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

The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3). However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO exhibited increased intracellular levels of sphingosine-1-phosphate (S1P). Furthermore, we found that concurrent ORMDL2 and ORMDL3 deficiency increased IκB-α phosphorylation, degranulation, and production of IL-4, IL-6, and TNF-α cytokines in antigen-activated mast cells. Interestingly, the chemotaxis towards antigen was increased in all mutant cell types analyzed. Experiments in vivo showed that passive cutaneous anaphylaxis (PCA), which is initiated by mast cell activation, was increased only in ORMDL2,3 double KO mice, supporting our in vitro observations with mast cells. On the other hand, ORMDL3 KO and ORMDL2,3 double KO mice showed faster recovery from passive systemic anaphylaxis, which could be mediated by increased levels of blood S1P presented in such mice. Our findings demonstrate that Ormdl2 deficiency potentiates the ORMDL3-dependent changes in mast cell signaling.

• Klíčová slova
• FcϵRI, ORMDL family, mast cells, passive cutaneous anaphylactic reaction, passive systemic anaphylaxis, sphingolipids, sphingosine-1-phosphate,
• MeSH
• anafylaxe etiologie   metabolismus   MeSH
• biologické markery MeSH
• chemotaxe imunologie   MeSH
• cytokiny metabolismus   MeSH
• exprese genu MeSH
• lysofosfolipidy krev   metabolismus   MeSH
• mastocyty imunologie   metabolismus   MeSH
• membránové proteiny chemie   nedostatek   genetika   metabolismus   MeSH
• multigenová rodina MeSH
• myši knockoutované MeSH
• myši MeSH
• náchylnost k nemoci MeSH
• pasivní kožní anafylaxe genetika   imunologie   MeSH
• sekvence aminokyselin MeSH
• sfingolipidy krev   metabolismus   MeSH
• sfingosin analogy a deriváty   krev   metabolismus   MeSH
• signální transdukce * MeSH
• vápník metabolismus   MeSH
• vápníková signalizace MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• cytokiny MeSH
• lysofosfolipidy MeSH
• membránové proteiny MeSH
• ORMDL3 protein, mouse MeSH Prohlížeč
• sfingolipidy MeSH
• sfingosin MeSH
• sphingosine 1-phosphate MeSH Prohlížeč
• vápník MeSH

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.

• MeSH
• anafylaxe genetika   MeSH
• buňky kostní dřeně metabolismus   fyziologie   MeSH
• C-terminální Src kinasa MeSH
• degranulace buněk MeSH
• fosfolipasy typu C metabolismus   MeSH
• fosfoproteiny genetika   MeSH
• fosforylace MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• kinasa Syk MeSH
• malá interferující RNA MeSH
• mastocyty metabolismus   fyziologie   MeSH
• membránové proteiny genetika   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• protoonkogenní proteiny c-fyn biosyntéza   MeSH
• protoonkogenní proteiny c-kit metabolismus   MeSH
• receptory IgE metabolismus   MeSH
• RNA interference MeSH
• signální transdukce MeSH
• skupina kinas odvozených od src-genu biosyntéza   metabolismus   MeSH
• tyrosinkinasy metabolismus   MeSH
• vápník metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• C-terminální Src kinasa MeSH
• fosfolipasy typu C MeSH
• fosfoproteiny MeSH
• Fyn protein, mouse MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• kinasa Syk MeSH
• lyn protein-tyrosine kinase MeSH Prohlížeč
• malá interferující RNA MeSH
• membránové proteiny MeSH
• Pag1 protein, mouse MeSH Prohlížeč
• protoonkogenní proteiny c-fyn MeSH
• protoonkogenní proteiny c-kit MeSH
• receptory IgE MeSH
• skupina kinas odvozených od src-genu MeSH
• Syk protein, mouse MeSH Prohlížeč
• tyrosinkinasy MeSH
• vápník 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