Regulation of the Calcium Signal by Phospholipase (PL) D, Sphingosine Kinase (SK), and Phosphatidylinositol 4‐Phosphate (PIP) 5‐Kinase in Mast Cells

Abstract

Activation of PLD and elevation of cytosolic Ca2+ are essential signals for degranulation in antigen-stimulated mast cells but the processes linking these events are unclear. Two potential downstream targets for the PLD product, phosphatidic acid, include SK and PIP 5-kinase both of which may regulate mobilization of Ca2+. To determine whether this is so, pharmacologic inhibitors as well as siRNAs directed against PLD1, PLD2, and PIP 5-kinase were tested for their effects on mobilization of Ca2+. All reagents suppressed Ca2+ mobilization and degranulation in antigen-stimulated cells. PLD appeared to be critical for sustaining elevated levels of cytosolic Ca2+ and for regulating production sphingosine 1 phosphate by SK but not of inositol 1,4,5-trisphosphate (IP3) by PLCγ 1/2. These studies suggested that in addition to release of intracellular Ca2+ by IP3, the PLD→SK pathway serves to sustain the calcium signal and thus degranulation. Interestingly, the studies with anti-PIP 5-kinase siRNA indicated that this enzyme regulated influx of Ca2+ not only in antigen-stimulated cells but also in cells stimulated with thapsigargin which is a potent stimulant of PLD but not of PLC. Therefore, PIP 5-kinase may regulate the calcium signal through provision of phosphatidylinositol 1,4-bisphosphate as a substrate for PLC and also as a necessary factor for facilitating Ca2+ influx.