Lubica Dráberová

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.

Mast Cell Chemotaxis – Chemoattractants and Signaling Pathways

Migration of mast cells is essential for their recruitment within target tissues where they play an important role in innate and adaptive immune responses. These processes rely on the ability of mast cells to recognize appropriate chemotactic stimuli and react to them by a chemotactic response. Another level of intercellular communication is attained by production of chemoattractants by activated mast cells, which results in accumulation of mast cells and other hematopoietic cells at the sites of inflammation. Mast cells express numerous surface receptors for various ligands with properties of potent chemoattractants. They include the stem cell factor (SCF) recognized by c-Kit, antigen, which binds to immunoglobulin E (IgE) anchored to the high affinity IgE receptor (FcεRI), highly cytokinergic (HC) IgE recognized by FcεRI, lipid mediator sphingosine-1-phosphate (S1P), which binds to G protein-coupled receptors (GPCRs). Other large groups of chemoattractants are eicosanoids [prostaglandin E2 and D2, leukotriene (LT) B4, LTD4, and LTC4, and others] and chemokines (CC, CXC, C, and CX3C), which also bind to various GPCRs. Further noteworthy chemoattractants are isoforms of transforming growth factor (TGF) β1–3, which are sensitively recognized by TGF-β serine/threonine type I and II β receptors, adenosine, C1q, C3a, and C5a components of the complement, 5-hydroxytryptamine, neuroendocrine peptide catestatin, tumor necrosis factor-α, and others. Here we discuss the major types of chemoattractants recognized by mast cells, their target receptors, as well as signaling pathways they utilize. We also briefly deal with methods used for studies of mast cell chemotaxis and with ways of how these studies profited from the results obtained in other cellular systems.

Lipid rafts in mast cell signaling.

Lipid rafts are defined as plasma membrane microdomains enriched with glycosphingolipids and cholesterol which render them insoluble in non-ionic detergents. Many surface receptors are constitutively or inducibly associated with lipid rafts, and it has been suggested that the rafts function as platforms regulating the induction of signaling pathways. The signaling capacity of lipid rafts has been extensively studied in rat basophilic leukemia cells. An aggregation of lipid raft components, such as glycosylphosphatidylinositol (GPI)-anchored glycoproteins (Thy-1 or TEC-21), triggers cell activation events which are similar to, but not identical with activation via the high-affinity IgE receptor (FcepsilonRI). Although FcepsilonRI in resting cells is not associated with lipid rafts, its aggregation induces a weak association with rafts and subsequent activation events. The properties of lipid rafts as well as the molecular mechanisms of their involvement in signal transduction are poorly understood. This review presents a critical analysis of recent results on structure-function relationship of lipid rafts and their regulatory role in signal transduction in mast cells.

Non-apoptotic phosphatidylserine externalization induced by engagement of glycosylphosphatidylinositol-anchored proteins.

The exposure of phosphatidylserine (PS) on the cell surface is a general marker of apoptotic cells. Non-apoptotic PS externalization is induced by several activation stimuli, including engagement of immunoreceptors. Immune cells can also be activated by aggregation of glycosylphosphatidylinositol-anchored proteins (GPI-APs). However, it is unknown whether cell triggering through these proteins, lacking transmembrane and cytoplasmic domains, also leads to PS externalization. Here we show that engagement of GPI-APs in rodent mast cells induces a rapid and reversible externalization of PS by a non-apoptotic mechanism. PS externalization triggered by GPI-AP-specific monoclonal antibodies was dependent on the activity of H+-ATP synthase and several other enzymes involved in mast cell signaling but was independent of cell degranulation, free cytoplasmic calcium up-regulation, and a decrease in lipid packing as determined by merocyanine 540 binding. Surprisingly, disruption of actin cytoskeleton by latrunculin B or plasma membrane integrity by methyl-β-cyclodextrin had opposite effects on PS externalization triggered through GPI-AP or the high affinity IgE receptor. We further show that PS externalization mediated by GPI-APs was also observed in some other cells, and its extent varied with antibodies used. Interestingly, effects of different antibodies on PS externalization were additive, indicating that independent stimuli converge onto a signaling pathways leading to PS externalization. Our findings identify the cell surface PS exposure induced through GPI-AP as a distinct mechanism of cell signaling. Such a mechanism could contribute to “inside-out” signaling in response to pathogens and other external activators and/or to initiation of other functions associated with PS externalization.

Differential sensitivity to acute cholesterol lowering of activation mediated via the high-affinity IgE receptor and Thy-1 glycoprotein

Lateral cross‐linking of transmembrane high‐affinity IgE receptors (FcϵRI) or glycosylphosphatidylinositol‐anchored Thy‐1 glycoproteins on the surface of rat mast cells and rat basophilic leukemia (RBL) cells triggers the signaling pathways that lead to the release of allergy mediators. Although both of these pathways are initiated by an increased activity of Lyn kinase, the exact mechanism by which Lyn kinase interacts with aggregated FcϵRI and Thy‐1 is not completely understood. Here we demonstrate that pretreatment of RBL cells with methyl‐β‐cyclodextrin (MBCD) resulted in a dose‐ and time‐dependent decrease in cellular cholesterol, increased detergent solubilization of Thy‐1 and Lyn kinase, and a transient increase in tyrosine phosphorylation of several proteins. Acute lowering of cholesterol suppressed the activation through Thy‐1, as determined by tyrosine phosphorylation of Syk kinase and some other proteins, and modulation of free cytoplasmic calcium. In contrast, the FcϵRI‐mediated activation events were more resistant. Thy‐1 and FcϵRI in MBCD‐pretreated cells also differed in the extent of aggregation after cross‐linking: Thy‐1 formed large caps, whereas FcϵRI accumulated in small patches. MBCD treatment induced an increased release of secretory components in both Thy‐1‐ and FcϵRI‐activated cells. The combined data indicate that cholesterol depletion does not merely block receptor signaling but has more complex consequences.

Regulation of microtubule nucleation from membranes by complexes of membrane-bound γ-tubulin with Fyn kinase and phosphoinositide 3-kinase

The molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is gamma-tubulin. Although previous results suggested that tyrosine kinases might serve as regulators of gamma-tubulin function, their exact roles remain enigmatic. In the present study, we show that a pool of gamma-tubulin associates with detergent-resistant membranes in differentiating P19 embryonal carcinoma cells, which exhibit elevated expression of the Src family kinase Fyn (protein tyrosine kinase p59(Fyn)). Microtubule-assembly assays demonstrated that membrane-associated gamma-tubulin complexes are capable of initiating the formation of microtubules. Pretreatment of the cells with Src family kinase inhibitors or wortmannin blocked the nucleation activity of the gamma-tubulin complexes. Immunoprecipitation experiments revealed that membrane-associated gamma-tubulin forms complexes with Fyn and PI3K (phosphoinositide 3-kinase). Furthermore, in vitro kinase assays showed that p85alpha (regulatory p85alpha subunit of PI3K) serves as a Fyn substrate. Direct interaction of gamma-tubulin with the C-terminal Src homology 2 domain of p85alpha was determined by pull-down experiments and immunoprecipitation experiments with cells expressing truncated forms of p85alpha. The combined results suggest that Fyn and PI3K might take part in the modulation of membrane-associated gamma-tubulin activities.

Involvement of filamentous actin in setting the threshold for degranulation in mast cells.

Previous studies using cytochalasins and latrunculin B, inhibitors of actin polymerization, showed that filamentous (F)‐actin had a negative regulatory role in Fcϵ receptor I (FcϵRI) signaling. How F‐actin is involved in regulating the activation of mast cells is unknown. In this study we investigated the role of F‐actin in mast cell activation induced by aggregation of the glycosylphosphatidylinositol (GPI)‐anchored proteins Thy‐1 and TEC‐21, and compared it to activation via FcϵRI. Pretreatment of rat basophilic leukemia cells with latrunculin B inhibited the Thy‐1‐induced actin polymerization and elevated the Thy‐1‐mediated secretory and calcium responses. Inhibition of actin polymerization followed by Thy‐1 aggregation resulted in an increased tyrosine phosphorylation of Syk, phospholipase Cγ (PLCγ), Gab2 and linker for activation of T cells (LAT) adapters, and some other signaling molecules. Enzymatic activities of phosphatidylinositol 3‐kinase, PLCγ, and phosphatase SHP‐2 were also up‐regulated, but tyrosine phosphorylation of ezrin was inhibited. Similar changes were observed in FcϵRI‐activated cells. Significant changes in intracellular distribution, tyrosine phosphorylation, and/or enzymatic activities of signaling molecules occurred in latrunculin‐pretreated cells before cell triggering. The combined data suggest that actin polymerization is critical for setting the thresholds for mast cell signaling via aggregation of both FcϵRI and GPI‐anchored proteins.

Functional heterogeneity of Thy-1 membrane microdomains in rat basophilic leukemia cells

Antibody‐mediated cross‐linking of Thy‐1 glycoprotein on the surface of rat mast cells and rat basophilic leukemia (RBL) cells initiates biochemical events which culminate in secretion of allergy mediators. Thy‐1, like some other glycosylphosphatidylinositol (GPI)‐anchored proteins, forms detergent‐insoluble complexes containing protein tyrosine kinases (PTK) and some other molecules which are implicated in the signaling pathway. On the surface of a rat mast cell there are more than 106 Thy‐1 molecules; however, it is not known which fraction of them is involved in transmembrane signaling, and what exactly is the heterogeneity of Thy‐1 complexes. Using sucrose density gradient ultracentrifugation of detergent‐lysed RBL cells we found that the density of Thy‐1 complexes depended on the detergent used and the lysis conditions employed. Sepharose 4B gel chromatography fractionation followed by density gradient ultracentrifugation revealed both size and density heterogeneity of Thy‐1 and Lyn PTK complexes. Cross‐linking of surface Thy‐1 caused significant changes in the density of these complexes, and an increase in Lyn kinase activity in low/medium‐density fractions. Thy‐1 in low‐density fractions was relatively resistant to cleavage with phosphatidylinositol‐specific phospholipase C (PI‐PLC). Interestingly, removal of only a small fraction of surface Thy‐1 by PI‐PLC abolished the cell activation as determined by tyrosine phosphorylation of certain proteins. When Triton X‐100 lysates were fractionated at 12 000 × g, about 50 % of Thy‐1 remained associated with the nuclear/cytoskeleton pellet; this fraction of Thy‐1 exhibited an increased sensitivity to PI‐PLC. Confocal laser scanning microscopy on fixed cells revealed that the total Thy‐1 was relatively homogeneously distributed over the plasma membrane, whereas the PI‐PLC‐resistant Thy‐1 was found mostly in small clusters. The combined data suggest that specialized membrane microdomains enriched in Thy‐1 with increased sensitivity to PI‐PLC are directly involved in coupling Thy‐1 aggregation to transmembrane signaling.

Engagement of phospholipid scramblase 1 in activated cells; Implication for phosphatidylserine externalization and exocytosis

Phosphatidylserine (PS) in quiescent cells is predominantly confined to the inner leaflet of the plasma membrane. Externalization of PS is a marker of apoptosis, exocytosis, and some nonapoptotic activation events. It has been proposed that PS externalization is regulated by the activity of PLSCR1 (phospholipid scramblase 1), a Ca2+-dependent endofacial plasma membrane protein, which is tyrosine-phosphorylated in activated cells. It is, however, unclear how the phosphorylation of PLSCR1 is related to its membrane topography, PS externalization, and exocytosis. Using rat basophilic leukemia cells as a model, we show that nonapoptotic PS externalization induced through the high affinity IgE receptor (FcϵRI) or the glycosylphosphatidylinositol-anchored protein Thy-1 does not correlate with enhanced tyrosine phosphorylation of PLSCR1. In addition, PS externalization in FcϵRI- or Thy-1-activated cells is not associated with alterations of PLSCR1 fine topography as detected by electron microscopy on isolated plasma membrane sheets. In contrast, activation by calcium ionophore A23187 induces changes in the cellular distribution of PLSCR1. We also show for the first time that in pervanadate-activated cells, exocytosis occurs even in the absence of PS externalization. Finally, we document here that tyrosine-phosphorylated PLSCR1 is preferentially located in detergent-insoluble membranes, suggesting its involvement in the formation of membrane-bound signaling assemblies. The combined data indicate that changes in the topography of PLSCR1 and its tyrosine phosphorylation, PS externalization, and exocytosis are independent phenomena that could be distinguished by employing specific conditions of activation.

STIM1-Directed Reorganization of Microtubules in Activated Mast Cells

Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca2+ controlled by store-operated Ca2+ entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca2+. Changes in cytosolic Ca2+concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca2+ response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca2+ plays an important role in the formation of microtubule protrusions in BMMCs.