ORMDL3 is a prominent member of a family of highly conserved endoplasmic reticulum resident proteins, ORMs (ORM1 and ORM2) in yeast, dORMDL in Drosophila and ORMDLs (ORMDL1, ORMDL2, and ORMDL3) in mammals. ORMDL3 mediates feedback inhibition of de novo sphingolipid synthesis. Expression levels of ORMDL3 are associated with the development of inflammatory and autoimmune diseases including asthma, systemic lupus erythematosus, type 1 diabetes mellitus and others. It has been shown that simultaneous deletions of other ORMDL family members could potentiate ORMDL3-induced phenotypes. To understand the complex function of ORMDL proteins in immunity in vivo, we analyzed mice with single or double deletions of Ormdl genes. In contrast to other single and double knockouts, simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupted blood homeostasis and reduced immune cell content in peripheral blood and spleens of mice. The reduced number of splenocytes was not caused by aberrant immune cell homing. A competitive bone marrow transplantation assay showed that the development of Ormdl1-/-/Ormdl3-/- B cells was dependent on lymphocyte intrinsic factors. Highly increased sphingolipid production was observed in the spleens and bone marrow of Ormdl1-/-/Ormdl3-/- mice. Slight, yet significant, increase in some sphingolipid species was also observed in the spleens of Ormdl3-/- mice and in the bone marrow of both, Ormdl1-/- and Ormdl3-/- single knockout mice. Taken together, our results demonstrate that the physiological expression of ORMDL proteins is critical for the proper development and circulation of lymphocytes. We also show cell-type specific roles of individual ORMDL family members in the production of different sphingolipid species.

• MeSH
• B-lymfocyty imunologie   metabolismus   MeSH
• delece genu * MeSH
• homeostáza * MeSH
• membránové proteiny * genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• sfingolipidy metabolismus   MeSH
• slezina imunologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

• MeSH
• cholesterol MeSH
• cytokiny MeSH
• heptanol MeSH
• mastocyty * MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• arachidonát-5-lipoxygenasa metabolismus   MeSH
• ikosanoidy analýza   metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• serin-C-palmitoyltransferasa metabolismus   MeSH
• sfingolipidy analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

SCOPE: The docosahexaenoic acid ester of hydroxy linoleic acid (13-DHAHLA) is a bioactive lipid with anti-inflammatory properties from the family of fatty acid esters of hydroxy fatty acids (FAHFA). METHODS AND RESULTS: To explore the biosynthesis of 13-DHAHLA from dietary oils, C57BL/6N mice are gavaged for 8 days with various corn oil/marine oil mixtures containing the same amount of DHA. Plasma levels of omega-3 FAHFAs are influenced by the lipid composition of the mixtures but do not reflect the changes in bioavailability of polyunsaturated fatty acids in plasma. Triacylglycerol-bound DHA and linoleic acid serve as more effective precursors for 13-DHAHLA synthesis than DHA bound in phospholipids or wax esters. Both 13(S)- and 13(R)-DHAHLA inhibit antigen and PGE2 -induced chemotaxis and degranulation of mast cells to a comparable extent and 13(S)-DHAHLA is identified as the predominant isomer in mouse adipose tissue. CONCLUSION: Here, the optimal nutritional source of DHA is identified, which supports production of anti-inflammatory FAHFAs, as triacylglycerol-based marine oil and also reveals a possible role of triacylglycerols in the synthesis of FAHFA lipokines.

• MeSH
• antiflogistika nesteroidní krev   farmakokinetika   MeSH
• biologická dostupnost MeSH
• chemotaxe účinky léků   MeSH
• kyseliny dokosahexaenové farmakokinetika   MeSH
• kyseliny linolové chemie   MeSH
• kyseliny mastné omega-3 farmakokinetika   farmakologie   MeSH
• mastocyty účinky léků   MeSH
• myši inbrední C57BL MeSH
• oleje chemie   farmakokinetika   MeSH
• stereoizomerie MeSH
• triglyceridy chemie   MeSH
• vodní organismy MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• 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

Chemotaxis of mast cells is one of the crucial steps in their development and function. Non-T cell activation linker (NTAL) is a transmembrane adaptor protein that inhibits the activation of mast cells and B cells in a phosphorylation-dependent manner. Here, we studied the role of NTAL in the migration of mouse mast cells stimulated by prostaglandin E2 (PGE2). Although PGE2 does not induce the tyrosine phosphorylation of NTAL, unlike IgE immune complex antigens, we found that loss of NTAL increased the chemotaxis of mast cells toward PGE2 Stimulation of mast cells that lacked NTAL with PGE2 enhanced the phosphorylation of AKT and the production of phosphatidylinositol 3,4,5-trisphosphate. In resting NTAL-deficient mast cells, phosphorylation of an inhibitory threonine in ERM family proteins accompanied increased activation of β1-containing integrins, which are features often associated with increased invasiveness in tumors. Rescue experiments indicated that only full-length, wild-type NTAL restored the chemotaxis of NTAL-deficient cells toward PGE2 Together, these data suggest that NTAL is a key inhibitor of mast cell chemotaxis toward PGE2, which may act through the RHOA/ERM/β1-integrin and PI3K/AKT axes.

• MeSH
• adaptorové proteiny signální transdukční metabolismus   MeSH
• aktiny metabolismus   MeSH
• antigeny CD29 metabolismus   MeSH
• bodová mutace MeSH
• chemotaxe * MeSH
• cholesterol metabolismus   MeSH
• dinoproston metabolismus   MeSH
• fosfatidylinositol-3-kinasy metabolismus   MeSH
• fosforylace MeSH
• integriny metabolismus   MeSH
• mastocyty metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• myši MeSH
• proteinové domény MeSH
• proteiny metabolismus   MeSH
• signální transdukce MeSH
• threonin chemie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Single-nucleotide polymorphism studies have linked the chromosome 17q12-q21 region, where the human orosomucoid-like (ORMDL)3 gene is localized, to the risk of asthma and several other inflammatory diseases. Although mast cells are involved in the development of these diseases, the contribution of ORMDL3 to the mast cell physiology is unknown. In this study, we examined the role of ORMDL3 in antigen-induced activation of murine mast cells with reduced or enhanced ORMDL3 expression. Our data show that in antigen-activated mast cells, reduced expression of the ORMDL3 protein had no effect on degranulation and calcium response, but significantly enhanced phosphorylation of AKT kinase at Ser 473 followed by enhanced phosphorylation and degradation of IκBα and translocation of the NF-κB p65 subunit into the nucleus. These events were associated with an increased expression of proinflammatory cytokines (TNF-α, IL-6, and IL-13), chemokines (CCL3 and CCL4), and cyclooxygenase-2 dependent synthesis of prostaglandin D2. Antigen-mediated chemotaxis was also enhanced in ORMDL3-deficient cells, whereas spreading on fibronectin was decreased. On the other hand, increased expression of ORMDL3 had no significant effect on the studied signaling events, except for reduced antigen-mediated chemotaxis. These data were corroborated by increased IgE-antigen-dependent passive cutaneous anaphylaxis in mice with locally silenced ORMDL3 using short interfering RNAs. Our data also show that antigen triggers suppression of ORMDL3 expression in the mast cells. In summary, we provide evidence that downregulation of ORMDL3 expression in mast cells enhances AKT and NF-κB-directed signaling pathways and chemotaxis and contributes to the development of mast cell-mediated local inflammation in vivo.

• MeSH
• chemotaxe * MeSH
• cytokiny genetika   imunologie   MeSH
• degranulace buněk * MeSH
• down regulace MeSH
• kultivované buňky MeSH
• mastocyty cytologie   imunologie   metabolismus   MeSH
• membránové proteiny genetika   imunologie   MeSH
• messenger RNA genetika   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• receptory IgE imunologie   MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

Ethanol has multiple effects on biochemical events in a variety of cell types, including the high-affinity immunoglobulin E receptor (FcεRI) signaling in antigen-activated mast cells. However, the underlying molecular mechanism remains unknown. To get better understanding of the effect of ethanol on FcεRI-mediated signaling we examined the effect of short-term treatment with non-toxic concentrations of ethanol on FcεRI signaling events in mouse bone marrow-derived mast cells. We found that 15 min exposure to ethanol inhibited antigen-induced degranulation, calcium mobilization, expression of proinflammatory cytokine genes (tumor necrosis factor-α, interleukin-6, and interleukin-13), and formation of reactive oxygen species in a dose-dependent manner. Removal of cellular cholesterol with methyl-β-cyclodextrin had a similar effect and potentiated some of the inhibitory effects of ethanol. In contrast, exposure of the cells to cholesterol-saturated methyl-β-cyclodextrin abolished in part the inhibitory effect of ethanol on calcium response and production of reactive oxygen species, supporting lipid-centric theories of ethanol action on the earliest stages of mast cell signaling. Further studies showed that exposure to ethanol and/or removal of cholesterol inhibited early FcεRI activation events, including tyrosine phosphorylation of the FcεRI β and γ subunits, SYK kinases, LAT adaptor protein, phospholipase Cγ, STAT5, and AKT and internalization of aggregated FcεRI. Interestingly, ethanol alone, and particularly in combination with methyl-β-cyclodextrin, enhanced phosphorylation of negative regulatory tyrosine 507 of LYN kinase. Finally, we found that ethanol reduced passive cutaneous anaphylactic reaction in mice, suggesting that ethanol also inhibits FcεRI signaling under in vivo conditions. The combined data indicate that ethanol interferes with early antigen-induced signaling events in mast cells by suppressing the function of FcεRI-cholesterol signalosomes at the plasma membrane.

• MeSH
• cholesterol metabolismus   MeSH
• cytokiny genetika   metabolismus   MeSH
• ethanol farmakologie   MeSH
• exprese genu účinky léků   MeSH
• fosforylace MeSH
• mastocyty účinky léků   metabolismus   MeSH
• myši MeSH
• receptory IgE metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• vápník metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Amplification of DNA templates from whole blood with Taq DNA polymerase remains a difficult task worldwide. Using a real-time PCR setup and a buffer supplemented with 1M 1,2-propanediol, 0.2M trehalose, and SYBR green I we show a reliable technique for genotyping in mice and detection of single-nucleotide polymorphisms/mutations in humans. Elimination of DNA extraction and use of the common Taq DNA polymerase and DNA dye bring about substantial savings in labor and cost.

• MeSH
• DNA chemie   MeSH
• genotyp MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• myši MeSH
• organické látky chemie   MeSH
• propylenglykol chemie   MeSH
• pufry MeSH
• Taq-polymerasa chemie   MeSH
• trehalosa chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH