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

Protein 4.1R, a member of the 4.1 family, functions as a bridge between cytoskeletal and plasma membrane proteins. It is expressed in T cells, where it binds to a linker for activation of T cell (LAT) family member 1 and inhibits its phosphorylation and downstream signaling events after T cell receptor triggering. The role of the 4.1R protein in cell activation through other immunoreceptors is not known. In this study, we used 4.1R-deficient (4.1R-KO) and 4.1R wild-type (WT) mice and explored the role of the 4.1R protein in the high-affinity IgE receptor (FcεRI) signaling in mast cells. We found that bone marrow mast cells (BMMCs) derived from 4.1R-KO mice showed normal growth in vitro and expressed FcεRI and c-KIT at levels comparable to WT cells. However, 4.1R-KO cells exhibited reduced antigen-induced degranulation, calcium response, and secretion of tumor necrosis factor-α. Chemotaxis toward antigen and stem cell factor (SCF) and spreading on fibronectin were also reduced in 4.1R-KO BMMCs, whereas prostaglandin E2-mediated chemotaxis was not affected. Antibody-induced aggregation of tetraspanin CD9 inhibited chemotaxis toward antigen in WT but not 4.1R-KO BMMCs, implying a CD9-4.1R protein cross-talk. Further studies documented that in the absence of 4.1R, antigen-mediated phosphorylation of FcεRI β and γ subunits was not affected, but phosphorylation of SYK and subsequent signaling events such as phosphorylation of LAT1, phospholipase Cγ1, phosphatases (SHP1 and SHIP), MAP family kinases (p38, ERK, JNK), STAT5, CBL, and mTOR were reduced. Immunoprecipitation studies showed the presence of both LAT1 and LAT2 (LAT, family member 2) in 4.1R immunocomplexes. The positive regulatory role of 4.1R protein in FcεRI-triggered activation was supported by in vivo experiments in which 4.1R-KO mice showed the normal presence of mast cells in the ears and peritoneum, but exhibited impaired passive cutaneous anaphylaxis. The combined data indicate that the 4.1R protein functions as a positive regulator in the early activation events after FcεRI triggering in mast cells.

• Keywords
• 4.1R protein, chemotaxis, degranulation, mast cell, passive cutaneous anaphylaxis,
• MeSH
• Chemotaxis immunology   MeSH
• Cell Degranulation immunology   MeSH
• Mast Cells immunology   metabolism   MeSH
• Microfilament Proteins immunology   metabolism   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Passive Cutaneous Anaphylaxis immunology   MeSH
• Receptors, IgE immunology   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
• Epb41 protein, mouse MeSH Browser
• Microfilament Proteins MeSH
• Receptors, IgE 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
• Enzyme Activation drug effects   genetics   immunology   MeSH
• Antigens immunology   metabolism   pharmacology   MeSH
• Phosphorylation drug effects   genetics   immunology   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Rats MeSH
• Mast Cells immunology   metabolism   MeSH
• Membrane Microdomains genetics   immunology   metabolism   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Oxidation-Reduction drug effects   MeSH
• Oxidants pharmacology   MeSH
• Hydrogen Peroxide pharmacology   MeSH
• Receptors, IgE genetics   immunology   metabolism   MeSH
• src-Family Kinases genetics   immunology   metabolism   MeSH
• Protein Transport drug effects   genetics   immunology   MeSH
• Protein Tyrosine Phosphatases antagonists & inhibitors   genetics   immunology   metabolism   MeSH
• Vanadates pharmacology   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
• Enzyme Inhibitors MeSH
• lyn protein-tyrosine kinase MeSH Browser
• Oxidants MeSH
• Hydrogen Peroxide MeSH
• pervanadate MeSH Browser
• Receptors, IgE MeSH
• src-Family Kinases MeSH
• Protein Tyrosine Phosphatases MeSH
• Vanadates 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
• Adaptor Proteins, Signal Transducing physiology   MeSH
• Adaptor Proteins, Vesicular Transport physiology   MeSH
• Cell Degranulation MeSH
• Phosphatidylinositol 3-Kinases physiology   MeSH
• Phospholipase C gamma MeSH
• Type C Phospholipases metabolism   MeSH
• Phosphoproteins physiology   MeSH
• Phosphorylation MeSH
• Mast Cells physiology   MeSH
• Membrane Proteins physiology   MeSH
• Mice MeSH
• Proteins physiology   MeSH
• Receptors, IgE physiology   MeSH
• Signal Transduction * MeSH
• Tyrosine 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
• Adaptor Proteins, Signal Transducing MeSH
• Adaptor Proteins, Vesicular Transport MeSH
• Phospholipase C gamma MeSH
• Type C Phospholipases MeSH
• Phosphoproteins MeSH
• LAB protein, mouse MeSH Browser
• Lat protein, mouse MeSH Browser
• LAT2 protein, mouse MeSH Browser
• Membrane Proteins MeSH
• Proteins MeSH
• Receptors, IgE MeSH
• Tyrosine MeSH
• Calcium MeSH