Ca2+ influx through CRAC channels activates cytosolic phospholipase A₂, leukotriene C₄secretion, and expression of c‐fos through ERK‐dependent and independent pathways in mast cells

Abstract

Cytosolic phospholipase A2 (cPLA2) is a Ca2+-dependent enzyme that mediates agonist-dependent arachidonic acid release in most cell types. Arachidonic acid can then be metabolized by the 5-lipoxygenase enzyme to generate the proinflammatory signal leukotriene C4 (LTC4). Here we report that Ca2+ entry through store-operated CRAC (Ca2+ release-activated Ca2+) channels activates the extracellular signal-regulated kinases (ERKs), members of the mitogen-activated protein kinase family, within minutes and this is necessary for stimulation of cPLA2. Ca2+ entry activates ERK indirectly, via recruitment of Ca2+-dependent protein kinase C alpha and betaI. Ca2+ influx also promotes translocation of cytosolic 5-lipoxygenase to the nuclear membrane, a key step in the activation of this enzyme. Translocation is dependent on ERK activation. A role for gene activation is shown by the finding that CRAC channel opening results in increased transcription and translation of c-fos. Inhibition of ERK activation failed to prevent c-fos expression. Our results show that CRAC channel activation elicits short-term effects through the co-coordinated regulation of two metabolic pathways (cPLA2 and 5-lipoxygenase), which results in the generation of both intra- and intercellular messengers within minutes, as well as longer term changes involving gene activation. These short-term effects are mediated via ERK, whereas, paradoxically, c-fos expression is not. Ca2+ influx through CRAC channels can therefore activate different signaling pathways at the same time, culminating in a range of temporally diverse responses.

Coupling of Ca2+ Microdomains to Spatially and Temporally Distinct Cellular Responses by the Tyrosine Kinase Syk

“…We previously found that activation of store-operated CRAC channels in mast cells triggered expression of the c-fos gene (20). To see whether such excitation-transcription coupling was driven by a local or global Ca 2ϩ signal, we stimulated a population of RBL-1 cells (a mast cell line) with thapsigargin, a Ca 2ϩ ATPase inhibitor on the endoplasmic reticulum, which depletes the Ca 2ϩ stores and thereby opens CRAC channels (6).…”

“…We have recently established that Ca 2ϩ microdomains near CRAC channels activate the cytoplasmic enzymes Ca 2ϩ -dependent phospholipase A 2 and 5-lipoxygenase via recruitment of the MEK/ERK pathway (20). This results in the generation of the intracellular messenger arachidonic acid, which is rapidly metabolized by 5-lipoxygenase to the proinflammatory paracrine signal leukotriene C 4 (18,20).…”

“…Reverse Transcription-PCR-Reverse transcription-PCR of c-fos expression was carried out as reported (20). Cells were stimulated with thapsigargin in 2 mM external Ca 2ϩ for 4 min, perfused with thapsigargin and Ca 2ϩ -free solution (containing 0.1 mM EGTA) for 41 min, and then RNA-extracted.…”

“…Western Blotting-All Western blots were carried out as reported (20). ERK2 expression was measured in order to normalize phosphorylated ERK expression to the number of cells used per gel lane.…”

Genetic polymorphisms in Kawasaki disease

“…IP 3 binds to its receptor (IP 3 R) on the endoplasmic reticulum (also called the calcium store) causing the release of calcium from stores [64] . An empty store results in the activation of both store-operated calcium channels and Ca 2+ -dependent signaling pathways, including inflammatory reactions [65,66] and apoptosis [64] . Onouchi et al reported that a G to A substitution in the 5'-untranslated region of CASP3 (rs72689236) is associated with susceptibility to Kawasaki disease in Japanese and in Americans of European descent [34] .…”