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 Cε (PLCε). Using primary astrocytes from wild-type and PLCε 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 PLCε. Interleukin-6 and interleukin-1β mRNA levels are also increased in a PLCε-dependent manner. Thrombin, lysophosphatidic acid, and S1P increase COX-2 protein expression through a mechanism involving RhoA, catalytically active PLCε, sustained activation of protein kinase D (PKD), and nuclear translocation of NF-κB. Endogenous ligands that are released from astrocytes in an in vitro wounding assay also induce COX-2 expression through a PLCε- and NF-κB-dependent pathway. Additionally, in vivo stab wound injury activates PKD and induces COX-2 and other inflammatory genes in WT but not in PLCε knockout mouse brain. Thus, PLCε links GPCRs to sustained PKD activation, providing a means for GPCR ligands that couple to RhoA to induce NF-κB signaling and promote neuroinflammation.

Fig. 1.

PLCɛ is required for induction of COX-2 in response to thrombin, LPA, and S1P. (A) COX-2 mRNA levels in primary WT and PLCɛ KO astrocytes treated with thrombin (5 nM) or vehicle (control) for 1 h were assessed by quantitative PCR. Fold increase is expressed relative to the WT or KO averaged controls. Data shown are the mean ± SEM of values (n = 6) from three independent experiments. (B) Western blot and quantification of COX-2 protein levels following 6-h vehicle (control), thrombin (5 nM), LPA (10 μM), S1P (5 μM), or carbachol (500 μM) treatment in WT and PLCɛ KO astrocytes. Western blots of triplicates from one representative experiment are shown. The bar graph is quantitated data representing the mean ± SEM of values (n = 9) from three independent experiments. COX-2 protein levels were normalized to GAPDH and expressed relative to the WT or KO averaged controls. *P < 0.01 between control and agonist treatment; ^#P < 0.05 and ^##P < 0.01 between agonist-treated groups, one-way ANOVA.

Fig. 2.

NF-κB is activated through PLCɛ and required for COX-2 expression. (A) NF-κB activation was assessed by measuring increases in p65 in the nuclear fraction of WT and PLCɛ KO astrocytes following 1-h vehicle, thrombin (5 nM), or carbachol (500 μM) treatment. Nuclear p65 protein levels were normalized to lamin A/C and expressed relative to the WT or KO averaged controls. Values from three independent experiments were quantitated as the mean ± SEM. (B) WT astrocytes were pretreated with BMS-345541 (5 μM) for 1 h before 6 h of treatment with vehicle, thrombin (5 nM), LPA (10 μM), or S1P (5 μM). COX-2 protein levels were normalized to GAPDH and expressed relative to the averaged ± inhibitor controls. Mean ± SEM of values (n = 9) from three independent experiments are shown. *P < 0.05 and **P < 0.01 between control and agonist treatment; ^#P < 0.05 and ^##P < 0.01 between agonist- ± drug-treated groups, one-way ANOVA.

Fig. 3.

Scratch wounding induces COX-2 through PLCɛ and NF-κB. (A) Confluent monolayers of WT and PLCɛ KO astrocytes were scratched for 8 h and COX-2 protein was assessed via Western blot. (B) WT astrocytes were pretreated with BMS-345541 (5 μM) before wounding and COX-2 detection. (C) Media from scratched WT astrocytes were applied to naïve WT and KO astrocytes and COX-2 expression was detected. COX-2 protein levels were normalized to GAPDH and expressed relative to the WT or KO averaged controls (A and C) or to the averaged ± inhibitor controls (B). Representative Western blots are shown and data were quantitated as the mean ± SEM of values (n = 9) from three independent experiments. *P < 0.01 between control and scratch wounding (A and B) or conditioned media (C); ^#P < 0.01 between scratch wounding (B) or conditioned media (C), one-way ANOVA.

Fig. 4.

PKD is activated through PLCɛ and mediates NF-κB activation and COX-2 expression. (A) Time course of phosphorylation of PKD (p-PKDS916) in WT (solid line) and PLCɛ KO (dotted line) astrocytes treated with vehicle (control, plotted as t = 0) and thrombin (5 nM), LPA (10 μM), S1P (5 μM), or carbachol (500 μM) for 1 and 6 h. Representative Western blots are shown for the 1-h time point for thrombin, LPA, and S1P and for the time course for carbachol. The p-PKDS916 protein levels were normalized to total PKD and expressed relative to the WT or KO averaged controls of values (n = 9) from three independent experiments quantitated as the mean ± SEM. (B) NF-κB activation was measured by detecting increases in p65 in the nuclear fraction of WT and PLCɛ KO astrocytes treated with thrombin (5 nM) for 1 h following knockdown of PKD with siRNA (2 μM). p65 protein levels were normalized to lamin A/C and expressed relative to the averaged siRNA control. Representative Western blots are shown and data were quantitated as the mean ± SEM of values from three independent experiments. (C) COX-2 levels were assessed by Western blotting after thrombin treatment (5 nM, 6 h) following knockdown of PKD with siRNA (2 μM). COX-2 protein levels were normalized to GAPDH and expressed relative to the averaged siRNA control. Representative Western blots are shown and data were quantitated as the mean ± SEM of values (n = 9) from three independent experiments. (D) PLCɛ KO astrocytes were infected with WT FLAG-tagged PLCɛ adenovirus or catalytically dead FLAG-tagged PLCɛ adenovirus before vehicle, thrombin (5 nM), or LPA (10 μM) treatment for 6 h. COX-2 protein levels were assessed by Western blotting. COX-2 protein levels were normalized to GAPDH and expressed relative to the EYFP control. Representative Western blots are shown and data were quantitated as the mean ± SEM of values (n = 6) from three independent experiments. *P < 0.05 and **P < 0.01 between control and agonist treatment; ^#P < 0.05 and ^##P < 0.01 between agonist-treated groups, one-way ANOVA.

Fig. 5.

In vivo stab wound injury induces COX-2 through PLCɛ. WT and PLCɛ KO mice at 8 wk of age were subjected to stab wound injury as described in Methods. Seven days following injury, brains were removed and lysates were prepared for Western blotting. (A) Representative Western blots of COX-2, MCP-1 (another inflammatory mediator), GFAP (a marker of astrogliosis), and phosphorylated PKD are shown for two animals per group. (B–E) Quantification of Western blot data from n = 8–10 mice per group. COX-2, GFAP, and MCP-1 protein levels were normalized to GAPDH, and p-PKDS916 was normalized to total PKD. *P < 0.05 and **P < 0.01 between control and stab wound injury; ^#P < 0.01 between stab wound injuries, one-way ANOVA.

Fig. 6.

Schematic of pathway by which PLCɛ mediates GPCR activation and inflammatory responses in astrocytes. CDC25 is the guanine nucleotide exchange factor domain, PH is the Pleckstrin Homology domain, X and Y comprise the catalytic domain, and the RA2 is the Ras association domain.