Calcium signalling in mast cells

Mast cells play a central role in many allergic and in ammatory conditions. These cells are activated following an intracellular rise in calcium, such as that which occurs after the activation of cell-surface receptors. One such important receptor is cysteinyl leukotriene (CysLT) receptor type 1 (CysLT1), which is activated by lipid mediators such as CysLTs LTC4, LTD4, and LTE4. CysLT1 stimulation leads to the hydrolysis of membrane phospholipids such as phosphatidylinositol 4 5-bisphosphate (PIP2) via phospholipase C-β , which results in the generation of diacylglycerol and inositol trisphosphate. Inositol trisphosphate transiently increases cytosolic calcium levels by releasing calcium from its internal stores. This transient phase is followed by an influx of external calcium caused by the opening of store-operated calcium release-activated calcium (CRAC) channels in the plasma membrane. To understand how CRAC channels are involved in receptor- driven calcium responses, I investigated whether the opening of CRAC channels regulates the production of cellular phosphoinositide. Using cytoplasmic calcium ion (Ca2+) imaging in the mast cell line RBL-2H3, I found that LTC4 induced repetitive calcium oscillations that ran down in the absence of external calcium and were sustained by calcium entry through CRAC channels. The molecular characterisation of CRAC channel components in RBL-2H3 cells revealed that LTC4 -mediated calcium oscillations were maintained through calcium entry via Orai1 and that the calcium signal could not be maintained by Orai3 or other calcium- permeable channels. Furthermore, STIM1 (but not STIM2) was the only homologue that supported calcium oscillations in RBL-2H3 cells. The inhibition of the cellular phosphoinositide pool by lithium chloride (LiCl) reduces calcium oscillations. Adding the substrate inositol rescued these oscillations, but only when external calcium was present. Pharmacologically blocking CRAC channels with a low concentration of CRAC channel blockers prevented the recovery of oscillations in LiCl-treated cells, even when inositol was present. To further understand how calcium entry contributes to the production of PIP2, I investigated whether PI4P- or PI5P-specific pools support the oscillatory calcium signal induced by LTC4. Accordingly, by using pharmacological blockers, concluded that PIP2 used in LTC4 -mediated calcium signalling is produced via the conversion of PI4P into PIP2 by PI5K1 kinases and that the cellular PI5P pool does not contribute to the calcium signal. Moreover, the conversion of PI4P into PIP2 was possible only when there was calcium entry via CRAC channels. Characterisation of the expressed PI5K1 kinases in RBL-2H3 cells revealed expression of only PIP5K1α and PIP5K1γ and that both kinases are needed to maintain the oscillatory calcium signal induced by LTC4 and to provide an overlapping function. To further expand current understanding of how calcium regulates PI5K1 kinases, I specifically investigated how calcium entry regulates PIP5K1γ. This was accomplished by looking into PIP5K1γ-regulating proteins, of which talin is a focal adhesion protein shown to activate PIP5K1γ. In this thesis, I show that the cleavage and activation of talin depend on calcium entry via CRAC channels, thereby elucidating a possible mechanism in how CRAC channels mediating calcium entry are involved in phosphoinositide production. This thesis identifies a new role for CRAC channels in mast cell activation. The opening of CRAC channels and calcium entry are required for PIP2 production and thus the maintenance of agonist-mediated calcium signalling.