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Phospholipase C epsilon links G protein-coupled receptor activation to inflammatory astrocytic responses.

ABSTRACT: Neuroinflammation plays a major role in the pathophysiology of diseases of the central nervous system, and the role of astroglial cells in this process is increasingly recognized. Thrombin and the lysophospholipids lysophosphatidic acid and sphingosine 1-phosphate (S1P) are generated during injury and can activate G protein-coupled receptors (GPCRs) on astrocytes. We postulated that GPCRs that couple to Ras homolog gene family, member A (RhoA) induce inflammatory gene expression in astrocytes through the small GTPase responsive phospholipase C? (PLC?). Using primary astrocytes from wild-type and PLC? knockout mice, we demonstrate that 1-h treatment with thrombin or S1P increases cyclooxygenase 2 (COX-2) mRNA levels ?10-fold and that this requires PLC?. Interleukin-6 and interleukin-1? mRNA levels are also increased in a PLC?-dependent manner. Thrombin, lysophosphatidic acid, and S1P increase COX-2 protein expression through a mechanism involving RhoA, catalytically active PLC?, sustained activation of protein kinase D (PKD), and nuclear translocation of NF-?B. Endogenous ligands that are released from astrocytes in an in vitro wounding assay also induce COX-2 expression through a PLC?- and NF-?B-dependent pathway. Additionally, in vivo stab wound injury activates PKD and induces COX-2 and other inflammatory genes in WT but not in PLC? knockout mouse brain. Thus, PLC? links GPCRs to sustained PKD activation, providing a means for GPCR ligands that couple to RhoA to induce NF-?B signaling and promote neuroinflammation.

SUBMITTER: Dusaban SS

PROVIDER: S-EPMC3587233 | biostudies-other | 2013 Feb

REPOSITORIES: biostudies-other

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Phospholipase C epsilon links G protein-coupled receptor activation to inflammatory astrocytic responses.

Dusaban Stephanie S SS Purcell Nicole H NH Rockenstein Edward E Masliah Eliezer E Cho Min Kyung MK Smrcka Alan V AV Brown Joan Heller JH

Proceedings of the National Academy of Sciences of the United States of America 20130211 9

Neuroinflammation plays a major role in the pathophysiology of diseases of the central nervous system, and the role of astroglial cells in this process is increasingly recognized. Thrombin and the lysophospholipids lysophosphatidic acid and sphingosine 1-phosphate (S1P) are generated during injury and can activate G protein-coupled receptors (GPCRs) on astrocytes. We postulated that GPCRs that couple to Ras homolog gene family, member A (RhoA) induce inflammatory gene expression in astrocytes th ...[more]

PMID: 23401561

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Project description:OBJECTIVE:The mechanism by which G-protein-coupled receptor 40 (GPR40) signaling amplifies glucose-stimulated insulin secretion through activation of protein kinase C (PKC) is unknown. We examined whether a GPR40 agonist, GW9508, could stimulate conventional and novel isoforms of PKC at two glucose concentrations (3 mM and 20 mM) in INS-1D cells. METHODS:Using epifluorescence microscopy, we monitored relative changes in the cytosolic fluorescence intensity of Fura2 as a marker of change in intracellular Ca2+ ([Ca2+]i) and relative increases in green fluorescent protein (GFP)-tagged myristoylated alanine-rich C kinase substrate (MARCKS-GFP) as a marker of PKC activation in response to GW9508 at 3 mM and 20 mM glucose. To assess the activation of the two PKC isoforms, relative increases in membrane fluorescence intensity of PKCα-GFP and PKCε-GFP were measured by total internal reflection fluorescence microscopy. Specific inhibitors of each PKC isotype were constructed and synthesized as peptide fusions with the third α-helix of the homeodomain of Antennapedia. RESULTS:At 3 mM glucose, GW9508 induced sustained MARCKS-GFP translocation to the cytosol, irrespective of changes in [Ca2+]i. At 20 mM glucose, GW9508 induced sustained MARCKS-GFP translocation but also transient translocation that followed sharp increases in [Ca2+]i. Although PKCα translocation was rarely observed, PKCε translocation to the plasma membrane was sustained by GW9508 at 3 mM glucose. At 20 mM glucose, GW9508 induced transient translocation of PKCα and sustained translocation as well as transient translocation of PKCε. While the inhibitors (75 μM) of each PKC isotype reduced GW9508-potentiated, glucose-stimulated insulin secretion in INS-1D cells, the PKCε inhibitor had a more potent effect. CONCLUSION:GW9508 activated PKCε but not PKCα at a substimulatory concentration of glucose. Both PKC isotypes were activated at a stimulatory concentration of glucose and contributed to glucose-stimulated insulin secretion in insulin-producing cells.

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Phospholipase C epsilon links G protein-coupled receptor activation to inflammatory astrocytic responses.

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Phospholipase C epsilon links G protein-coupled receptor activation to inflammatory astrocytic responses.

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