Studies on the regulation mechanisms of diverse phospholipase C isotypes

Abstract

Phospholipase C (PLC) is a key enzyme in signal transduction by catalyzing the formation of inositol-1,4,5-triphosphates (IP3) and diacylglycerol (DAG) from phosphatidylinositol-4,5-bisphosphates (PIP2), which are implicated in intracellular Ca2+ mobilization and protein kinase C (PKC) activation, respectively. To date, 13 members of the mammalian PLC family have been identified and they are classified into 6 isotypes based on their primary structures : β, γ, δ, ε, ζ, and η. Although all PLC isotypes show similar enzymatic activity, they display distinct knock-out phenotypes. Given that PLC isotypes possess unique domain structures and show different tissue expression patterns, it seems likely that they are influenced by different signaling pathway to regulate different physiologic functions. Therefore, elucidating the regulation mechanisms of each PLC isotype is prerequisite to understand its physiological functions.PLC-β is a key effector in G-protein coupled receptor (GPCR)-mediated signaling. Many studies have shown that the four PLC-β subtypes (β1~β4) have different physiological functions despite their similar structures. Because the PLC-β subtypes possess different PDZ-binding motifs, they have the potential to interact with different PDZ proteins. In this study, I identified PDZ domain-containing 1 (PDZK1) as a PDZ protein that specifically interacts with PLC-β3. To elucidate the functional roles of PDZK1, I next screened for potential interacting proteins of PDZK1 and identified the somatostatin receptors (SSTRs) as another protein that interacts with PDZK1. Through these interactions, PDZK1 assembles as a ternary complex with PLC-β3 and SSTRs. Interestingly, the expression of PDZK1 and PLC-β3, but not PLC-β1, markedly potentiated SST-induced PLC activation. However, disruption of the ternary complex inhibited SST-induced PLC activation, which suggests that PDZK1-mediated complex formation is required for the specific activation of PLC-β3 by SST. Consistent with this observation, the knockdown of PDZK1 or PLC-β3, but not that of PLC-β1, significantly inhibited SST-induced intracellular Ca2+ mobilization, which further attenuated subsequent ERK1/2 phosphorylation. From these results, I strongly suggest that the formation of a complex between SSTRs, PDZK1, and PLC-β3 is essential for the specific activation of PLC-β3 and the subsequent physiologic responses by SST.PLC-η1 is the most recently identified PLC isotype and is primarily expressed in nerve tissue. However, its functional role is still unclear. In this study, I found for the first time that PLC-η1 acts as a signal amplifier in G protein-coupled receptor (GPCR)-mediated PLC/Ca2+ signaling. Short-hairpin RNA (shRNA)-mediated knockdown of endogenous PLC-η1 reduced lysophosphatidic acid (LPA)- , bradykinin (BK)- , and PACAP-induced PLC activity in mouse neuroblastoma Neuro2A (N2A) cells, indicating that PLC-η1 participates in GPCR-mediated PLC activation. Interestingly, ionomycin-induced PLC activity was significantly decreased by PLC-η1, but not PLC-η2, knockdown. In addition, I found that intracellular Ca2+ source is enough for PLC-η1 activation. Furthermore, the IP3 receptor inhibitor, 2-APB, inhibited LPA-induced PLC activation in control N2A cells, whereas this effect was not observed in PLC-η1 knockdown N2A cells, suggesting a pivotal role of intracellular Ca2+ mobilization in PLC-η1 activation. Finally, LPA-induced ERK1/2 phosphorylation and expression of the downstream target gene, krox-24, were significantly decreased by PLC-η1 knockdown and these knockdown effects were abolished by 2-APB. From these results, I strongly suggest that PLC-η1 is secondarily activated by intracellular Ca2+ mobilization from the ER and therefore amplifies GPCR-mediated PLC/Ca2+ signaling.Mast cell is responsible for IgE-mediated allergic responses through secretion of various inflammatory cytokines and mediators. Thus, pharmacological regulation of mast cell activation is significant for the development of novel anti-allergic drugs. In this study, I found that spiraeoside (SP) inhibits mast cell activation and allergic response in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effect. At the molecular level, SP reduced Ag-induced phosphorylation and activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited phosphosrylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPK such as ERK1/2, p38, and JNK, which eventually attenuated expression of TNF-α and IL-4. Finally, I found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. From these results, I strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic response by primarily inhibiting Lyn-induced PLC-γ2 / MAPK signaling in mast cell.