New Regulatory Roles of Galectin-3 in High-Affinity IgE Receptor Signaling

Abstract

c Aggregation of the high-affinity receptor for IgE (FcRI) 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 FcRI-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. FcRI-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-FcRI complexes; this may be related to our finding that Gal3 is a positive regulator of FcRI 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 FcRI-mediated signaling events in mast cells. M ast cells are important immune cells involved in multiple biological processes (1, 2). Under pathological conditions, they are responsible for IgE-mediated hyperreactivity and participate in severe diseases, such as allergy and asthma (3). Antigen (Ag)-mediated mast cell activation leads to the release of secretory granules containing a variety of preformed mediators (e.g., histamine and proteases), de novo synthesis of cytokines and chemokines, and enhanced production of arachidonic acid metabolites (4, 5). The principal surface receptor involved in mast cell activation is the high-affinity receptor for IgE (FcεRI), which belongs to the family of multichain immune recognition receptors. FcεRI is a tetrameric complex formed by an IgE-binding ␣ subunit, a signalamplifying ␤ subunit, and a homodimer of disulfide-linked ␥ subunits. Each FcεRI ␤ and ␥ subunit contains one immunoreceptor tyrosine-based activation motif (ITAM), which, after tyrosine phosphorylation, serves as a docking site for other signaling molecules, such as the SRC family kinase LYN or spleen tyrosine kinase (SYK). These two enzymes, together with other kinases, then phosphorylate various adaptor proteins, including linker of activated T cells 1 (LAT1) and LAT2 (also known as non-T cell activation linker [NTAL]). These adaptors are involved in activation of phospholipase C␥ (PLC␥) and subsequent signal transduction events, leading to calcium response and degranulation (6). FcεRI signaling is a complex process that depends on the magnitude of receptor aggregation and a balance between positive and negative signals that determine the extent of the response (7, 8). Although signaling pathways leading to mast cell activation have been extensively...

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