GMCSF activates NF-κB via direct interaction of the GMCSF receptor with IκB kinase β

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 192-199 ◽  
Author(s):  
Karin Ebner ◽  
Alexander Bandion ◽  
Bernd R. Binder ◽  
Rainer de Martin ◽  
Johannes A. Schmid
Keyword(s):  
Endothelial Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Nuclear Factor Κb ◽  
Janus Kinase ◽  
Cell Extracts ◽  
Iκb Kinase ◽  
Α Chain ◽  
Β Chain

Abstract Granulocyte-macrophage colony-stimulating factor (GMCSF) has a central role in proliferation and differentiation of hematopoetic cells. Furthermore, it influences the proliferation and migration of endothelial cells. GMCSF elicits these functions by activating a receptor consisting of a ligand-specific α-chain and a β-chain, which is common for GMCSF, interleukin-3 (IL-3), and IL-5. It is known that various signaling molecules such as Janus kinase 2 or transcription factors of the signal transducer and activator of transcription (STAT) family bind to the common β-chain and initiate signaling cascades. However, α-chain—specific signal transduction adapters have to be postulated given that IL-3, IL-5, and GMCSF induce partly distinct biologic responses. Using a yeast 2-hybrid system, we identified the α-chain of the GMCSF receptor (GMRα) as putative interaction partner of IκB kinase β, one of the central signaling kinases activating the transcription factor nuclear factor—κB (NF-κB). Using endogenous protein levels of endothelial cell extracts, we could verify the interaction by coimmunoprecipitation experiments. Fluorescence resonance energy transfer (FRET) microscopy confirmed the direct interaction of CFP-IKKβ and YFPGMRα in living cells. Functional studies demonstrated GMCSF-dependent activation of IκB kinase activity in endothelial cells, degradation of IκB, and activation of NF-κB. Further biologic studies using GMCSF-dependent TF-1 cells indicated that GMCSF-triggered activation of NF-κB is important for cell survival and proliferation. (Blood. 2003;102:192-199)

Mechanotransduction by integrin is essential for IL-6 secretion from endothelial cells in response to uniaxial continuous stretch

2005 ◽  
Vol 288 (5) ◽  
pp. C1012-C1022 ◽  
Author(s):  
Akitoshi Sasamoto ◽  
Masato Nagino ◽  
Satoshi Kobayashi ◽  
Keiji Naruse ◽  
Yuji Nimura ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Kinase Inhibitor ◽  
Umbilical Vein ◽  
Nuclear Factor Κb ◽  
Inhibitory Peptide ◽  
Signaling Mechanism ◽  
Iκb Kinase ◽  
Intracellular Ca ◽  
Protein Kinase C Inhibitor ◽  
Umbilical Vein Endothelial Cells

We previously reported that uniaxial continuous stretch in human umbilical vein endothelial cells (HUVECs) induced interleukin-6 (IL-6) secretion via IκB kinase (IKK)/nuclear factor-κB (NF-κB) activation. The aim of the present study was to clarify the upstream signaling mechanism responsible for this phenomenon. Stretch-induced IKK activation and IL-6 secretion were inhibited by application of α5β1 integrin-inhibitory peptide (GRGDNP), phosphatidylinositol 3-kinase inhibitor (LY-294002), phospholipase C-γ inhibitor (U-73122), or protein kinase C inhibitor (H7). Although depletion of intra- or extracellular Ca2 + pool using thapsigargin (TG) or EGTA, respectively, showed little effect, a TG-EGTA mixture significantly inhibited stretch-induced IKK activation and IL-6 secretion. An increase in the intracellular Ca2 + concentration ([Ca2 +]i) upon continuous stretch was observed even in the presence of TG, EGTA, or GRGDNP, but not in a solution containing the TG-EGTA mixture, indicating that both integrin activation and [Ca2 +]i rise are crucial factors for stretch-induced IKK activation and after IL-6 secretion in HUVECs. Furthermore, while PKC activity was inhibited by the TG-EGTA mixture, GRGDNP, LY-294002, or U-73122, PLC-γ activity was retarded by GRGDNP or LY-294002. These results indicate that continuous stretch-induced IL-6 secretion in HUVECs depends on outside-in signaling via integrins followed by a PI3-K-PLC-γ-PKC-IKK-NF-κB signaling cascade. Another crucial factor, [Ca2 +]i increase, may at least be required to activate PKC needed for NF-κB activation.

scholarly journals Direct visualization of interaction between calmodulin and connexin45

2017 ◽  
Vol 474 (24) ◽  
pp. 4035-4051 ◽  
Author(s):  
Juan Zou ◽  
Mani Salarian ◽  
Yanyi Chen ◽  
You Zhuo ◽  
Nicole E. Brown ◽  
...  
Keyword(s):  
Peptide Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Resonance Spectroscopy ◽  
Direct Visualization ◽  
Bioluminescence Resonance Energy Transfer ◽  
Binding Motifs ◽  
Physiological Conditions ◽  
Interaction Mode

Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164–186) of Cx45 using a peptide model. The strong binding (Kd ∼ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.

scholarly journals Activation of JAK2 in Human Vascular Endothelial Cells by Granulocyte-Macrophage Colony-Stimulating Factor

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 863-872 ◽  
Author(s):  
Raffaella Soldi ◽  
Luca Primo ◽  
Maria Felice Brizzi ◽  
Fiorella Sanavio ◽  
Massimo Aglietta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tyrosine Kinase ◽  
Janus Kinase ◽  
Colony Stimulating Factor ◽  
Functional Program ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Β Chain ◽  
Stimulating Factor

Abstract Besides the regulation of hematopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the expression of a functional program in endothelial cells (ECs) related to angiogenesis and to their survival in the bone marrow microenvironment. ECs express specific GM-CSF high-affinity binding sites, which mediate the proliferative and migratory response. We now report that ECs express the α and β subunits of GM-CSF receptor (GM-CSFR), and that GM-CSF is able to activate the Janus kinase (JAK)2, a member of the cytosolic tyrosine kinase family, which is known to mediate signals of several non–tyrosine kinase receptors. JAK2 tyrosine phoshorylation, as well as activation of its catalytic activity, is induced by subnanomolar concentrations of GM-CSF and occurs within 3 minutes of stimulation and persists at least for 10 minutes. The effect is specific as inferred by the lack of effect of heat-inactivated GM-CSF or neutralized by specific antibodies and by the finding that interleukin-5, which utilizes a specific α chain and the same β chain of GM-CSFR, does not phosphorylate JAK2. Furthermore, we show that the amount of JAK2 physically associated with GM-CSFR β chain is increased after GM-CSF stimulation and that GM-CSF triggers both β chain and JAK2 tyrosine phosphorylation. Taken together, these results suggest that biologic activities of GM-CSF in vascular endothelium may, in part, be elicited by GM-CSFR–mediated JAK2 activation.

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin

2001 ◽  
Vol 357 (3) ◽  
pp. 687-697 ◽  
Author(s):  
Jonathan P. WAUD ◽  
Alexandra BERMÚDEZ FAJARDO ◽  
Thankiah SUDHAHARAN ◽  
Andrew R. TRIMBY ◽  
Jinny JEFFERY ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Donor And Acceptor ◽  
Green Fluorescent

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

scholarly journals Investigation of a Direct Interaction between miR4749 and the Tumor Suppressor p53 by Fluorescence, FRET and Molecular Modeling

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 346
Author(s):  
Anna Rita Bizzarri ◽  
Salvatore Cannistraro
Keyword(s):  
Tumor Suppressor ◽  
Computational Modeling ◽  
Binding Free Energy ◽  
Sequence Similarity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Free Energy Calculations ◽  
Tumor Suppressor P53 ◽  
High Sequence Similarity

The interactions between the DNA binding domain (DBD) of the tumor suppressor p53 and miR4749, characterized by a high sequence similarity with the DNA Response Element (RE) of p53, was investigated by fluorescence spectroscopy combined with computational modeling and docking. Fluorescence quenching experiments witnessed the formation of a specific complex between DBD and miR4749 with an affinity of about 105 M. Förster Resonance Energy Transfer (FRET) allowed us to measure a distance of 3.9 ± 0.3 nm, between the lone tryptophan of DBD and an acceptor dye suitably bound to miR4749. Such information, combined with a computational modeling approach, allowed us to predict possible structures for the DBD-miR4749 complex. A successive docking refinement, complemented with binding free energy calculations, led us to single out a best model for the DBD-miR4749 complex. We found that the interaction of miR4749 involves the DBD L3 loop and the H1 helix, close to the Zn-finger motif; with this suggesting that miR4749 could directly inhibit the p53 interaction with DNA. These results might inspire new therapeutic strategies finalized to restore the p53 functional activity.

scholarly journals Identification of a Selective RelA Inhibitor Based on DSE-FRET Screening Methods

2020 ◽  
Vol 21 (23) ◽  
pp. 9150
Author(s):  
Yoshitomo Shiroma ◽  
Go Fujita ◽  
Takuya Yamamoto ◽  
Ryou-u Takahashi ◽  
Ashutosh Kumar ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclear Factor Κb ◽  
Screening Methods ◽  
Biological Functions ◽  
Mobility Shift ◽  
Dna Strand Exchange ◽  
Mobility Shift Assay ◽  
Dna Strand

Nuclear factor-κB (NF-κB) is an important transcription factor involved in various biological functions, including tumorigenesis. Hence, NF-κB has attracted attention as a target factor for cancer treatment, leading to the development of several inhibitors. However, existing NF-κB inhibitors do not discriminate between its subunits, namely, RelA, RelB, cRel, p50, and p52. Conventional methods used to evaluate interactions between transcription factors and DNA, such as electrophoretic mobility shift assay and luciferase assays, are unsuitable for high-throughput screening (HTS) and cannot distinguish NF-κB subunits. We developed a HTS method named DNA strand exchange fluorescence resonance energy transfer (DSE-FRET). This assay is suitable for HTS and can discriminate a NF-κB subunit. Using DSE-FRET, we searched for RelA-specific inhibitors and verified RelA inhibition for 32,955 compounds. The compound A55 (2-(3-carbamoyl-6-hydroxy-4-methyl-2-oxopyridin-1(2H)-yl) acetic acid) selectively inhibited RelA–DNA binding. We propose that A55 is a seed compound for RelA-specific inhibition and could be used in clinical applications.

Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production

2008 ◽  
Vol 294 (4) ◽  
pp. C985-C993 ◽  
Author(s):  
Emmanuel Lorne ◽  
Jaroslaw W. Zmijewski ◽  
Xia Zhao ◽  
Gang Liu ◽  
Yuko Tsuruta ◽  
...  
Keyword(s):  
Xanthine Oxidase ◽  
Nuclear Translocation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nuclear Factor Κb ◽  
Neutrophil Activation ◽  
Limited Information ◽  
Inhibitory Protein ◽  
Tnf Α ◽  
Factor Α

Reactive oxygen species (ROS) contribute to neutrophil activation and the development of acute inflammatory processes in which neutrophils play a central role. However, there is only limited information concerning the mechanisms through which extracellular ROS, and particularly cell membrane-impermeable species, such as superoxide, enhance the proinflammatory properties of neutrophils. To address this issue, neutrophils were exposed to superoxide generating combinations of xanthine oxidase and hypoxanthine or lumazine. Extracellular superoxide generation induced nuclear translocation of nuclear factor-κB (NF-κB) and increased neutrophil production of the NF-κB-dependent cytokines tumor necrosis factor-α (TNF-α) and macrophage inhibitory protein-2 (MIP-2). In contrast, there were no changes in TNF-α or MIP-2 expression when neutrophils lacking Toll-like receptor-4 (TLR4) were exposed to extracellular superoxide. Immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET) studies demonstrated association between TLR4 and xanthine oxidase. Exposure of neutrophils to heparin attenuated binding of xanthine oxidase to the cell surface as well as interactions with TLR4. Heparin also decreased xanthine oxidase-induced nuclear translocation of NF-κB as well as production of proinflammatory cytokines. These results demonstrate that extracellular superoxide has proinflammatory effects on neutrophils, predominantly acting through an TLR4-dependent mechanism that enhances nuclear translocation of NF-κB and increases expression of NF-κB-dependent cytokines.

scholarly journals Rotaviruses Associate with Cellular Lipid Droplet Components To Replicate in Viroplasms, and Compounds Disrupting or Blocking Lipid Droplets Inhibit Viroplasm Formation and Viral Replication

2010 ◽  
Vol 84 (13) ◽  
pp. 6782-6798 ◽  
Author(s):  
Winsome Cheung ◽  
Michael Gill ◽  
Alessandro Esposito ◽  
Clemens F. Kaminski ◽  
Nathalie Courousse ◽  
...  
Keyword(s):  
Lipid Droplets ◽  
Time Course ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatial Proximity ◽  
Progeny Virus ◽  
Virus Family ◽  
Cell Extracts ◽  
Perilipin A

ABSTRACT Rotaviruses are a major cause of acute gastroenteritis in children worldwide. Early stages of rotavirus assembly in infected cells occur in viroplasms. Confocal microscopy demonstrated that viroplasms associate with lipids and proteins (perilipin A, ADRP) characteristic of lipid droplets (LDs). LD-associated proteins were also found to colocalize with viroplasms containing a rotaviral NSP5-enhanced green fluorescent protein (EGFP) fusion protein and with viroplasm-like structures in uninfected cells coexpressing viral NSP2 and NSP5. Close spatial proximity of NSP5-EGFP and cellular perilipin A was confirmed by fluorescence resonance energy transfer. Viroplasms appear to recruit LD components during the time course of rotavirus infection. NSP5-specific siRNA blocked association of perilipin A with NSP5 in viroplasms. Viral double-stranded RNA (dsRNA), NSP5, and perilipin A cosedimented in low-density gradient fractions of rotavirus-infected cell extracts. Chemical compounds interfering with LD formation (isoproterenol plus isobutylmethylxanthine; triacsin C) decreased the number of viroplasms and inhibited dsRNA replication and the production of infectious progeny virus; this effect correlated with significant protection of cells from virus-associated cytopathicity. Rotaviruses represent a genus of another virus family utilizing LD components for replication, pointing at novel therapeutic targets for these pathogens.

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