Phospholipase Cε is a nexus for Rho and Rap-mediated G protein-coupled receptor-induced astrocyte proliferation

Abstract

Phospholipase C (PLC ) has been suggested to transduce signals from small GTPases, but its biological function has not yet been clarified. Using astrocytes from PLC -deficient mice, we demonstrate that endogenous G protein-coupled receptors (GPCRs) for lysophosphatidic acid, sphingosine 1-phosphate, and thrombin regulate phosphoinositide hydrolysis primarily through PLC . Stimulation by lysophospholipids occurs through Gi, whereas thrombin activates PLC through Rho. Further studies reveal that PLC is required for thrombin-but not LPA-induced sustained ERK activation and DNA synthesis, providing a novel mechanism for GPCR and Rho signaling to cell proliferation. The requirement for PLC in this pathway can be explained by its role as a guanine nucleotide exchange factor for Rap1. Thus, PLC serves to transduce mitogenic signals through a mechanism distinct from its role in generation of PLC-derived second messengers.mitogenesis ͉ small GTPases ͉ thrombin ͉ lysophospholipids ͉ guanine nucleotide exchange factor S timulation of a variety of cell surface receptor types, most prominently G protein-coupled receptors (GPCRs), leads to the activation of phospholipase C (PLC). Hydrolysis of phosphatidylinositol (4,5)bisphosphate (PIP 2 ) generates the second messengers inositol (1,4,5)trisphosphate (InsP 3 ) and diacylglycerol (DAG) (1). Thirteen mammalian PLC isozymes, divided into six families, have been identified. All contain conserved catalytic regions as well as subtype-specific domains that allow for a plethora of specific regulatory mechanisms. The best characterized of these are the PLC␤ family, regulated by direct binding of the heterotrimeric G protein subunits ␣ q and ␤␥, and PLC␥ regulated by receptor and nonreceptor protein tyrosine kinases (2).The same second messengers are generated by all of the PLCs. Thus, regardless of the extracellular signal-and PLC isoform-activated, InsP 3 will be formed and trigger Ca 2ϩ release from intracellular stores, and DAG accumulation will lead to activation of protein kinase C (3). Undoubtedly, there are distinct spatiotemporal aspects to the signals elicited by different receptors and classes of PLC, but it remains unclear why so many PLC isoforms, capable of generating the same signal, should exist.An emerging area in biology has been the recognition that many proteins identified on the basis of a particular activity serve additional functions. The regulators of G protein signaling (RGS) proteins are paradigmatic in this regard. These proteins contain not only canonical RGS sequences dedicated to GTP hydrolysis but also non-RGS domains and motifs with alternative functions. Thus, not only do they turn off GPCR signaling but in addition they can enhance small G protein activation, serve as effectors, and act as scaffold proteins (4). PLC , an isoform of PLC that was discovered fewer than 6 years ago, likewise has the structure of a multifunctional signaling protein (5-7). It contains not only PLC catalytic regions but also, in its N-terminal region, a CDC25 homology domain...

Multiple roles of phosphoinositide-specific phospholipase C isozymes

“…PLC-ε induced PDGF-dependent cell growth in BaF3 cells overexpressing PDGF receptor (198) or EGF-dependent cell growth in HEK293 cells or MEF cells (197,206). A recent report indicates that thrombin-dependent astrocyte proliferation is mediated by PLC-ε (207). In that report, GEF activity of PLC-ε was required for the Erk activation and cell astrocyte proliferation.…”

Activity of PLCε contributes to chemotaxis of fibroblasts towards PDGF

“…4C,D). Next, we used adenoviruses expressing PLCe variants deficient either in PLC activity or in Ras-binding (previously described in Citro et al, 2007;Oestreich et al, 2007) to rescue PDGF-BB-mediated chemotaxis in PLCe null fibroblasts. We observed that the expression of a PLCe variant that cannot bind Ras (Ad-RAm) restored chemotaxix of PLCe KO cells as well as the expression WT PLCe (Fig.…”

Transient Receptor Potential Canonical 3 (TRPC3) Mediates Thrombin-Induced Astrocyte Activation and Upregulates Its Own Expression in Cortical Astrocytes

“…On the other hand, Wang and Reiser (2003) reported that thrombin/PAR-1 uses Ca 2ϩ and PKC-dependent Pyk2 to activate Src, thereby leading to ERK1/2 activation in rat astrocytes, suggesting that Ca 2ϩ signaling may regulate MAPK pathway. In this context, Citro et al (2007) reported that there are two pathways in thrombininduced astrocytic ERK activation; G q/11 /PLC␤/DAG/PKC pathway and G 12/13 /Rap1/B-Raf/PLC pathway, in which the former is involved in transient ERK phosphorylation and the latter in long-term sustained ERK phosphorylation, implying that the relationship between thrombin/PAR-1-induced MAPKs activation and resultant Ca 2ϩ signaling in astrocytes could be more complicated. Future studies are needed to determine the detailed molecular pathways that TRPC3-mediated Ca 2ϩ signaling may regulate.…”

“…In this context, Sorensen et al (2003) reported that PAR-1 agonist peptide-induced astrocytic proliferation is inhibited 80% in the presence of Y27632, a specific inhibitor of Rho kinase activity that is profoundly involved in the regulation of astrocytic morphology. Exoenzyme C3 from Clostridium botulinum, a cell-permeable Rho inhibitor, inhibits both astrocyte morphological changes (Höltje et al, 2005) and thrombininduced astrocyte proliferation (Citro et al, 2007). Moreover, Y27632 inhibits both morphological changes and proliferation induced by thromboxane A 2 receptor agonist in astrocytoma cells (Honma et al, 2006), suggesting that the morphological change is an initial phenomenon and a universal feature of proliferating astrocytes.…”