Phospholipase Ce Links G Protein-Coupled Receptor Activation to Inﬂammatory Astrocytic Responses

Phospholipase Ce links G protein-coupled receptor activation to inﬂammatory astrocytic responses

Stephanie S. Dusabana,b, Nicole H. Purcella, Edward Rockensteinc,d, Eliezer Masliahc,d, Min Kyung Choe, Alan V. Smrckaf, and Joan Heller Browna,1

Departments of aPharmacology, cNeurosciences, and dPathology, School of Medicine, and bBiomedical Sciences Graduate Program, University of California at San Diego, La Jolla, CA 92093; eCollege of Oriental Medicine, Dongguk University, Gyeongju 780-714, Korea; and fDepartment of Pharmacology and Physiology, University of Rochester, Rochester, NY 14642

Edited* by Melvin I. Simon, University of California at San Diego, La Jolla, CA, and approved January 23, 2013 (received for review October 5, 2012)

Neuroinflammation plays a major role in the pathophysiology of members of the PLC family in that it is predominantly activated diseases of the central nervous system, and the role of astroglial by small GTPases including Ras family members and Ras ho- cells in this process is increasingly recognized. Thrombin and the molog gene family, member A (RhoA) (12–19). The more widely lysophospholipids lysophosphatidic acid and sphingosine 1-phos- studied PLCβ is regulated by GPCRs that activate Gαq and phate (S1P) are generated during injury and can activate G protein- transduce physiological responses via rapid inositol (1,4,5)tri- coupled receptors (GPCRs) on astrocytes. We postulated that sphosphate-mediated calcium mobilization (20, 21). In contrast, GPCRs that couple to Ras homolog gene family, member A (RhoA) PLCe appears to mediate more-sustained phosphoinositide hy- induce inflammatory gene expression in astrocytes through the drolysis (22) and does so through its regulation by a different set α small GTPase responsive phospholipase Ce (PLCe). Using primary of GPCRs, particularly those that couple to G 12/13 to activate astrocytes from wild-type and PLCe knockout mice, we demon- RhoA (23–30). A physiological role of this more-sustained sig- strate that 1-h treatment with thrombin or S1P increases cycloox- naling is suggested by studies from our laboratory demonstrating e ygenase 2 (COX-2) mRNA levels ∼10-fold and that this requires that activation of PLC by thrombin leads to prolonged Ras- PLCe. Interleukin-6 and interleukin-1β mRNA levels are also in- related protein 1 (Rap1) activation, ERK signaling, and DNA e creased in a PLCe-dependent manner. Thrombin, lysophosphatidic synthesis in astrocytes (11). In addition, there is a role for PLC in GPCR-mediated hypertrophic growth of cardiomyocytes (31, acid, and S1P increase COX-2 protein expression through a mecha- e PHARMACOLOGY nism involving RhoA, catalytically active PLCe, sustained activation 32). PLC has also been implicated in the induction of the in- κ flammatory mediators TGF-β and cyclooxygenase 2 (COX-2) in of protein kinase D (PKD), and nuclear translocation of NF- B. – Endogenous ligands that are released from astrocytes in an in vitro response to chemical carcinogens or UV irradiation (33 36). e There is, however, no available information on whether GPCRs wounding assay also induce COX-2 expression through a PLC - and e fl NF-κB–dependent pathway. Additionally, in vivo stab wound in- use PLC signaling to regulate in ammatory gene expression. We hypothesized that GPCR agonists including thrombin, jury activates PKD and induces COX-2 and other inflammatory LPA, and S1P contribute to the pathophysiological role of astro- genes in WT but not in PLCe knockout mouse brain. Thus, PLCe cytes by activating PLCe and hence inflammatory gene expression. links GPCRs to sustained PKD activation, providing a means for We examined this hypothesis using astrocytes derived from brains GPCR ligands that couple to RhoA to induce NF-κB signaling and e fl of WT and PLC KO mice. The studies presented here demon- promote neuroin ammation. strate that PLCe is required to transduce astrocyte activation by GPCRs to induction of COX-2 gene expression. The molec- astrogliosis | inflammatory signaling cascades ular mechanisms elucidated in this work reveal that the diac- ylglycerol (DAG)-regulated kinase, protein kinase D (PKD), is any diseases of the central nervous system (CNS) are initi- activated in a sustained manner through PLCe and that both PLCe Mated or exacerbated by the activation of inflammatory sig- and PKD are required to activate NF-κB and regulate proin- naling cascades. Microglial and astroglial cells participate in this flammatory gene expression. PLCe is also shown to contribute process by both producing and responding to proinflammatory to inflammatory signaling in astrocytes in response to in vitro cytokines and mediators including IL-1β, IL-6, and inducible nitric wounding and in vivo brain injury. oxide synthase (1–5). Astrocytes also respond to ligands for G protein-coupled receptors (GPCRs), including thrombin and the Results lysophospholipids lysophosphatidic acid (LPA) and sphingosine 1- PLCe Is Required for Induction of COX-2 in Response to Thrombin, LPA, phosphate (S1P) (5). These ligands can be generated within or and S1P. Activation of PAR1, LPA, and S1P receptors has been supplied to the injured brain as a result of breakdown of the blood– linked to astrogliosis (4, 5, 9, 10). Because we have shown that brain barrier (6, 7). Signaling through these GPCRs has been these receptors stimulate phosphoinositide hydrolysis in astro- implicated in CNS injury and disease. For example, genetic de- cytes through PLCe (11), we asked whether PLCe could be the letion of the protease-activated receptor 1 (PAR1) thrombin re- link between these GPCRs and downstream inflammatory pro- ceptor inhibits astrocyte proliferation and neuronal cell death cesses associated with astrogliosis. COX-2 and other cytokines induced by transient cerebral ischemia or stab wound injury (8, 9). were used as markers of inflammation. In WT astrocytes treated Ligand-mediated activation of LPA and S1P receptors has also with 5 nM thrombin for 1 h, COX-2 mRNA increased nearly 10- been shown to stimulate astrocyte proliferation (5). Additionally, fold. This response was markedly attenuated in astrocytes from recent studies implicate S1P receptors on astrocytes in the astrogliosis and inflammation observed in a model of multiple sclerosis (10). Despite considerable evidence that signaling via PAR1, LPA, Author contributions: S.S.D. and J.H.B. designed research; S.S.D., E.R., and M.K.C. per- and S1P receptors stimulates astrogliosis, the molecular mecha- formed research; E.M. and A.V.S. contributed new reagents/analytic tools; S.S.D. analyzed nism by which GPCRs activated by these ligands contribute to data; and S.S.D., N.H.P., A.V.S., and J.H.B. wrote the paper. CNS pathophysiology is largely unknown. The authors declare no conflict of interest. We previously reported that phospholipase Ce (PLCe) is the *This Direct Submission article had a prearranged editor. PLC isoform responsible for mediating phosphoinositide hy- 1To whom correspondence should be addressed. E-mail: [email protected]. drolysis in astrocytes stimulated with thrombin, LPA, or S1P This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (11). PLCe was discovered a decade ago and differs from other 1073/pnas.1217355110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1217355110 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 Fig. 1. PLCe is required for induction of COX-2 in response to thrombin, LPA, and S1P. (A) COX-2 mRNA levels in primary WT and PLCe 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 PLCe 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.

PLCe KO mice (Fig. 1A). The increase in COX-2 mRNA was treatment with BMS-345541 (Fig. 2B). COX-2 expression in accompanied by a threefold increase in COX-2 protein in response to LPA and S1P was likewise abolished when NF-κB thrombin-treated WT astrocytes, which was also significantly activation was inhibited with BMS-345541 (Fig. 2B). diminished in PLCe KO astrocytes (Fig. 1B). S1P also increased mRNA levels for COX-2, as well as for IL-1β and IL-6, and these Scratch Wounding Induces COX-2 Through PLCe and NF-κB. To ex- responses were significantly attenuated by deletion of PLCe (Fig. plore the functional implications of this pathway in the astrocytic S1). The ability of S1P and LPA to increase COX-2 protein was response to injury in vitro, we applied multidirectional scratch to also attenuated in PLCe KO astrocytes (Fig. 1B). In contrast, monolayers of WT and PLCe KO astrocytes. Expression of carbachol, which activates muscarinic cholinergic receptors that COX-2 protein was increased nearly 2.5-fold at 8 h after scratch do not signal through PLCe (11), did not induce COX-2. in WT astrocytes but not in PLCe KO astrocytes (Fig. 3A). Blocking NF-κB activation with BMS-345541 fully prevented NF-κB Is Activated Through PLCe and Required for COX-2 Expression. COX-2 induction, implicating NF-κB activation in this in vitro NF-κB is known to regulate a number of inflammatory genes wounding response (Fig. 3B). Because no exogenous agonist was including COX-2. We examined translocation of the p65 subunit added to activate PLCe, we surmised that scratch elicits release of NF-κB from the cytosol to the nucleus as a measure of of GPCR ligands or other soluble activators of PLCe into the cell thrombin-stimulated NF-κB activation. Thrombin induced a sig- medium. This was tested by applying media collected from nificant increase in nuclear p65 in WT astrocytes, but this re- scratched cells to naïve cells. Conditioned medium from sponse was absent in PLCe KO cells (Fig. 2A). Carbachol, which scratched cells significantly increased COX-2 protein expression did not induce COX-2 expression, also failed to induce NF-κB in naïve astrocytes from WT mice but not in those from PLCe activation (Fig. 2A). To demonstrate that NF-κB activation KO mice (Fig. 3C). These data indicate that factors released mediates thrombin-induced COX-2 expression, we examined the during scratch injury also signal through PLCe to induce COX-2. effects of BMS-345541, an inhibitor of the upstream IκB kinase (IKK). BMS-345541 has been shown to selectively inhibit IKK PKD Is Activated Through PLCe and Mediates NF-κB Activation and relative to a panel of other serine/threonine and tyrosine kinases COX-2 Expression. Phosphoinositide hydrolysis leads to generation and prevent activation of NFκB (37). The ability of thrombin to of DAG, which in turn regulates DAG-sensitive protein kinases increase COX-2 expression was inhibited by 80% following including protein kinase C (PKC) and its downstream target

Fig. 2. NF-κB is activated through PLCe and required for COX-2 expression. (A) NF-κB activation was assessed by measuring increases in p65 in the nuclear fraction of WT and PLCe 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.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1217355110 Dusaban et al. Downloaded by guest on September 28, 2021 Fig. 3. Scratch wounding induces COX-2 through PLCe and NF-κB. (A) Conﬂuent monolayers of WT and PLCe 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.

PKD. We examined the involvement of PLCe in the activation of to WT and PLCe KO mice as described (38). Mice were killed at PKD by thrombin, LPA, and S1P. PKD activation was assessed day 7 after wounding and the cortex was removed for Western by measuring its phosphorylation at the S916 autophosphor- blotting. We observed significant increases in COX-2 expression ylation site. Thrombin increased PKD phosphorylation in WT and PKD activation in the cortical region of injured WT mice (Fig. astrocytes 2.5-fold at 1 h, and phosphorylation was sustained for 5 A, B, and E). Monocyte chemotactic protein-1 (MCP-1), another PHARMACOLOGY up to 6 h after thrombin treatment (Fig. 4A). PKD activation was marker of inflammation, and GFAP, a marker of astrogliosis, were increased to an even greater extent in WT astrocytes treated for also significantly increased in the WT mouse cortex following stab 1 h with LPA and S1P (5.5- and 7.5-fold, respectively) and injury (Fig. 5 A, C,andD). Neither of these markers was increased remained elevated above basal at 6 h (Fig. 4A). The activation of following stab wound injury in brains of PLCe KO mice (Fig. 5). PKD by all three ligands was dramatically reduced in astrocytes from PLCe KO mice. Carbachol also induced PKD activation at Discussion early times (15 min), but the response was more modest and PAR1, LPA, and S1P receptor activation contribute to astro- neither sustained nor PLCe-dependent (Fig. 4A). gliosis, a process associated with inflammation (4, 5, 9, 10). The We previously showed that RhoA is required for PLCe acti- mechanism by which signaling through these GPCRs induces vation by thrombin (11). To determine whether PKD activation neuroinflammation and participates in pathophysiological re- and subsequent COX-2 expression are also downstream effectors sponse to ischemia or brain injury is not well-understood. PAR1, of RhoA activation, we treated WT astrocytes with C3 exo- LPA, and S1P receptors are among the most efficacious of the enzyme, an agent that ribosylates and prevents RhoA signaling. GPCRs in their ability to couple to Gα12/13 and activate the low- Thrombin was unable to increase PKD activation or COX-2 molecular weight G protein RhoA (23–30). RhoA signaling has expression when RhoA function was blocked (Fig. S2 A and B). been implicated in the regulation of inflammatory genes, in- Inhibition of RhoA also prevented COX-2 induction by scratch cluding COX-2, in endothelial and NIH 3T3 cells (39–43). wounding (Fig. S2C). However, the molecular mechanisms by which GPCRs that acti- To determine whether PKD mediates NF-κB activation and vate RhoA lead to NF-κB and inflammatory gene expression have subsequent COX-2 induction, we used siRNA to knock down not been fully elucidated. The studies presented here demon- PKD in WT astrocytes. Treatment with siRNA significantly de- strate that stimulation of PLCe and the subsequent sustained creased PKD expression (Fig. S3). In astrocytes transfected with activation of PKD transduce GPCR activation to inflammatory control scrambled siRNA, thrombin induced a marked increase in gene expression (Fig. 6). nuclear p65; this response was abolished in cells transfected with PKD, a downstream target for regulation by PKC, also requires PKD siRNA (Fig. 4B). The ability of thrombin to increase COX-2 DAG for its activation (44–46). Because DAG generation and expression was likewise abolished by PKD knockdown (Fig. 4C). PKC activation occur downstream of phosphoinositide hydrolysis, Last, to demonstrate that PLCe and its catalytic activity are it is generally assumed that PKD is activated through the ca- required for COX-2 expression, we performed a rescue experi- nonical Gαq/PLCβ signaling cascade (47, 48). We show here, ment. In PLCe KO astrocytes infected with WT PLCe adenovi- however, that carbachol, which activates muscarinic receptors rus, the induction of COX-2 expression by thrombin and LPA that couple through Gαq/PLCβ to regulate phosphoinositide hy- treatment was restored (Fig. 4D). In contrast, PLCe KO astro- drolysis, induces only transient PKD activation. In contrast, cytes infected with catalytically dead PLCe adenovirus still failed thrombin, LPA, and S1P, which activate PLCe (11), lead to sus- to respond to thrombin or LPA with significant increases in tained PKD activation. This is consistent with previous data COX-2 (Fig. 4D). showing that PLCβ knockdown significantly reduces short-term inositol phosphate production, whereas knockdown of PLCe In Vivo Stab Wound Injury Induces COX-2 Through PLCe. Penetrating affects the longer-term agonist-induced generation of inositol head injury destroys neurons and leads to the generation of re- phosphates (22). PLCe is unique relative to other isoforms of active astrocytes. Our findings suggest that GPCR agonists present PLC in that it has an extended carboxyl terminus that contains at sites of injury could act through PLCe to drive astrogliosis. To a CDC25 domain that functions as a Rap guanine nucleotide extend the observation that PLCe mediates inflammatory signaling exchange factor (11, 15, 49). We have suggested that Rap1, ac- from GPCRs to an in vivo setting, cortical stab wound was applied tivated by PLCe, feeds forward through the RA2 domain on PLCe

Dusaban et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 Fig. 4. PKD is activated through PLCe and mediates NF-κB activation and COX-2 expression. (A) Time course of phosphorylation of PKD (p-PKDS916) in WT (solid line) and PLCe 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 PLCe 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)PLCe KO astrocytes were infected with WT FLAG- tagged PLCe adenovirus or catalytically dead FLAG-tagged PLCe 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.

to further activate and prolong generation of second messengers of PLCe to support sustained signals defines its unique role in such as DAG (12). The ability of PLCe to mediate sustained regulation of astrocyte function. signaling could be of particular importance with regard to its The physiological significance of our observations is supported proposed role in pathophysiology. by results from an in vitro wounding assay and an in vivo cortical The data presented here support the hypothesis that activation of stab wound model. These studies confirm that PLCe is a mediator PLCe and sustained PKD signaling are required for NF-κBacti- of inflammation and in addition provide indirect evidence that vation and COX-2 expression. In this regard, we show that transient PLCe is activated by endogenous mediators released in response PKD activation, as observed in response to carbachol, fails to in- κ to stress. The scratch wounding response could be recapitulated crease NF- B activation and subsequent COX-2 expression. In by the addition of conditioned medium to unscratched cells. addition, we demonstrate that in the absence of sustained PKD Similarly, during in vivo stab wound, breakdown of the blood– activation (i.e., in the PLCe KO or when PKD is down-regulated), brain barrier or generation of endogenous ligands would appear thrombin treatment fails to activate NF-κB or increase COX-2 to signal via PLCe to elicit PKD activation and induce COX-2, expression. Previous studies have shown that Gα12/13 and Rho signaling can lead to PKD or NF-κB activation and COX-2 ex- GFAP, and MCP-1 expression. Future studies, beyond the scope pression (43, 48, 50, 51), but the molecular mechanism by which of this manuscript, will be needed to determine what agonists are e Rho signaling is transduced into these responses has not been released to activate PLC in vitro and in vivo. Thrombin, LPA, delineated. These studies show that PLCe, and its catalytic activity, and S1P are possible candidates because injection of these ago- are required for induction of COX-2 expression, and that this nists into the striatum of mice induces astrogliosis (5, 9). Of ad- occurs through PKD and NF-κB activation, defining a critical and ditional interest, ligands for orphan GPCRs could signal through previously unknown link between GPCR-mediated Rho activation receptor coupling to Gα12/13 to converge on RhoA and PLCe and and inflammatory processes. Our data further argue that the ability induce inflammatory gene expression.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1217355110 Dusaban et al. Downloaded by guest on September 28, 2021 DharmaFECT 3, in a 1:3 ratio, respectively, were individually incubated in Opti- MEM media (Gibco) at room temperature for 10 min, mixed, and incubated further for 20 min. The siRNA/DharmaFECT 3 mixture was added to plates containing fresh media. Following overnight incubation, astrocytes were serum-starved for 18–24 h before agonist treatment and analysis by Western blot.

Transduction of Astrocytes with Adenovirus. PLCe KO astrocytes were plated and infected the next day for 4–6 h in complete media with 200 multi- plicity of infection of adenovirus expressing FLAG-tagged WT PLCe, FLAG-tagged catalytically dead PLCe, or enhanced yellow ﬂuorescent protein (EYFP) as previously described (11, 32, 55). Following 4–6hof infection, astrocytes were washed and serum-starved for 18–24 h before agonist treatment.

Quantitative PCR. Total RNA was extracted from agonist-treated WT and PLCe KO astrocytes using an RNeasy Kit (Invitrogen) as previously described (11). cDNA was ampliﬁed using TaqMan Universal Master Mix in the presence of gene-speciﬁc primers for COX-2, IL-1β, and IL-6, with GAPDH used as an internal control (Applied Biosystems). Data were normalized to internal GAPDH, and fold change was determined according to a published protocol (56).

Nuclear Fractionation Experiments. WT and PLCe KO astrocytes were grown to conﬂuence in 10-cm dishes. Before agonist treatment, cells were serum- starved for 18–24 h. Cells were fractionated according to a previously reported protocol (57). p65 expression in the nuclear fraction was assessed by Western blotting, and purity was determined with the cytosolic marker Rho GDP dissociation inhibitor (RhoGDI) and the nuclear marker lamin A/C.

Western Blotting. Freshly removed brain cortices were snap-frozen in liquid

nitrogen and lysates were prepared with RIPA buffer. Astrocyte lysates were PHARMACOLOGY Fig. 5. In vivo stab wound injury induces COX-2 through PLCe. WT and PLCe prepared with the same lysis buffer. Western blot analysis was performed KO mice at 8 wk of age were subjected to stab wound injury as described in according to the previous described protocol (58). The antibodies used for Methods. Seven days following injury, brains were removed and lysates were immunoblotting were as follows: NF-κB p65 (H-286) from Santa Cruz Bio- prepared for Western blotting. (A) Representative Western blots of COX-2, technology; RhoGDI, p-PKD (Ser916), PKD, MCP-1, FLAG tag (DYKDDDDK), MCP-1 (another inflammatory mediator), GFAP (a marker of astrogliosis), and lamin A/C from Cell Signaling Technology; and COX-2 from Cayman. 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, Wounding Assays. WT and PLCe KO astrocytes were plated in six-well plates, GFAP, and MCP-1 protein levels were normalized to GAPDH, and p-PKDS916 grown to confluence, and serum-starved 18–24 h before wounding as pre- was normalized to total PKD. *P < 0.05 and **P < 0.01 between control and viously described (59). The wound was made by scraping astrocytes with stab wound injury; #P < 0.01 between stab wound injuries, one-way ANOVA. a 200-μL pipette tip six to eight times bidirectionally across the dish. Eight or 24 h after wounding, cells were harvested for analysis by Western blotting or conditioned media were collected. Up-regulation of IL-1β, IL-6, and COX-2 has important disease implications: COX-2 plays a proinflammatory role in neurodegenerative disease and is increased in cerebral ischemia and traumatic brain injury (52–54), whereas IL-1β and IL-6 are up-regulated and contribute to damage of the myelin sheath during multiple sclerosis (10). Delineating a signaling pathway that links GPCRs through RhoA to PLCe and astrogliosis has the potential to reveal new therapeutic targets to limit neuroinflammation. Methods Agonists and Inhibitors. Sources were as follows: thrombin (Enzyme Research Laboratories), LPA (Avanti Polar Lipids), S1P (Avanti Polar Lipids), BMS-345541 (Sigma-Aldrich), and C3 Rho inhibitor cell-permeable (Cytoskeleton).

Animals. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of California at San Diego. Generation of homozygous C57BL6/ Sv129 PLC« KO mice has been described previously (31). PLC« heterozygous mice were bred to generate homozygous animals.

Primary Culture of Astrocytes. Astrocytes were isolated from postnatal days 1–3 WT and KO mice as previously described (11). Purity of astrocytes was determined to be ∼95% based on GFAP staining. In all experiments, WT and PLCe KO astrocytes were used at passage 2. Fig. 6. Schematic of pathway by which PLCe mediates GPCR activation and PKD siRNA Transfections. Predesigned mouse siRNA for PKD and control siRNA inﬂammatory responses in astrocytes. CDC25 is the guanine nucleotide ex- were purchased from Bioneer. WT astrocytes were transfected with 2 μMsiRNA change factor domain, PH is the Pleckstrin Homology domain, X and Y using DharmaFECT 3 transfection reagent (Thermo Scientiﬁc). siRNA and comprise the catalytic domain, and the RA2 is the Ras association domain.

Dusaban et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Cortical Stab Wound. Bilateral cortical stab wound was performed in WT and Statistical Analysis. Statistical differences were determined using Tukey’s PLCe KO (8 wk of age) as previously described (38). Brieﬂy, beginning at the level multicomparison analysis after one-way ANOVA with Prism software − of bregma, a 26-gauge needle was inserted 2.0 mm, and 1.5 mm lateral of the (GraphPad). P < 0.05 was considered signiﬁcant. midline, to a depth of 1.4 mm and extended three times in the cerebral cortex. Following 7 d of injury, animals were killed and the brain was removed. The ACKNOWLEDGMENTS. We thank Dr. Shigeki Miyamoto for technical assistance, brain hemisphere was removed and snap-frozen in liquid nitrogen and lysates helpful discussion, and review of the manuscript. This work was supported by were prepared with RIPA buffer. Sham-operated and noninjured mice were National Institutes of Health Grants GM 36927 (to S.S.D.), AG 18440 (to E.M.), determined to have equivalent basal levels of COX-2 expression, and thus the GM 53536 (to A.V.S.), and GM 36927 (to J.H.B.), and Tobacco-Related Disease latter group was used as the control in most experiments. Research Program 20KT-0048 (to N.H.P.).

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