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.

• Keywords
• immunoglobulin E, lipid raft, mast cell, plasma membrane, signal transduction, tumor necrosis factor, tyrosine kinase,
• MeSH
• Antigens metabolism   MeSH
• Cell Degranulation MeSH
• Ursolic Acid MeSH
• Lipids pharmacology   MeSH
• Mast Cells metabolism   MeSH
• Mice MeSH
• Receptors, IgE * metabolism   MeSH
• Triterpenes * pharmacology   metabolism   MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens MeSH
• Lipids MeSH
• Receptors, IgE * MeSH
• Triterpenes * MeSH
• Calcium 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.

• Keywords
• FcϵRI, ORMDL family, mast cells, passive cutaneous anaphylactic reaction, passive systemic anaphylaxis, sphingolipids, sphingosine-1-phosphate,
• MeSH
• Anaphylaxis etiology   metabolism   MeSH
• Biomarkers MeSH
• Chemotaxis immunology   MeSH
• Cytokines metabolism   MeSH
• Gene Expression MeSH
• Lysophospholipids blood   metabolism   MeSH
• Mast Cells immunology   metabolism   MeSH
• Membrane Proteins chemistry   deficiency   genetics   metabolism   MeSH
• Multigene Family MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Disease Susceptibility MeSH
• Passive Cutaneous Anaphylaxis genetics   immunology   MeSH
• Amino Acid Sequence MeSH
• Sphingolipids blood   metabolism   MeSH
• Sphingosine analogs & derivatives   blood   metabolism   MeSH
• Signal Transduction * MeSH
• Calcium metabolism   MeSH
• Calcium Signaling MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biomarkers MeSH
• Cytokines MeSH
• Lysophospholipids MeSH
• Membrane Proteins MeSH
• ORMDL3 protein, mouse MeSH Browser
• Sphingolipids MeSH
• Sphingosine MeSH
• sphingosine 1-phosphate MeSH Browser
• Calcium 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
• Anaphylaxis genetics   MeSH
• Bone Marrow Cells metabolism   physiology   MeSH
• CSK Tyrosine-Protein Kinase MeSH
• Cell Degranulation MeSH
• Type C Phospholipases metabolism   MeSH
• Phosphoproteins genetics   MeSH
• Phosphorylation MeSH
• Intracellular Signaling Peptides and Proteins metabolism   MeSH
• Syk Kinase MeSH
• RNA, Small Interfering MeSH
• Mast Cells metabolism   physiology   MeSH
• Membrane Proteins genetics   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Proto-Oncogene Proteins c-fyn biosynthesis   MeSH
• Proto-Oncogene Proteins c-kit metabolism   MeSH
• Receptors, IgE metabolism   MeSH
• RNA Interference MeSH
• Signal Transduction MeSH
• src-Family Kinases biosynthesis   metabolism   MeSH
• Protein-Tyrosine Kinases metabolism   MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CSK Tyrosine-Protein Kinase MeSH
• Type C Phospholipases MeSH
• Phosphoproteins MeSH
• Fyn protein, mouse MeSH Browser
• Intracellular Signaling Peptides and Proteins MeSH
• Syk Kinase MeSH
• lyn protein-tyrosine kinase MeSH Browser
• RNA, Small Interfering MeSH
• Membrane Proteins MeSH
• Pag1 protein, mouse MeSH Browser
• Proto-Oncogene Proteins c-fyn MeSH
• Proto-Oncogene Proteins c-kit MeSH
• Receptors, IgE MeSH
• src-Family Kinases MeSH
• Syk protein, mouse MeSH Browser
• Protein-Tyrosine Kinases MeSH
• Calcium 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
• Enzyme Activation MeSH
• Antigens metabolism   MeSH
• Cell Membrane metabolism   MeSH
• Time Factors MeSH
• Cholesterol metabolism   MeSH
• Detergents pharmacology   MeSH
• DNA metabolism   MeSH
• Phosphorylation MeSH
• Phosphotyrosine metabolism   MeSH
• DNA Fragmentation MeSH
• Immunoblotting MeSH
• Microscopy, Confocal MeSH
• Protein Conformation MeSH
• Rats MeSH
• Myristic Acid metabolism   MeSH
• Palmitic Acid metabolism   MeSH
• Luminescent Proteins metabolism   MeSH
• Membrane Microdomains metabolism   MeSH
• Lipid Metabolism MeSH
• Mice MeSH
• Octoxynol pharmacology   MeSH
• Precipitin Tests MeSH
• Receptors, IgE metabolism   MeSH
• Recombinant Proteins metabolism   MeSH
• Sphingolipids metabolism   MeSH
• Signal Transduction MeSH
• src-Family Kinases chemistry   metabolism   physiology   MeSH
• Protein Structure, Tertiary MeSH
• Transfection MeSH
• Tyrosine metabolism   MeSH
• Calcium metabolism   MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Green Fluorescent Proteins MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Antigens MeSH
• Cholesterol MeSH
• Detergents MeSH
• DNA MeSH
• Phosphotyrosine MeSH
• Myristic Acid MeSH
• Palmitic Acid MeSH
• Luminescent Proteins MeSH
• lyn protein-tyrosine kinase MeSH Browser
• Octoxynol MeSH
• Receptors, IgE MeSH
• Recombinant Proteins MeSH
• Sphingolipids MeSH
• src-Family Kinases MeSH
• Tyrosine MeSH
• Calcium MeSH
• Green Fluorescent Proteins MeSH