Negative Regulation of Mast Cell Signaling and Function by the Adaptor LAB/NTAL

Engagement of the Fcɛ receptor I (FcɛRI) 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 FcɛRI-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 γ1 and phospholipase C γ2, 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

Regulation of mast cell activation at the level of the high-affinity IgE receptor and STIM1

(EN) This thesis is focused on two important gate-keepers of mast cell signaling. The first is the complex of the high-affinity receptor for immunoglobulin E (IgE) (FcεRI) associated with Lck/Yes- related novel tyrosine kinase (Lyn), which is involved in acquired immune responses and the second is the stromal interaction molecule (STIM)1, which senses calcium levels in endoplasmic reticulum (ER) and upon depletion of ER Ca2+ stores participates in opening of the plasma membrane Ca2+ release- activated Ca2+ (CRAC) channels. Although the structure of FcεRI is known for many years and numerous molecules associated with the receptor have been described, the exact molecular mechanism of initiation and termination of the FcεRI signaling is elusive. Therefore, we evaluated the current knowledge on the molecular mechanisms of FcεRI phosphorylation with emphasis on the newly described model according to which cross-talk between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) sets the threshold for FcεRI tyrosine phosphorylation (PTK-PTP interplay model). Furthermore, we extended the knowledge about topography of active phosphatases which are prone to oxidation within the clusters of transmembrane adaptor proteins non-T cell activation linker (NTAL) and linker for activation of T..

Regulace aktivace žírných buněk na úrovni vysokoafinitního receptoru pro IgE a STIM1

(EN) This thesis is focused on two important gate-keepers of mast cell signaling. The first is the complex of the high-affinity receptor for immunoglobulin E (IgE) (FcεRI) associated with Lck/Yes- related novel tyrosine kinase (Lyn), which is involved in acquired immune responses and the second is the stromal interaction molecule (STIM)1, which senses calcium levels in endoplasmic reticulum (ER) and upon depletion of ER Ca2+ stores participates in opening of the plasma membrane Ca2+ release- activated Ca2+ (CRAC) channels. Although the structure of FcεRI is known for many years and numerous molecules associated with the receptor have been described, the exact molecular mechanism of initiation and termination of the FcεRI signaling is elusive. Therefore, we evaluated the current knowledge on the molecular mechanisms of FcεRI phosphorylation with emphasis on the newly described model according to which cross-talk between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) sets the threshold for FcεRI tyrosine phosphorylation (PTK-PTP interplay model). Furthermore, we extended the knowledge about topography of active phosphatases which are prone to oxidation within the clusters of transmembrane adaptor proteins non-T cell activation linker (NTAL) and linker for activation of T...(CZ) Tato disertační práce je zaměřena na dva důležité regulátory signalizace žírných buněk. Prvním z nich je komplex vysoce afinního receptoru pro imunoglobulin E (IgE) (FcεRI), který se podílí na získaných (adaptivních) imunitních odpovědí a druhý je stromální interakční molekula 1 (STIM1), která monitoruje hladiny vápníku v endoplasmickém retikulu (ER) a po uvolnění Ca2+ z ER se podílí na otevření vápníkovým uvolněním aktivovaných vápníkových (CRAC) kanálů. I když je struktura FcεRI známá již mnoho let a byla popsána řada molekul asociovaných s tímto receptorem, přesný molekulární mechanismus zahájení a ukončení FcεRI signalizace zůstává nejasný. V této studii jsme vyhodnotili dosavadní poznatky o molekulárních mechanismech iniciace fosforylace FcεRI s důrazem na nově popsaný model, podle kterého vzájemné interakce mezi protein tyrosin fosfatázami (PTPs) a protein tyrosin kinázami (PTKs) nastaví práh tyrosinové fosforylace FcεRI (PTK-PTP interakční model). Dále jsme rozšířili poznatky týkající se topografie fosfatáz citlivých k oxidaci po stimulaci FcεRI v rámci klastrů transmembrálních adaptorových proteinů: T buňky neaktivující "linker" (NTAL) a "linker" aktivovaných T lymfocytů (LAT). Žírné buňky původem z kostní dřeně (BMMC) jsme použili jako modelový systém pro získání nových poznatků o...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions

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

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (FcεRI) Signaling in Mast Cells by Suppressing the Function of FcεRI-Cholesterol Signalosome.

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

Short-term exposure to ethanol inhibits antigen-induced degranulation and calcium response in BMMCs.

<p>IgE-sensitized cells were preincubated for 15 min with various concentrations of ethanol (0–1%), which was also present during antigen-mediated activation. (A, B) Degranulation (release of β-glucuronidase) was measured 5 min (A) or 15 min (B) after exposure of the cells to the indicated concentrations of antigen. (C, D) Calcium response after addition of antigen (arrow, Ag, 100 ng/ml) was measured in the presence of 1 mM extracellular calcium (C), or in its absence (D), followed by addition of 1 mM calcium (arrow, Ca²⁺ in D). Calcium levels in the absence of antigen activation but in the presence of 1% ethanol is also shown in C and D (empty circles). Data are means ± SEs (n = 6–8). Statistical significance of intergroup differences is shown in A and B. In C and D, statistical significance of differences between control cells (0% ethanol) and cells exposed to 0.2% ethanol (violet line), 0.5% ethanol (green line) or 1% ethanol (red line) calculated for the corresponding time intervals (coloured lines) are also indicated.</p

Pretreatment with ethanol inhibits tyrosine phosphorylation of FcεRI β and γ subunits and some other proteins involved in FcεRI signaling.

<p>(A) IgE-sensitized cells were preincubated for 15 min with BSS-BSA alone (Ctrl) or supplemented with ethanol (0.5%) and/or Mβ and then activated or not with antigen (100 ng/ml) in the presence or absence of the compounds. After 5 min the cells were solubilized in 0.2% Triton X-100 and FcεRI was immunoprecipitated (IP) from postnuclear supernatants. The immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting with PY-20-HRP conjugate. For loading controls, the same membrane was stripped and re-blotted with FcεRI-β-chain-specific antibody. Representative immunoblots from three to five independent experiments are shown on the left. The immunoblots were analyzed by densitometry and the fold increase in tyrosine FcεRI-β and -γ chain phosphorylation, normalized to non-activated cells and the amount of FcεRI-β chain, is also shown on the right. (B) IgE-sensitized cells were incubated and activated as above. Five min after triggering the cells were solubilized, size fractionated, and tyrosine phosphorylated proteins were detected by immunoblotting with the phosphoprotein-specific antibodies. Antibodies for the corresponding proteins were used for detection of loading controls. Representative immunoblots from three to four independent experiments are shown. (C) The immunoblots were analyzed by densitometry. Fold increases of protein tyrosine phosphorylation, normalized to control (Ctrl) non-activated cells and the corresponding protein loads are shown. (D and E) IgE-sensitized cells were incubated with the drugs as in A and then activated with antigen (100 ng/ml) in the presence of the drugs for the indicated time intervals. The cells were solubilized, size fractionated, and LYN phosphorylated on Tyr 507 (D) or Tyr 416 (E) was detected by immunoblotting with the corresponding antibodies. After stripping, the membranes were developed for LYN used as a loading control. Fold increase in protein tyrosine phosphorylation, normalized to non-activated cells (Ctrl) and protein load, is also shown. Means ± SEs and the statistical significance of differences in A, C, E, and D were calculated from three to five independent experiments.</p

Model of FcεRI-mediated activation in ethanol-pretreated mast cells.

<p>In nonactivated cells (A), the topography of FcεRI and other signaling molecules, such as SRC family kinase LYN, protein tyrosine phosphatase (PTP), and adaptor proteins (LAT, PAG, and NTAL), prevents signaling. An important role in this process is played by the plasma membrane cholesterol. Aggregation of the FcεRI-IgE complexes by multivalent antigen (B) induces topographical changes that lead to formation of the FcεRI signalosome and enhanced tyrosine phosphorylation of the FcεRI β and γ subunits by LYN and SYK kinases. This results in enhanced degranulation, calcium response, cytokine production and numerous other events. In the cells exposed to ethanol and/or with reduced amount of cholesterol (C), the topography of plasma membrane molecules is slightly modified, resulting in increased tyrosine phosphorylation of some signaling molecules even in nonactivated cells. Aggregation of the receptor in ethanol-treated cells leads to suboptimal topographical changes resulting in reduced tyrosine phosphorylation of the FcεRI β and γ subunits by LYN and SYK kinases and/or enhanced activity of the corresponding phosphatases (D). This leads to reduced degranulation, calcium response, cytokine production and other events. Ethanol could also bind directly to some cytoplasmic or plasma membrane proteins, such as ion channel proteins, and in this way inhibit the cell signaling.</p