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1.
Cells ; 12(16)2023 08 15.
Article in English | MEDLINE | ID: mdl-37626879

ABSTRACT

Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-ß chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-ß chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.


Subject(s)
Mast Cells , Signal Transduction , Heptanol , Cholesterol , Cytokines
2.
Int J Mol Sci ; 25(1)2023 Dec 26.
Article in English | MEDLINE | ID: mdl-38203527

ABSTRACT

Due to their unique three-dimensional structure, DNA or RNA oligonucleotide aptamers bind to various molecules with high affinity and specificity. Aptamers, alone or in combination with antibodies, can be used to sensitively quantify target molecules by quantitative real-time polymerase chain reaction (qPCR). However, the assays are often complicated and unreliable. In this study, we explored the feasibility of performing the entire assay on wells of routinely used polypropylene PCR plates. We found that polypropylene wells efficiently bind proteins. This allows the entire assay to be run in a single well. To minimize nonspecific binding of the assay components to the polypropylene wells, we tested various blocking agents and identified methylcellulose as an effective alternative to the commonly used BSA. Methylcellulose not only demonstrates comparable or superior blocking capabilities but also offers the advantage of a well-defined composition and non-animal origin. Our findings support the utilization of aptamers, either alone or in combination with antibodies, for sensitive quantification of selected molecules immobilized in polypropylene PCR wells in a streamlined one-well qPCR assay under well-defined conditions.


Subject(s)
Aptamers, Nucleotide , Polypropylenes , Antibodies , Methylcellulose , Real-Time Polymerase Chain Reaction
3.
J Biol Chem ; 298(11): 102497, 2022 11.
Article in English | MEDLINE | ID: mdl-36115460

ABSTRACT

Pentacyclic triterpenoids, including ursolic acid (UA), are bioactive compounds with multiple biological activities involving anti-inflammatory effects. However, the mode of their action on mast cells, key players in the early stages of allergic inflammation, and underlying molecular mechanisms remain enigmatic. To better understand the effect of UA on mast cell signaling, here we examined the consequences of short-term treatment of mouse bone marrow-derived mast cells with UA. Using IgE-sensitized and antigen- or thapsigargin-activated cells, we found that 15 min exposure to UA inhibited high affinity IgE receptor (FcεRI)-mediated degranulation, calcium response, and extracellular calcium uptake. We also found that UA inhibited migration of mouse bone marrow-derived mast cells toward antigen but not toward prostaglandin E2 and stem cell factor. Compared to control antigen-activated cells, UA enhanced the production of tumor necrosis factor-α at the mRNA and protein levels. However, secretion of this cytokine was inhibited. Further analysis showed that UA enhanced tyrosine phosphorylation of the SYK kinase and several other proteins involved in the early stages of FcεRI signaling, even in the absence of antigen activation, but inhibited or reduced their further phosphorylation at later stages. In addition, we show that UA induced changes in the properties of detergent-resistant plasma membrane microdomains and reduced antibody-mediated clustering of the FcεRI and glycosylphosphatidylinositol-anchored protein Thy-1. Finally, UA inhibited mobility of the FcεRI and cholesterol. These combined data suggest that UA exerts its effects, at least in part, via lipid-centric plasma membrane perturbations, hence affecting the functions of the FcεRI signalosome.


Subject(s)
Receptors, IgE , Triterpenes , Mice , Animals , Receptors, IgE/metabolism , Mast Cells/metabolism , Cell Degranulation , Calcium/metabolism , Triterpenes/pharmacology , Triterpenes/metabolism , Antigens/metabolism , Lipids/pharmacology , Ursolic Acid
4.
Front Immunol ; 12: 670205, 2021.
Article in English | MEDLINE | ID: mdl-34248949

ABSTRACT

Mast cells are potent immune sensors of the tissue microenvironment. Within seconds of activation, they release various preformed biologically active products and initiate the process of de novo synthesis of cytokines, chemokines, and other inflammatory mediators. This process is regulated at multiple levels. Besides the extensively studied IgE and IgG receptors, toll-like receptors, MRGPR, and other protein receptor signaling pathways, there is a critical activation pathway based on cholesterol-dependent, pore-forming cytolytic exotoxins produced by Gram-positive bacterial pathogens. This pathway is initiated by binding the exotoxins to the cholesterol-rich membrane, followed by their dimerization, multimerization, pre-pore formation, and pore formation. At low sublytic concentrations, the exotoxins induce mast cell activation, including degranulation, intracellular calcium concentration changes, and transcriptional activation, resulting in production of cytokines and other inflammatory mediators. Higher toxin concentrations lead to cell death. Similar activation events are observed when mast cells are exposed to sublytic concentrations of saponins or some other compounds interfering with the membrane integrity. We review the molecular mechanisms of mast cell activation by pore-forming bacterial exotoxins, and other compounds inducing cholesterol-dependent plasma membrane perturbations. We discuss the importance of these signaling pathways in innate and acquired immunity.


Subject(s)
Cell Membrane/metabolism , Cholesterol/metabolism , Cytotoxins/metabolism , Gram-Positive Bacteria/metabolism , Gram-Positive Bacterial Infections/metabolism , Mast Cells/metabolism , Animals , Calcium Signaling , Cell Death , Cell Degranulation , Cell Membrane/immunology , Cell Membrane/microbiology , Cell Membrane/pathology , Cellular Microenvironment , Cytokines/metabolism , Gram-Positive Bacteria/immunology , Gram-Positive Bacterial Infections/immunology , Gram-Positive Bacterial Infections/microbiology , Gram-Positive Bacterial Infections/pathology , Host-Pathogen Interactions , Humans , Inflammation Mediators/metabolism , Mast Cells/immunology , Mast Cells/microbiology , Mast Cells/pathology
5.
PLoS One ; 10(12): e0144596, 2015.
Article in English | MEDLINE | ID: mdl-26658290

ABSTRACT

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-ß-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-ß-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI ß and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-ß-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane.


Subject(s)
Cholesterol/metabolism , Ethanol/pharmacology , Mast Cells/drug effects , Receptors, IgE/metabolism , Signal Transduction/drug effects , Animals , Calcium/metabolism , Cytokines/genetics , Cytokines/metabolism , Dose-Response Relationship, Drug , Gene Expression/drug effects , Mast Cells/metabolism , Mice , Phosphorylation
6.
Eur J Immunol ; 40(11): 3235-45, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21061444

ABSTRACT

The transmembrane adaptor protein NTAL (non-T-cell activation linker) participates in signalosome assembly in hematopoietic cells, but its exact role in cell physiology remains enigmatic. We report here that BM-derived mast cells from NTAL-deficient mice, responding to Ag alone or in combination with SCF, exhibit reduced spreading on fibronectin, enhanced filamentous actin depolymerization and enhanced migration towards Ag relative to WT cells. No such differences between WT and NTAL(-/-) BM-derived mast cells were observed when SCF alone was used as activator. We have examined the activities of two small GTPases, Rac and Rho, which are important regulators of actin polymerization. Stimulation with Ag and/or SCF enhanced activity of Rac(1,2,3) in both NTAL(-/-) and WT cells. In contrast, RhoA activity decreased and this trend was much faster and more extensive in NTAL(-/-) cells, indicating a positive regulatory role of NTAL in the recovery of RhoA activity. After restoring NTAL into NTAL(-/-) cells, both spreading and actin responses were rescued. This is the first report of a crucial role of NTAL in signaling, via RhoA, to mast cell cytoskeleton.


Subject(s)
Bone Marrow Cells/immunology , Cytoskeleton/immunology , Mast Cells/immunology , Proteins/immunology , Signal Transduction/immunology , rho GTP-Binding Proteins/immunology , Actins/genetics , Actins/immunology , Actins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Antigens/immunology , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , Cytoskeleton/genetics , Cytoskeleton/metabolism , Mast Cells/cytology , Mast Cells/metabolism , Mice , Mice, Knockout , Proteins/genetics , Proteins/metabolism , Signal Transduction/genetics , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/immunology , rac GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein
7.
J Immunol ; 179(8): 5169-80, 2007 Oct 15.
Article in English | MEDLINE | ID: mdl-17911602

ABSTRACT

Engagement of the FcepsilonRI in mast cells and basophils leads to a rapid tyrosine phosphorylation of the transmembrane adaptors LAT (linker for activation of T cells) and NTAL (non-T cell activation linker, also called LAB or LAT2). NTAL regulates activation of mast cells by a mechanism, which is incompletely understood. Here we report properties of rat basophilic leukemia cells with enhanced or reduced NTAL expression. Overexpression of NTAL led to changes in cell morphology, enhanced formation of actin filaments and inhibition of the FcepsilonRI-induced tyrosine phosphorylation of the FcepsilonRI subunits, Syk kinase and LAT and all downstream activation events, including calcium and secretory responses. In contrast, reduced expression of NTAL had little effect on early FcepsilonRI-induced signaling events but inhibited calcium mobilization and secretory response. Calcium response was also repressed in Ag-activated cells defective in Grb2, a major target of phosphorylated NTAL. Unexpectedly, in cells stimulated with thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) ATPase, the amount of cellular NTAL directly correlated with the uptake of extracellular calcium even though no enhanced tyrosine phosphorylation of NTAL was observed. The combined data indicate that NTAL regulates FcepsilonRI-mediated signaling at multiple steps and by different mechanisms. At early stages NTAL interferes with tyrosine phosphorylation of several substrates and formation of signaling assemblies, whereas at later stages it regulates the activity of store-operated calcium channels through a distinct mechanism independent of enhanced NTAL tyrosine phosphorylation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Transport System y+/physiology , Calcium Signaling/immunology , Fusion Regulatory Protein 1, Light Chains/physiology , Mast Cells/immunology , Mast Cells/metabolism , Tyrosine/metabolism , Amino Acid Sequence , Animals , Calcium/metabolism , Cell Line, Tumor , Intracellular Fluid/metabolism , Molecular Sequence Data , Phosphorylation , Rats , Receptors, IgE/physiology
8.
J Immunol Methods ; 328(1-2): 139-51, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-17900607

ABSTRACT

Immunolabeling of isolated plasma membrane (PM) sheets combined with high-resolution electron microscopy is a powerful technique for understanding the topography of PM-bound signaling molecules. However, this technique has been mostly confined to analysis of membrane sheets from adherent cells. Here we present a rapid, simple and versatile method for isolation of PM sheets from non-adherent cells, and show its use for examination of the topography of Fcepsilon receptor I (FcepsilonRI) and transmembrane adaptors, LAT (linker for activation of T cells) and NTAL (non-T cell activation linker), in murine bone marrow-derived mast cells (BMMC). The data were compared with those obtained from widely used but tumor-derived rat basophilic leukemia (RBL) cells. In non-activated cells, FcepsilonRI was distributed either individually or in small clusters of comparable size in both cell types. In multivalent antigen-activated BMMC as well as RBL cells, FcepsilonRI was internalized to a similar extent, but, strikingly, internalization in BMMC was not preceded by formation of large (~200 nm) aggregates of FcepsilonRI, described previously in activated RBL cells. On the other hand, downstream adaptor proteins, LAT and NTAL, were localized in independent domains in both BMMC and RBL cells before and after FcepsilonRI triggering. The combined data demonstrate unexpected properties of FcepsilonRI signaling assemblies in BMMC and emphasize the importance of studies of PM sheets isolated from non-tumor cells.


Subject(s)
Cell Membrane/ultrastructure , Clinical Laboratory Techniques , Mast Cells/ultrastructure , Membrane Proteins/ultrastructure , Receptors, IgE/ultrastructure , Animals , Flow Cytometry , Immunoblotting , Mast Cells/immunology , Membrane Proteins/immunology , Mice , Microscopy, Electron, Transmission , Rats , Receptors, IgE/immunology
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