Dioleoyl phosphatidic acid increases intracellular Ca2+ through endogenous LPA receptors in C6 glioma and L2071 fibroblasts

Prostaglandins & other Lipid Mediators 83 (2007) 268–276

Dioleoyl phosphatidic acid increases intracellular Ca2+ through endogenous LPA receptors in C6 glioma and L2071 fibroblasts Young-Ja Chang a , Yu-Lee Kim a , Yun-Kyung Lee a , Santosh J. Sacket a , Kyeok Kim a , Hyo-Lim Kim a , Mijin Han a , Yoe-Sik Bae b , Fumikazu Okajima c , Dong-Soon Im a,∗ a

Laboratory of Pharmacology, College of Pharmacy and Research Institute for Drug Development, Pusan National University, San 30, Jang-Jun-dong, Geum-Jung-gu, Busan 609-735, Republic of Korea b Department of Biochemistry, College of Medicine, Dong-A University, Busan 602-714, Republic of Korea c Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma 371, Japan Received 13 October 2006; received in revised form 21 January 2007; accepted 26 January 2007 Available online 3 February 2007

Abstract Phosphatidic acid (PA) increased intracellular Ca2+ concentration ([Ca2+ ]i ) in C6 rat glioma and L2071 mouse fibroblast cells. Dioleoyl PA (PA, 18:1) was the most efficacious, followed by dipalmitoyl PA (16:0 PA) and dimyristoyl PA (14:0 PA). Lysophosphatidic acid (LPA) also increased the [Ca2+ ]i in the both cells. PA desensitized LPA-induced Ca2+ response completely in C6 cells, but partly in L2071 cells. Treatment of pertussis toxin (PTX), a specific inhibitor of Gi/o -type G proteins, completely ameliorated LPA- and PA-induced Ca2+ response in C6 cells. However, in L2071 cells, PTX inhibited PA-induced Ca2+ increase by 80% and LPA-induced one by 20%. Ki16425, a specific inhibitor of LPA1 /LPA3 receptors, completely inhibited both LPA- and PA-induced Ca2+ responses in C6 cells. On the other hand, in L2071 cells, Ki16425 completely inhibited PA-induced Ca2+ response, but partly LPA-induced one. VPC32183, another specific inhibitor of LPA1 /LPA3 receptors, completely inhibited LPA- and PA-induced Ca2+ responses in both C6 and L2071 cells. Therefore, PA and LPA appear to increase [Ca2+ ]i through Ki16425/VPC32183-sensitive LPA receptor coupled to PTX-sensitive G proteins in C6 cells. In L2071 cells, however, LPA increases [Ca2+ ]i through Ki16425insensitive LPA receptor coupled to PTX-insensitive G proteins and Ki16425-sensitive LPA receptor coupled to PTX-sensitive G protein, whereas PA utilized only the latter pathway. Our results suggest that PA acts as a partial agonist on endogenous LPA receptors, which are sensitive to Ki16425 and coupled to PTX-sensitive G protein, but not on LPA receptors, which are not sensitive to Ki16425 and coupled to PTX-insensitive G protein. © 2007 Elsevier Inc. All rights reserved. Keywords: Phosphatidic acid; Lysophosphatidic acid; G-protein-coupled receptor; Calcium; LPA1 ; LPA3

Abbreviations: PA, dioleoyl phosphatidic acid; LPA, lysophosphatidic acid; VPC32183, (S)-phosphoric acid mono-{2-octadec9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl} ester; Ki16425, 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonylamino)-3-methyl-5isoxazolyl]benzylthio) propanoic acid; PTX, pertussis toxin; GPCR, G-protein-coupled receptor; LPA1 , lysophosphatidic acid receptor type 1 (EDG2); LPA2 , lysophosphatidic acid receptor type 2 (EDG4); LPA3 , lysophosphatidic acid receptor 3 (EDG7); LPA4 , lysophosphatidic acid receptor type 4 (GPR23); LPA5 , lysophosphatidic acid receptor 5 (GPR92); PLD, phospholipase D; PLA2 , phospholipase A2 ; AACOCF3 , arachidonyltrifluoromethyl ketone; MAFP, methylarachidonyl fluorophosphonate; BEL, bromoenol lactone; DDT, dl-dithiothreitol ∗ Corresponding author. Tel.: +82 51 510 2817; fax: +82 51 513 6754. E-mail address: [email protected] (D.-S. Im). 1098-8823/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2007.01.014

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1. Introduction Dioleoyl phosphatidic acid (PA) comprises a minor fraction of the total phospholipid pool, however, it is involved in lipid biosynthesis and a substrate for potent cellular phosphatases which rapidly generate diacylglycerides [1,2]. Upon stimulation of cells with certain metabolic agonists, PA levels increase rapidly through activation of phospholipase D (PLD) [2]. PA has been proposed as a second messenger for many stimuli, and its action on intracellular signaling molecules such as mTOR has intensively been studied [3–5]. On the other hand, a glycosylphosphatidylinositol-specific PLD has been found in serum [6], and an ecto-PA phosphohydrolase and PA-specific phospholipase A2 (PLA2 ) have been reported [7–9], suggesting external exposure of PA. In fact, exogenous PA has been demonstrated to induce a calcium influx in a number of biological tissues, including isolated platelets [10–12], parotid gland [13,14], liver cells [15,16], nerve terminals [17], epithelium [18], osteoblasts [19,20], and cardiac myocytes [21]. PA exerts potent mitogenic effects [22–27]. Nevertheless, the finding of lysophosphatidic acid (LPA) as a potent mitogenic lipid [28] and the fact that commercial preparation of PA was contaminated with LPA shifted the importance of PA to LPA [29]. Contrary to the above, however, growth factor induces cellular proliferation through PA dependent activation of mitogen-activated protein (MAP) kinase, an event resulting from upstream induction of tyrosine kinase, MAP kinase or MAP kinase kinase [30] and, in this system, LPA was without effect. Furthermore, several reports excluded the LPA contamination as the cause of the PA response [21,24,25,30–33]. The binding of specific PA receptors in phosphoinositide metabolism and mitogenesis induced by exposure of cortical astrocytes to the phospholipid was implicated, and actin polymerization in fibroblasts has been suggested to result from specific binding of PA to its receptor [33,34]. Furthermore, in recombinant LPA receptorexpressing cells, PA has been shown as a partial agonist of LPA1 , LPA2 and LPA3 receptors, even though others could not observe such agonism in LPA receptor-overexpression systems [35–39]. Therefore, we asked questions of whether PA could act as a ligand of G-protein coupled receptor (GPCR) on the plasma membrane and also whether its action was mediated through LPA receptors. By employing pharmacological inhibitors of LPA receptors, we observed that PA increased [Ca2+ ]i through LPA receptors, which were endogenously expressed, in rat glioma and mouse fibroblasts, suggesting the role of first messenger and partial agonism of PA on endogenous LPA receptors. 2. Materials and methods 2.1. Materials 1,2-Dioleoyl-sn-glycero-3-phosphate (18:1 PA), 1,2-dioctanoyl-sn-glycero-3-phosphate (8:0 PA), 1,2-didecanoylsn-glycero-3-phosphate (10:0 PA), 1,2-dilauroyl-sn-glycero-3-phosphate (12:0 PA), 1,2-dimyristoyl-sn-glycero-3phosphate (14:0 PA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (16:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), and VPC32183 were purchased from Avanti Polar Lipids (Alsbaster, AL, USA). Fura 2-AM/AM, quinacrine, arachidonyltrifluoromethyl ketone (AACOCF3 ), and methylarachidonyl fluorophosphonate (MAFP) were obtained from Calbiochem (Darmstadt, Germany). Pertussis toxin (PTX), bromoenol lactone (BEL), dl-dithiothreitol (DDT), and other materials were purchased from Sigma–Aldrich (St. Louis, MO, USA). 2.2. Cell culture Rat C6 glioma cells were maintained in high glucose DMEM, containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 50 ␮g/ml streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate, at 37 ◦ C in a humidified 5% CO2 incubator [40]. L2071 mouse fibroblasts were cultured in RPMI 1640 media, containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, 50 ␮g/ml streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate, at 37 ◦ C in a humidified 5% CO2 incubator [41]. 2.3. Measurement of intracellular Ca2+ concentration Cells were trypsin-digested, sedimented, resuspended in Hepes-buffered medium (HBM), consisting of 20 mM Hepes (pH 7.4), 103 mM NaCl, 4.8 mM KCl, 1.2 mM KH2 PO4 , 1.2 mM MgSO4 , 0.5 mM CaCl2 , 25 mM NaHCO3 , 15 mM glucose and 0.1% bovine serum albumin (fatty acid free), and then incubated for 40 min with 5 ␮M fura 2-

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AM for Ca2+ measurement. The amount of [Ca2+ ]i was estimated from the change in the fluorescence of the fura 2-loaded cells [42]. Fluorescence emission at 510 nm wavelength from two excitation wavelengths (340 and 380 nm) were measured every 0.1 s by F4500 fluorescence spectrophotometer (Hitachi, Japan), and the ratio of fluorescence intensities from the two wavelengths was monitored as an estimate of [Ca2+ ]i [42]. 2.4. Reverse transcriptase-PCR To identify expression of LPA receptors in C6 glioma cells and L2071 fibroblasts by RT-PCR, first strand cDNA was synthesized with total RNA isolated by using the Promega ImProm-II Reverse Transcription System (Madison, WI, USA). Synthesized cDNA products along with primers for LPA1–5 were used for PCR by using Promega Go-Taq DNA polymerase (Madison, WI, USA). Specific primers for LPA1 (sense 5 -TCT TCT GGG CCA TTT TCA AC-3 , antisense 5 -TGC CTG AAG GTG GCG CTC AT-3 ), LPA2 (sense 5 -ACC AAC CTG CTG GTT ATT GC-3 , antisense 5 -TCC AAG TCA CAG AGG CAG TG-3 ), LPA3 (sense 5 -GGA ATT GCC TCT GCA ACA TCT-3 , antisense 5 GAG TAG ATG ATG GGG TTC A-3 ), LPA4 (sense 5 -ACC ACC ACT TGC TTT GAA GG-3 , antisense 5 -AGA GTT GCA AGG CAC AAG GT-3 ) for C6 or LPA4 (sense 5 -AAC CTG GCC CTC TCT GAT TT-3 , antisense 5 -CCT TCA AAG CAA GTG GTG GT-3 ) for L2071, and LPA5 (sense 5 -TCC TAC TGG CCA ACC TCA TC-3 , antisense 5 -GAA GTA GCC TCT GGC TGG TG-3 ) were used to amplify 349, 383, 382, 353/335 and 348 bp of fragments of LPA1–5 , respectively. The PCR reaction was performed by 30 cycles of denaturation at 95 ◦ C for 1 min, annealing at 55 ◦ C for 1 min, and elongation at 72 ◦ C for 2 min for LPA2 , LPA4 , and LPA5 or denaturation at 95 ◦ C for 30 s, annealing at 56 ◦ C for 30 s, and elongation at 72 ◦ C for 2 min for LPA1 and LPA3 in an Eppendorf Mastcycler gradient PCR machine (Hamburg, Germany) [42]. Ten microliters of aliquots were electrophoresed in a 1.2% agarose gel, and stained with ethidium bromide. 2.5. Statistics The results are expressed as means ± S.E. of the number of determinations indicated. Statistical significance of differences was determined by ANOVA. Significance was accepted when P < 0.05. 3. Results 3.1. PA and LPA increase intracellular Ca2+ concentration in C6 rat glioma and L2071 mouse fibroblasts PA and LPA increased [Ca2+ ]i in C6 rat glioma and L2071 mouse fibroblasts (Fig. 1). Efficacy order of PAs was 18:1 PA > 18:0 PA > 16:0 PA > 14:0 PA in C6 glioma cells (Fig. 1A), and the efficacy order in L2071 fibroblasts was 18:1 PA > 16:0 PA > 14:0 PA > 18:0 PA (Fig. 1B). Dilauroyl PA (12:0), 10:0 PA and 8:0 PA were not effective on the Ca2+ response in both cell types (Fig. 1A and B). Concentration-dependence of PA and LPA was investigated in both

Fig. 1. Ca2+ increase by LPA and PAs in C6 glioma and L2071 fibroblasts cells. Representative Ca2+ traces with 20 ␮M each of LPA, 18:1 PA, 18:0 PA, 16:0 PA, or 14:0 PA in C6 glioma cells (A) or in L2071 fibroblast cells (B). Each lipid was added at the arrow indicated. The data shown are representative of three independent experiments.

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Fig. 2. Concentration-dependence of LPA- and PA-induced Ca2+ increase in C6 glioma and L2071 fibroblasts cells. Concentration–response curves of LPA-induced intracellular Ca2+ increase (closed circle) and 18:1 PA-induced intracellular Ca2+ increase (closed triangle) in C6 glioma cells (A) or in L2071 fibroblast cells (B) are made by percentages to 20 ␮M LPA-induced maximum response. The results are expressed as means ± S.E. of three independent experiments.

cell lines. In C6 glioma cells, EC50 values for LPA and PA were about 68 nM and 1.63 ␮M, respectively (Fig. 2A). Therefore, PA was about 24 times less potent than LPA, but the efficacy was not significantly different, that is the maximum effects by two lipids were similar. In L2071 cells, EC50 values for LPA and PA were about 119 nM and 2.19 ␮M, respectively (Fig. 2B). Therefore, PA was about 18 times less potent than LPA, but the efficacy was also not significantly different. 3.2. Desensitization of the Ca2+ increase by PA and LPA Next, desensitization experiments were conducted. As shown in Fig. 3, LPA desensitized PA response in both cells (Fig. 3A and C). On the other hand, PA desensitized LPA response in C6 cells, but only partly in L2071 cells (Fig. 3B and D), implying that PA-induced response belongs to LPA-induced broader response in L2071 cells.

Fig. 3. Desensitization by PA and LPA on the Ca2+ increase. Representative Ca2+ traces with 20 ␮M each of LPA and 18:1 PA in C6 glioma cells (A and B) or in L2071 fibroblast cells (C and D). Each lipid was sequentially added at the arrows indicated. The data shown are representative of three independent experiments (A–D).

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Fig. 4. Effect of PTX on PA- and LPA-induced Ca2+ increase. Cells treated with PTX (100 ng/ml, 24 h) or without were used for Ca2+ measurements with 20 ␮M each of LPA or 18:1 PA in C6 glioma cells (A and B) or in L2071 fibroblast cells (C). Each lipid was added at the arrow indicated (A and B). Histogram shows percentage of Ca2+ increase by 20 ␮M each of LPA, 18:1 PA and 16:0 PA in PTX-treated L2071 cells, compared to the increases in non-treated cells (C). The data shown are representative of three independent experiments (A and B). Data are presented as means ± S.E. of three independent experiments (C). Statistical significance: **P < 0.01 vs. PTX non-treated cells.

3.3. Effect of pertussis toxin on PA- and LPA-induced Ca2+ response Using pertussis toxin (PTX), a specific inhibitor of Gi/o -type G proteins, involvement of Gi/o protein-coupled receptors was investigated. As shown in Fig. 4, treatment of PTX (100 ng/ml, 24 h) completely ameliorated PA- and LPA-induced Ca2+ increases in C6 cells, suggesting that Gi/o protein-coupled receptors are activated by PA and LPA. However, PTX treatment inhibited PA-induced Ca2+ increase by 80%, but 20% of LPA-induced one in L2071 cells (Fig. 4C), suggesting two different receptors for PA and LPA in L2071 cells; PTX-sensitive receptor and PTX-insensitive receptor. 3.4. Effects of Ki16425 and VPC32183 on PA- and LPA-induced Ca2+ response To elucidate whether LPA receptors are involved in PA-induced response, Ki16425 and VPC32183, structurally different antagonists for LPA1 /LPA3 receptors, were applied. As shown in Fig. 5, Ki16425 completely ameliorated PA- and LPA-induced Ca2+ increases in C6 cells, whereas it completely inhibited PA-induced Ca2+ increase, but partly LPA-induced one in L2071 cells (Fig. 5). This suggests that LPA-induced Ca2+ increase is mediated in L2071 cells through two receptors; Ki16425-sensitive LPA receptor and Ki16425-insensitive LPA receptor, whereas PA-induced Ca2+ response in these cells is mediated only through Ki16425-sensitive LPA receptor. In contrast to Ki16425, another antagonist, VPC32183, completely antagonized PA- and LPA-induced Ca2+ increases in both C6 and L2071 cells (Fig. 6), although susceptibility was different. Treatment of Ki16425 or VPC32183 did not inhibit ATP-induced Ca2+ increases in the both cell lines, confirming the selectivity of the antagonists and excluding a possibility of cell damage by the antagonists (data not shown). Differential susceptibility of LPA and PA responses to both antagonists may imply different specificity of the antagonists for each LPA receptor.

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Fig. 5. Effect of Ki16425 on PA- and LPA-induced Ca2+ increase. Representative Ca2+ traces with 20 ␮M LPA (A and C) or 20 ␮M 18:1 PA (B and D) in C6 glioma cells (A and B) or in L2071 fibroblast cells (C and D). Ki16425 (100 ␮M) or vehicle was added at the first arrow, and 20 ␮M LPA or 18:1 PA was added at the arrow indicated after 30 min of incubation. The data shown are representative of three independent experiments (A–D).

Fig. 6. Effect of VPC32183 on PA- and LPA-induced Ca2+ increase. Percentage of Ca2+ increase in C6 glioma cells by 300 nM LPA (A) or 10 ␮M 18:1 PA (B) in the presence of different concentrations of VPC32183 is shown, compared to the increases without VPC32183. Data are presented as means ± S.E. of three independent experiments (A and B). Representative Ca2+ traces with 50 nM LPA (C) or 1 ␮M 18:1 PA (D) in L2071 fibroblast cells are shown. VPC32183 (20 ␮M) or vehicle was added at the first arrow, and 50 nM LPA or 1 ␮M 18:1 PA was added at the second arrow. The data shown are representative of three independent experiments (C and D).

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Fig. 7. Expression analysis of five LPA receptors in C6 glioma and L2071 fibroblasts cells. RT-PCR was performed using mRNAs extracted from C6 glioma and L2071 fibroblasts cells. The data is a representative of three independent experiments with similar results.

3.5. Expression of LPA receptors in C6 and L2071 cells RT-PCR was conducted to analyze expression levels of five LPA receptors, including recently identified LPA receptors, LPA4 (GPR23) and LPA5 (GPR92) [43–47]. In C6 cells, transcripts of LPA1 , LPA2 , LPA4 , and LPA5 were detected and in L2071 cells all five LPA receptors were shown to be expressed (Fig. 7). Only obvious difference was the presence of LPA3 receptor expression in L2071 cells and the absence of it in C6 cells, although expression levels of other LPA receptors were shown to be little different. 4. Discussion Although many researches have been focused on the second messenger roles of PA as a signaling product of PLD [3–5], a first messenger role of PA on GPCR needs also to be considered [2], since the presence of several metabolic enzymes for PA such as glycosylphosphatidylinositol-specific PLD in serum [6] and ecto-PA phosphohydrolase [7] and PA-specific PLA2 [8,9] in the plasma membrane suggests the production and degradation of PA in the extracellular milieu. In recombinant LPA receptor-expressing cells, PA has been shown as a partial agonist for LPA1 , LPA2 and LPA3 receptors [35–37]. However, the action of PA on endogenous LPA receptors has not yet been reported. Furthermore, partial agonism of PA in LPA receptor-overexpression systems remains largely controversial [38,39]. Therefore, we questioned whether PA could act as a ligand of GPCR on the plasma membrane and also whether its action was mediated through endogenous LPA receptors. By employing pharmacological inhibitors of LPA receptors, we found that PA increased [Ca2+ ]i through PTX-sensitive G proteins and Ki16425/VPC32183-sensitive LPA receptors endogenously expressed in rat glioma and mouse fibroblasts, this confirming partial agonism of PA on LPA receptors. And we also observed differential susceptibility of LPA receptors in C6 cells and L2071 cells to treatment of PA, PTX, Ki16425, and VPC32183; partial desensitization by PA, PTX-insensitivity, and partial inhibition by Ki16425 were observed in L2071 cells, however, complete desensitization by PA, PTX-sensitivity, and complete inhibition by Ki16425 were observed in C6 cells. It is highly likely in L2071 cells that mainly PTX- and Ki16425-insensitive LPA receptor type mediates Ca2+ increase, and that this receptor is not much activated by PA. On the other hand, PTX- and Ki16425-sensitive LPA receptor type mediates the whole LPA-induced Ca2+ response in C6 glioma cells and this receptor is easily activated by PA. Currently, information on the susceptibility of LPA4 and LPA5 to both antagonists is not available [43–47]. Based on the expression levels of LPA receptors in two cell lines and antagonist susceptibility of PA- and LPA-induced Ca2+ responses, however, LPA1 receptor is likely to be responsible for Ki16425-sensitive and PTX-sensitive Ca2+ increases by PA and LPA. On the other hand, LPA3 receptor expressed only in L2071 cells might be responsible for Ki16425-insensitive and PTX-insensitive Ca2+ increase by LPA in L2071 cells. Although further investigation is necessary to confirm which receptor type is responsible and the production of PA in the extracellular milieu, our present results on the first messenger role of PA and its agonism on endogenous LPA receptor strongly suggest it as an intercellular bioactive lipid mediator.

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Two possibilities also need to be considered when we interpret PA actions. One is contamination of LPA in PA preparation. However, current PA preparations are chemically synthesized and over 99% pure (Avanti Polar Lipids). Even through 1% contamination of LPA are present in PA preparation, PA response observed in our study could not be explained by the contaminated LPA, because LPA was about 20 times more potent than PA in the Ca2+ response. If the 1% LPA contaminant is the active material in PA preparation, LPA potency should be two log orders better than PA potency. The other possibility is conversion of PA to LPA by PLA2 in the assay media. However, this possibility is also quite low in our study, because we measured intracellular Ca2+ increase which occurred within several seconds and PA-induced Ca2+ increase was not ameliorated by the presence of specific PLA2 inhibitors such as BEL, AACOCF3 , MAFP, quinacrine, and DDT (data not shown). Acknowledgement This work was supported by the Korea Science and Engineering Foundation Grant (R01-2005-000-10011-02005). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43]

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