whether macrophage survival after LPS encounter was due to their inability to activate the Ca2+ pathway. As matter of fact, bone marrow-derived macrophages were unable to mobilize Ca2+ and to translocate NFAT to the. To further investigate whether the Ca2+-NFAT pathway played any role in LPS-stimulated macrophages, we performed a comparative kinetic microarray analysis in conditions allowing or inhibiting NFAT activation. We show here that no modulation of gene expression can be attributed to NFAT. Gene expression analyses were performed using Affymetrix GeneChips in the following groups of murine macrophaghes: 1) CD14-deficient macrophages stimulated with LPS; 2) wt macrophages stimulated with LPS in presence of EGTA; 3) wt macrophages stimulated with LPS. The following kinetic time points were examined: 0, 6 and 24 hours following LPS activation. This experimental setting allowed us to select for effects due to Ca2+ fluxes and exclude the effects due to other causes, particularly the block of TRIF recruitment in CD14-deficient cells and the EGTA effects unrelated to Ca2+ chelation.

Project description:Dendritic cells (DCs) are a special class of leukocytes able to activate both innate and adaptive immune responses. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. We have observed that following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. Indeed, LPS induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. According with this observation CD14-deficient DCs do not die following LPS stimulation. Nevertheless, CD14-deficient DC death following LPS activation can be restored by co-stimulating DCs with LPS and thapsigargin. Thapsigargin empties the intracellular calcium stores by blocking calcium pumping into the sarcoplasmic and endoplasmic reticulum and thereby activates plasma membrane calcium channels. This, in turn, allows an influx of calcium into the cytosol and NFAT activation. To identify the NFAT controlled apoptosis genes in LPS activated DCs we have performed a kinetic microarray analysis (0, 48 and 60 h) in conditions allowing or inhibiting NFAT activation. Four genes have been selected: Nur77, Gadd45g, Ddit3/Gadd153/Chop-10 and Tia1. To identify apoptosis genes selectively modulated by NFAT, a comparative kinetic (time points 0, 48 and 60 h) microarray analysis was performed in the following conditions: 1) wild type bone marrow derived DCs (wtBMDCs) stimulated with LPS; 2) CD14-deficient BMDCs stimulated with LPS; 3) wtBMDCs stimulated with LPS in presence of thapsigargin; 4) CD14-deficient BMDCs stimulated with LPS in presence of thapsigargin.

Dataset Information

T Cell Receptor-Induced NF-kB Signaling and Transcriptional Activation Are Regulated by STIM1- and Orai1-Mediated Calcium Entry

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure