Synergistic activation of phospholipases Cγ and Cβ: A novel mechanism for PI3K-independent enhancement of FcεRI-induced mast cell mediator release

Abstract

Antigen/IgE-mediated mast cell activation via FcεRI can be markedly enhanced by the activation of other receptors expressed on mast cells and these receptors may thus contribute to the allergic response in vivo. One such receptor family is the G protein-coupled receptors (GPCRs). Although the signaling cascade linking FcεRI aggregation to mast cell activation has been extensively investigated, the mechanisms by which GPCRs amplify this response are relatively unknown. To investigate this, we utilized prostaglandin (PG)E 2 based on initial studies demonstrating its greater ability to augment antigen-mediated degranulation in mouse mast cells than other GPCR agonists examined. This enhancement, and the ability of PGE 2 to amplify antigen-induced calcium mobilization, was independent of phosphoinositide 3-kinase but was linked to a pertussis toxinsensitive synergistic translocation to the membrane of phospholipase (PL)Cγ and PLCβ and to an enhancement of PLCγ phosphorylation. This "trans-synergistic" activation of PLCβ and γ, in turn, enhanced production of inositol 1,4,5-trisphosphate, store operated calcium entry, and activation of protein kinase C (PKC) (α and β). These responses were critical for the promotion of degranulation. This is the first report of synergistic activation between PLCγ and PLCβ that permits reinforcement of signals for degranulation in mast cells.

Btk-dependent Rac activation and actin rearrangement following FcεRI aggregation promotes enhanced chemotactic responses of mast cells

“…Consistent with previous results (Kuehn et al, 2008a), and the ability of wortmannin to block BMMC chemotaxis (Fig. 4A), antigen-mediated phosphorylation of AKT was enhanced by PGE 2 and this enhancement was most prominent at the 2 minute time point (Fig.…”

“…1). Therefore, in addition to their ability to potentiate antigen-mediated activation of mast cells (Ali et al, 1990;Hundley et al, 2004;Kuehn et al, 2008a;Zhong et al, 2003) already present in tissues, KIT and GPCR ligands might also enable expansion of mast-cell populations at sites of antigen infiltration. We note, however, that the effects of SCF noted here were a consequence of short-term exposure to SCF.…”

“…Our previous observation that PGE 2 enhances antigen-induced PLC 1 -dependent Ins(1,4,5)P 3 production and the associated Ca 2+ signal in BMMCs (Kuehn et al, 2008a), suggests that the Ca 2+ signal could be one candidate for propagation of synergistic signals. The absolute requirement for a PLC-or Ins(1,4,5)P 3 -mediated Ca 2+ signal was verified by use of U73122 (a PLC inhibitor), 2APB [an Ins(1,4,5)P 3 -receptor antagonist and inhibitor of storeoperated Ca…”

“…We hypothesized that PI3K might be a common upstream molecule that regulates these processes. Our previous studies have shown that 100 nM wortmannin effectively blocks AKT phosphorylation induced by antigen (Kim et al, 2008), SCF (Kim et al, 2008) and PGE 2 (Kuehn et al, 2008a), which acts as a surrogate marker for PI3K activation. As shown in Fig.…”

“…1B). GPCR agonists were examined in a similar manner, and, on the basis of their efficacy in enhancing antigen-mediated degranulation in BMMCs (Kuehn et al, 2008a;Kuehn and Gilfillan, 2007;Zhong et al, 2003), PGE 2 and adenosine were selected as examples for study. From Fig.…”