Aggregation of the high-affinity receptor for IgE (FcεRI) in mast cells initiates activation events that lead to degranulation and release of inflammatory mediators. To better understand the signaling pathways and genes involved in mast cell activation, we developed a high-throughput mast cell degranulation assay suitable for RNA interference experiments using lentivirus-based short hairpin RNA (shRNA) delivery. We tested 432 shRNAs specific for 144 selected genes for effects on FcεRI-mediated mast cell degranulation and identified 15 potential regulators. In further studies, we focused on galectin-3 (Gal3), identified in this study as a negative regulator of mast cell degranulation. FcεRI-activated cells with Gal3 knockdown exhibited upregulated tyrosine phosphorylation of spleen tyrosine kinase and several other signal transduction molecules and enhanced calcium response. We show that Gal3 promotes internalization of IgE-FcεRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcεRI ubiquitination. Furthermore, we found that Gal3 facilitates mast cell adhesion and motility on fibronectin but negatively regulates antigen-induced chemotaxis. The combined data indicate that Gal3 is involved in both positive and negative regulation of FcεRI-mediated signaling events in mast cells.

• MeSH
• aktiny metabolismus   MeSH
• buněčná adheze MeSH
• chemotaxe MeSH
• cytokiny genetika   metabolismus   MeSH
• fosforylace MeSH
• galektin 3 genetika   metabolismus   MeSH
• lyzozomy metabolismus   MeSH
• malá interferující RNA MeSH
• mastocyty cytologie   fyziologie   MeSH
• myši inbrední BALB C MeSH
• prostaglandin D2 metabolismus   MeSH
• receptory IgE genetika   metabolismus   MeSH
• signální transdukce MeSH
• ubikvitinace MeSH
• vápník metabolismus   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aktiny MeSH
• cytokiny MeSH
• galektin 3 MeSH
• Lgals3 protein, mouse MeSH Prohlížeč
• malá interferující RNA MeSH
• prostaglandin D2 MeSH
• receptory IgE MeSH
• vápník MeSH

Non-T cell activation linker (NTAL; also called LAB or LAT2) is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI) signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO) and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD) in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD) have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs) with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.

• MeSH
• adaptorové proteiny vezikulární transportní genetika   metabolismus   MeSH
• genetická transkripce fyziologie   MeSH
• mastocyty metabolismus   MeSH
• myši knockoutované MeSH
• myši MeSH
• receptory IgE metabolismus   MeSH
• signální transdukce imunologie   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny vezikulární transportní MeSH
• LAB protein, mouse MeSH Prohlížeč
• receptory IgE MeSH
• vápník MeSH

Mast cell activation mediated by the high affinity receptor for IgE (FcεRI) is a key event in allergic response and inflammation. Other receptors on mast cells, as c-Kit for stem cell factor and G protein-coupled receptors (GPCRs) synergistically enhance the FcεRI-mediated release of inflammatory mediators. Activation of various signaling pathways in mast cells results in changes in cell morphology, adhesion to substrate, exocytosis, and migration. Reorganization of cytoskeleton is pivotal in all these processes. Cytoskeletal proteins also play an important role in initial stages of FcεRI and other surface receptors induced triggering. Highly dynamic microtubules formed by αβ-tubulin dimers as well as microfilaments build up from polymerized actin are affected in activated cells by kinases/phosphatases, Rho GTPases and changes in concentration of cytosolic Ca(2+). Also important are nucleation proteins; the γ-tubulin complexes in case of microtubules or Arp 2/3 complex with its nucleation promoting factors and formins in case of microfilaments. The dynamic nature of microtubules and microfilaments in activated cells depends on many associated/regulatory proteins. Changes in rigidity of activated mast cells reflect changes in intermediate filaments build up from vimentin. This review offers a critical appraisal of current knowledge on the role of cytoskeleton in mast cells signaling.

• Klíčová slova
• actins, intermediate filaments, mast cell activation, microfilaments, microtubules, signal transduction, tubulins, vimentin,
• Publikační typ
• časopisecké články MeSH

The earliest known biochemical step that occurs after ligand binding to the multichain immune recognition receptor is tyrosine phosphorylation of the receptor subunits. In mast cells and basophils activated by multivalent antigen-IgE complexes, this step is mediated by Src family kinase Lyn, which phosphorylates the high affinity IgE receptor (Fc epsilonRI). However, the exact molecular mechanism of this phosphorylation step is incompletely understood. In this study, we tested the hypothesis that changes in activity and/or topography of protein-tyrosine phosphatases (PTPs) could play a major role in the Fc epsilonRI triggering. We found that exposure of rat basophilic leukemia cells or mouse bone marrow-derived mast cells to PTP inhibitors, H(2)O(2) or pervanadate, induced phosphorylation of the Fc epsilonRI subunits, similarly as Fc epsilonRI triggering. Interestingly, and in sharp contrast to antigen-induced activation, neither H(2)O(2) nor pervanadate induced any changes in the association of Fc epsilonRI with detergent-resistant membranes and in the topography of Fc epsilonRI detectable by electron microscopy on isolated plasma membrane sheets. In cells stimulated with pervanadate, H(2)O(2) or antigen, enhanced oxidation of active site cysteine of several PTPs was detected. Unexpectedly, most of oxidized phosphatases bound to the plasma membrane were associated with the actin cytoskeleton. Several PTPs (SHP-1, SHP-2, hematopoietic PTP, and PTP-MEG2) showed changes in their enzymatic activity and/or oxidation state during activation. Based on these and other data, we propose that down-regulation of enzymatic activity of PTPs and/or changes in their accessibility to the substrates play a key role in initial tyrosine phosphorylation of the Fc epsilonRI and other multichain immune receptors.

• MeSH
• aktivace enzymů účinky léků   genetika   imunologie   MeSH
• antigeny imunologie   metabolismus   farmakologie   MeSH
• fosforylace účinky léků   genetika   imunologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• krysa rodu Rattus MeSH
• mastocyty imunologie   metabolismus   MeSH
• membránové mikrodomény genetika   imunologie   metabolismus   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oxidace-redukce účinky léků   MeSH
• oxidancia farmakologie   MeSH
• peroxid vodíku farmakologie   MeSH
• receptory IgE genetika   imunologie   metabolismus   MeSH
• skupina kinas odvozených od src-genu genetika   imunologie   metabolismus   MeSH
• transport proteinů účinky léků   genetika   imunologie   MeSH
• tyrosinfosfatasy antagonisté a inhibitory   genetika   imunologie   metabolismus   MeSH
• vanadáty farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• inhibitory enzymů MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• oxidancia MeSH
• peroxid vodíku MeSH
• pervanadate MeSH Prohlížeč
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosinfosfatasy MeSH
• vanadáty MeSH

Engagement of the Fcepsilon receptor I (FcepsilonRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non-T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow-derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcepsilonRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C gamma1 and phospholipase C gamma2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain-containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.

• MeSH
• adaptorové proteiny signální transdukční fyziologie   MeSH
• adaptorové proteiny vezikulární transportní fyziologie   MeSH
• degranulace buněk MeSH
• fosfatidylinositol-3-kinasy fyziologie   MeSH
• fosfolipasa C gama MeSH
• fosfolipasy typu C metabolismus   MeSH
• fosfoproteiny fyziologie   MeSH
• fosforylace MeSH
• mastocyty fyziologie   MeSH
• membránové proteiny fyziologie   MeSH
• myši MeSH
• proteiny fyziologie   MeSH
• receptory IgE fyziologie   MeSH
• signální transdukce * MeSH
• tyrosin metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• adaptorové proteiny vezikulární transportní MeSH
• fosfolipasa C gama MeSH
• fosfolipasy typu C MeSH
• fosfoproteiny MeSH
• LAB protein, mouse MeSH Prohlížeč
• Lat protein, mouse MeSH Prohlížeč
• LAT2 protein, mouse MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny MeSH
• receptory IgE MeSH
• tyrosin MeSH
• vápník MeSH