Cross-linking the high affinity
IgE receptor on the rat basophil
leukemia clone 2H3 (RBL-2H3) cell line, an vitro model for mast cell signaling, results in granule release. A great deal of research has focused on the earliest steps in this signaling cascade resulting in models which include the participation of lyn, syk,
phospholipase C (PLC),
protein kinase C (PKC) and intracellular
calcium mobilization. In an effort to look at pathways downstream of
calcium mobilization,
ionomycin-mediated granule release was studied. The
kinase inhibitors PP1 (src family),
GF109203X (PKC),
PD98059 (MEK1/2), and
U0126 (MEK1/2) substantially inhibited
ionomycin-mediated granule release, while the p38
kinase inhibitor
SB203580 did not. Both p38 and erk were phosphorylated upon
ionomycin treatment, but only extracellular regulated
kinase (erk) activation was completely inhibited by PP1 treatment and partially inhibited by the
MEK inhibitors, thus, correlating with the granule release data. Interestingly, while
GF109203X alone had no affect on erk activation, combining it with
U0126 completely blocked this response. This suggests the existence an alternate pathway for erk activation that is
MEK independent and PKC dependent. Experiments in which
ionomycin and PP1 were titrated (independently) demonstrated a correlation between erk phosphorylation and granule release, implicating erk in a PP1-inhibitable pathway operating downstream of
calcium and controlling mast cell degranulation.