Antigen stimulation of mast cells through high affinity receptors for IgE (FcepsilonRI) leads to Ca2+ mobilization and, as a consequence, the release of inflammatory mediators that are responsible for allergic diseases such as asthma. It is generally believed that inositol 1,4,5-trisphoshate (IP3) is responsible for mobilizing Ca2+ from the intracellular endoplasmic reticulum (ER) stores. Dr. Choi's laboratory has shown that the amount of IP3 produced by FcepsilonRI stimulation is not sufficient to cause Ca2+ mobilization through FcepsilonRI and sphingosine 1-phosphate (SIP), a product of sphingosine kinase (SK) may play a major role in Ca2+ mobilization through FcepsilonRI. Currently cytosolic form of SK has been cloned and in Dr. Choi's laboratory cloned a putative membrane-associated SK. The goal of this proposal is to test the hypothesis that the membrane-associated, not cytosolic, SK is responsible for Ca2+ mobilization through FcepsilonRI in mast cells. In these studies, a mast cell line, RBL-2H3 cells will be used as a model system as FcepsilonRI-mediated signaling has been extensively studied in this cell line. Strategies will be 1) To establish stable RBL-2H3 cell lines overexpressing the cytosolic or membrane-associated SK. 2) To determine which form of SK, cytosolic or membrane-associated, is responsible for S1P production in the endoplasmic reticulum (ER) membrane after FcepsilonRI clustering and Ca2+ mobilization. 3) To measure functional responses such as Ca2+ response, IP3 production and hexosaminidase release after FcepsilonRI clustering in the two cell lines.
