Pentacyclic triterpenoid ursolic acid interferes with mast cell activation via a lipid-centric mechanism affecting FcεRI signalosome functions.

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.

Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways.

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.

ORMDL2 Deficiency Potentiates the ORMDL3-Dependent Changes in Mast Cell Signaling.

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.