Aggregation of high-affinity IgE receptors (FcϵRIs) on granulated mast cells triggers signaling pathways leading to a calcium response and release of inflammatory mediators from secretory granules. While microtubules play a role in the degranulation process, the complex molecular mechanisms regulating microtubule remodeling in activated mast cells are only partially understood. Here, we demonstrate that the activation of bone marrow mast cells induced by FcϵRI aggregation increases centrosomal microtubule nucleation, with G protein-coupled receptor kinase-interacting protein 2 (GIT2) playing a vital role in this process. Both endogenous and exogenous GIT2 were associated with centrosomes and γ-tubulin complex proteins. Depletion of GIT2 enhanced centrosomal microtubule nucleation, and phenotypic rescue experiments revealed that GIT2, unlike GIT1, acts as a negative regulator of microtubule nucleation in mast cells. GIT2 also participated in the regulation of antigen-induced degranulation and chemotaxis. Further experiments showed that phosphorylation affected the centrosomal localization of GIT2 and that during antigen-induced activation, GIT2 was phosphorylated by conventional protein kinase C, which promoted microtubule nucleation. We propose that GIT2 is a novel regulator of microtubule organization in activated mast cells by modulating centrosomal microtubule nucleation.

• Klíčová slova
• G protein-coupled receptor kinase-interacting protein 2 (GIT2), centrosome, mast cells, microtubule nucleation, protein kinase C (PKC),
• MeSH
• centrozom metabolismus   MeSH
• kostní dřeň * MeSH
• mastocyty * metabolismus   MeSH
• mikrotubuly * metabolismus   MeSH
• myši MeSH
• proteiny aktivující GTPasu * metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Git2 protein, mouse MeSH Prohlížeč
• proteiny aktivující GTPasu * MeSH

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.

• Klíčová slova
• immunoglobulin E, lipid raft, mast cell, plasma membrane, signal transduction, tumor necrosis factor, tyrosine kinase,
• MeSH
• antigeny metabolismus   MeSH
• degranulace buněk MeSH
• kyselina ursolová MeSH
• lipidy farmakologie   MeSH
• mastocyty metabolismus   MeSH
• myši MeSH
• receptory IgE * metabolismus   MeSH
• triterpeny * farmakologie   metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny MeSH
• lipidy MeSH
• receptory IgE * MeSH
• triterpeny * MeSH
• vápník MeSH

A better understanding of the molecular mechanisms leading to mast cell migration and chemotaxis is the long-term goal in mast cell research and is essential for comprehension of mast cell function in health and disease. Various techniques have been developed in recent decades for in vitro and in vivo assessment of mast cell motility and chemotaxis. In this chapter, three microscopy assays facilitating real-time quantification of mast cell chemotaxis and migration are described, focusing on individual cell tracking and data analysis.

• Klíčová slova
• Cell migration, Cell tracking, Chemoattractant, Chemokine, Chemotaxis, Mast cells,
• MeSH
• analýza buněčné migrace metody   MeSH
• biotest metody   MeSH
• buněčný tracking metody   MeSH
• chemotaxe fyziologie   MeSH
• fibronektiny metabolismus   MeSH
• lidé MeSH
• mastocyty cytologie   fyziologie   MeSH
• mikroskopie metody   MeSH
• myši MeSH
• počítačové systémy MeSH
• pohyb buněk fyziologie   MeSH
• prostředí kontrolované MeSH
• sefarosa MeSH
• software MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fibronektiny MeSH
• sefarosa MeSH

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

• Klíčová slova
• SHP-1 tyrosine phosphatase, bone marrow-derived mast cells, cell activation, microtubule nucleation, γ-tubulin complexes,
• MeSH
• degranulace buněk MeSH
• HEK293 buňky MeSH
• kinasa Syk metabolismus   MeSH
• lidé MeSH
• mastocyty cytologie   metabolismus   MeSH
• MFC-7 buňky MeSH
• mikrotubuly metabolismus   MeSH
• myši MeSH
• tubulin metabolismus   MeSH
• tyrosinfosfatasa nereceptorového typu 6 antagonisté a inhibitory   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kinasa Syk MeSH
• Ptpn6 protein, mouse MeSH Prohlížeč
• Syk protein, mouse MeSH Prohlížeč
• tubulin MeSH
• tyrosinfosfatasa nereceptorového typu 6 MeSH

Mast cells play an effector role in innate immunity, allergy, and inflammation. Antigen-mediated activation of mast cells initiates signaling events leading to Ca2+ response and the release of inflammatory and allergic mediators from granules. Diseases associated with deregulated mast cell functions are hard to treat and there is an increasing demand for new therapeutic strategies. Miltefosine (hexadecylphosphocholine) is a new candidate for treatment of mast cell-driven diseases as it inhibits activation of mast cells. It has been proposed that miltefosine acts as a lipid raft modulator through its interference with the structural organization of surface receptors in the cell membrane. However, molecular mechanisms of its action are not fully understood. Here, we report that in antigen-activated bone marrow-derived mast cells (BMMCs), miltefosine inhibits degranulation, reorganization of microtubules, as well as antigen-induced chemotaxis. While aggregation and tyrosine phosphorylation of IgE receptors were suppressed in activated cells pre-treated with miltefosine, overall tyrosine phosphorylation levels of Lyn and Syk kinases, and Ca2+ influx were not inhibited. In contrast, lipid raft disruptor methyl-β-cyclodextrin attenuated the Ca2+ influx. Tagged-miltefosine rapidly localized into the cell interior, and live-cell imaging of BMMCs with labeled intracellular granules disclosed that miltefosine inhibited movement of some granules. Immunoprecipitation and in vitro kinase assays revealed that miltefosine inhibited Ca2+- and diacylglycerol-regulated conventional protein kinase C (cPKC) isoforms that are important for mast cell degranulation. Inhibition of cPKCs by specific inhibitor Ly333531 affected activation of BMMCs in the same way as miltefosine. Collectively, our data suggest that miltefosine modulates mast cells both at the plasma membrane and in the cytosol by inhibition of cPKCs. This alters intracellular signaling pathway(s) directed to microtubules, degranulation, and migration.

• Klíčová slova
• bone marrow-derived mast cells, cell activation, microtubules, miltefosine, protein kinase C,
• Publikační typ
• časopisecké články MeSH

Mast cells play crucial roles in both innate and adaptive arms of the immune system. Along with basophils, mast cells are essential effector cells for allergic inflammation that causes asthma, allergic rhinitis, food allergy and atopic dermatitis. Mast cells are usually increased in inflammatory sites of allergy and, upon activation, release various chemical, lipid, peptide and protein mediators of allergic reactions. Since antigen/immunoglobulin E (IgE)-mediated activation of these cells is a central event to trigger allergic reactions, innumerable studies have been conducted on how these cells are activated through cross-linking of the high-affinity IgE receptor (FcεRI). Development of mature mast cells from their progenitor cells is under the influence of several growth factors, of which the stem cell factor (SCF) seems to be the most important. Therefore, how SCF induces mast cell development and activation via its receptor, KIT, has been studied extensively, including a cross-talk between KIT and FcεRI signaling pathways. Although our understanding of the signaling mechanisms of the FcεRI and KIT pathways is far from complete, pharmaceutical applications of the knowledge about these pathways are underway. This review will focus on recent progresses in FcεRI and KIT signaling and chemotaxis.

• Klíčová slova
• Chemotaxis, IgE receptor, KIT receptor, Mast cell, Plasma membrane, Signal transduction,
• MeSH
• chemotaxe * účinky léků   MeSH
• lidé MeSH
• mastocyty cytologie   účinky léků   MeSH
• signální transdukce * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH

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

Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca(2+) response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)(2) or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcεRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)(2) fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcεRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• antigeny CD9 fyziologie   MeSH
• antigeny CD98 - lehké řetězce metabolismus   MeSH
• antigeny metabolismus   MeSH
• biologické modely MeSH
• buněčná membrána metabolismus   MeSH
• chemotaxe MeSH
• cytoskelet metabolismus   MeSH
• fosfoproteiny metabolismus   MeSH
• fosforylace MeSH
• glukuronidasa metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   MeSH
• krysa rodu Rattus MeSH
• mastocyty cytologie   MeSH
• membránové proteiny metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• potkani Wistar MeSH
• receptory IgE metabolismus   MeSH
• transportní systém aminokyselin y+ metabolismus   MeSH
• tyrosin chemie   MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční MeSH
• antigeny CD9 MeSH
• antigeny CD98 - lehké řetězce MeSH
• antigeny MeSH
• fosfoproteiny MeSH
• glukuronidasa MeSH
• imunoglobuliny - Fab fragmenty MeSH
• Lat protein, mouse MeSH Prohlížeč
• Lat protein, rat MeSH Prohlížeč
• membránové proteiny MeSH
• receptory IgE MeSH
• SLC7A8 protein, mouse MeSH Prohlížeč
• Slc7a8 protein, rat MeSH Prohlížeč
• transportní systém aminokyselin y+ MeSH
• tyrosin MeSH
• vápník MeSH

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

• MeSH
• adaptorové proteiny signální transdukční * MeSH
• aktivace lymfocytů MeSH
• B-lymfocyty imunologie   metabolismus   MeSH
• buněčné linie MeSH
• buňky NK imunologie   metabolismus   MeSH
• fosfoproteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• lymfoidní tkáň cytologie   metabolismus   MeSH
• membránové mikrodomény chemie   metabolismus   MeSH
• membránové proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• monocyty imunologie   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• proteiny * MeSH
• receptory antigenů B-buněk metabolismus   MeSH
• receptory Fc metabolismus   MeSH
• receptory IgE metabolismus   MeSH
• receptory IgG metabolismus   MeSH
• sekvence aminokyselin MeSH
• signální transdukce * MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• transportní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adaptorové proteiny signální transdukční * MeSH
• fosfoproteiny MeSH
• LAT protein, human MeSH Prohlížeč
• Lat protein, mouse MeSH Prohlížeč
• LAT2 protein, human MeSH Prohlížeč
• LAT2 protein, mouse MeSH Prohlížeč
• membránové proteiny MeSH
• proteiny * MeSH
• receptory antigenů B-buněk MeSH
• receptory Fc MeSH
• receptory IgE MeSH
• receptory IgG MeSH
• transportní proteiny MeSH

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.

• MeSH
• aktivace enzymů MeSH
• antigeny metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• časové faktory MeSH
• cholesterol metabolismus   MeSH
• detergenty farmakologie   MeSH
• DNA metabolismus   MeSH
• fosforylace MeSH
• fosfotyrosin metabolismus   MeSH
• fragmentace DNA MeSH
• imunoblotting MeSH
• konfokální mikroskopie MeSH
• konformace proteinů MeSH
• krysa rodu Rattus MeSH
• kyselina myristová metabolismus   MeSH
• kyselina palmitová metabolismus   MeSH
• luminescentní proteiny metabolismus   MeSH
• membránové mikrodomény metabolismus   MeSH
• metabolismus lipidů MeSH
• myši MeSH
• oktoxynol farmakologie   MeSH
• precipitinové testy MeSH
• receptory IgE metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• sfingolipidy metabolismus   MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu chemie   metabolismus   fyziologie   MeSH
• terciární struktura proteinů MeSH
• transfekce MeSH
• tyrosin metabolismus   MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zelené fluorescenční proteiny 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
• cholesterol MeSH
• detergenty MeSH
• DNA MeSH
• fosfotyrosin MeSH
• kyselina myristová MeSH
• kyselina palmitová MeSH
• luminescentní proteiny MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• oktoxynol MeSH
• receptory IgE MeSH
• rekombinantní proteiny MeSH
• sfingolipidy MeSH
• skupina kinas odvozených od src-genu MeSH
• tyrosin MeSH
• vápník MeSH
• zelené fluorescenční proteiny MeSH