Lysophosphatidylethanolamine (LPE), a lyso-type metabolite of phosphatidylethanolamine, has been reported to be an intercellular signaling molecule. LPE mobilizes intracellular Ca2+ through G-protein-coupled receptor (GPCR) in some cells types. However, GPCRs for lysophosphatidic acid (LPA) were not implicated in the LPE-mediated activities in LPA GPCR overexpression systems or in SK-OV3 ovarian cancer cells. In the present study, in human SH-SY5Y neuroblastoma cells, experiments with LPA1 antagonists showed LPE induced intracellular Ca2+ increases in an LPA1 GPCR-dependent manner. Furthermore, LPE increased intracellular Ca2+ through pertussis-sensitive G proteins, edelfosine-sensitive-phospholipase C, 2-APB-sensitive IP3 receptors, Ca2+ release from intracellular Ca2+ stores, and subsequent Ca2+ influx across plasma membranes, and LPA acted on LPA1 and LPA2 receptors to induce Ca2+ response in a 2-APB-sensitive and insensitive manner. These findings suggest novel involvements for LPE and LPA in calcium signaling in human SH-SY5Y neuroblastoma cells.
Lysophosphatidylethanolamine (LPE) is a metabolic product from phosphatidylethanolamine (a minor constituent of cell membranes) by phospholipase A2. LPE has an ethanolamine head group linked to a lysophosphatidic acid. LPE is commercially used as a plant bio-regulator to delay leaf and fruit senescence, improve product shelf-life post harvest, and mitigate ethylene-induced process (Cowan, 2009). In addition, LPE appears to have certain roles in organisms other than mammals, for example, in the housefly, LPE has antimicrobial activity (Meylaers
LPE has been detected in human serum at concentrations of about several hundreds nanograms per ml (Misra, 1965; Makide
In SK-OV3 and OVCAR-3 ovarian cancer cells, LPE induces several responses, which include increasing intracellular Ca2+ concentration ([Ca2+]i) (Park
Human SH-SY5Y neuroblastoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured at 37°C in a 5% CO2 humidified incubator, and maintained in RPMI 1640 medium (GenDEPOT, Barker, TX, USA) supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 50 μg/mL streptomycin, 2 mM glutamine, and 1 mM sodium pyruvate.
Cells were trypsin-digested, allowed to sediment, resuspended in HEPES-buffered medium (HBM), consisting of 20 mM HEPES (pH 7.4), 103 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 0.5 mM CaCl2, 25 mM NaHCO3, and 15 mM glucose, and then incubated for 40 min with 5 μM fura 2-AM. [Ca2+]i levels were estimated by measuring changes in fura-2 fluorescence at an emission wavelength of 510 nm and excitation wavelengths of 340 nm and 380 nm every 0.1 sec using a F4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) (Park
To detect the expressions of LPA receptors in SH-SY5Y cells by RT-PCR, first strand cDNA was synthesized using total RNA isolated using Trizol reagent (Invitrogen, Waltham, MA, USA). Synthesized cDNA products and primers for LPA1–6 were subjected to PCR using Promega Go-Taq DNA polymerase (Madison, WI, USA). The primers used to amplify 317, 317, 321, 341, 308, and 247 bps fragments of LPA1–6 and GAPDH were as follows: LPA1 (sense 5′-CAG GAC CCA ATA CTC GGA GA-3′, antisense 5′-GTT GAA AAT GGC CCA GAA GA-3′), LPA2 (sense 5′-TTT CAC TTG AGG GCT GGT TC-3′, antisense 5′-CAT GAG CAG GAA GAC AAG CA-3′), LPA3 (sense 5′-CTC ATG GCC TTC CTC ATC AT-3′, antisense 5′-GCC ATA CAT GTC CTC GTC CT-3′), LPA4 (sense 5′-CTT CGC AAG CCT GCT ACT CT-3′, antisense 5′-GGC TTT GTG GTC AAA GGT GT-3′), LPA5 (sense 5′-TCT CCC GTG TCC TGA CTA CC-3′, antisense 5′-TGA GCA TCA GGA AGA TGC AG-3′), and LPA6 (sense 5′-TGC TCA GTA GTG GCA GCA GT-3′, antisense 5′-CAG GCA GCA GAT TCA TTG TC-3′), and GAPDH (sense 5′-GAG TCA ACG GAT TTG GTC GT-3′, antisense 5′-TTG ATT TTG GAG GGA TCT CG-3′). PCR reactions were performed over 30 cycles of 95°C for 30 s (denaturation), 57°C for 30 s (annealing) for LPA1–6, and 72°C for 30 s (elongation) for GAPDH in an Eppendorf Mastcycler gradient unit (Hamburg, Germany) (Park
The results are expressed as means ± SEs for the indicated numbers of determinations. Significances of differences were determined using the student t test, and significance was accepted for
Synthetic oleoyl LPE (18:1 LPE) increased [Ca2+]i levels in SH-SY5Y neuroblastoma cells (Fig. 1A) in a concentration-dependent manner (Fig. 1C), and response to LPA was greater than to LPE, but in SH-SY5Y cells LPA and LPE had similar efficacies (Fig. 1B, 1D). Responses were also studied using structurally different LPEs, that is, stearoyl LPE (18:0 LPE), octadecanyl LPE (ether-linked 18:0 LPE), and palmitoyl LPE (16:0 LPE). As shown in Fig. 2, 18:1 LPE, 18:0 LPE, ether-linked 18:0 LPE, and 16:0 LPE induced a [Ca2+]i increase in SY-SY5Y cells, which contrasted to that observed in MDAMB-231 cells, in which oleoyl LPE (18:1 LPE) was the only active LPE. Structure-activity relationships in LPE-responsive cells are addressed in the Discussion.
Because previous studies have implicated LPA receptor in LPE-induced Ca2+ signaling in certain cell types, we investigated homologous and heterologous desensitizations of LPE- and LPA-induced [Ca2+]i increases in SH-SY5Y cells. In desensitization experiments, LPE or LPA were pre-treated for 1 min before adding LPE (10 μM) or LPA (10 μM). As shown in Fig. 3A, 3B, LPE pre-treatment blocked LPE-induced [Ca2+]i response by 100%, and LPA pre-treatment attenuated LPA-induced response by 90%, implying homologous desensitization. In addition, LPA pre-treatment attenuated LPE-induced [Ca2+]i response by 90%, and LPE pre-treatment attenuated LPA-induced [Ca2+]i response by 63%, implying heterologous desensitization (Fig. 3). In addition, we examined the expression levels of the six known LPA receptors by RT-PCR in human SH-SY5Y cells. LPA1 and LPA2 were found to be strongly expressed, whereas the other four LPA receptors were not detected (Fig. 3C). These results suggest that LPE acts on LPA1 and/or LPA2 receptors in SH-SY5Y cells.
Three pharmacological tools were applied to investigate the involvements of LPA receptors in SH-SY5Y cells, that is, structurally different antagonists of LPA1 and LPA3 (Ki16425 and VPC32183) (Heise
To investigate cascades signaling LPE and LPA [Ca2+]i responses, SH-SY5Y cells were treated with specific inhibitors or blockers of Gi/o-type G proteins, phospholipase C, inositol 1,4,5-trisphosphate receptor (IP3R), extracellular Ca2+, or autotaxin, that is, pertussis toxin (PTX), edelfosine, 2-APB, EGTA, and HA130, respectively (Park
To determine whether LPE is converted to LPA by autotaxin (also known as lysophospholipase D), and this LPA mediates the action of LPE, we pretreated SH-SY5Y cells with HA130 (a specific inhibitor of autotaxin). However, HA130 did not inhibit LPE-induced Ca2+ increase, indicating that autotaxin was not responsible for the observed effects of LPE (Fig. 6).
In the present study, LPE-induced [Ca2+]i increase was found to be mediated through LPA1 in SH-SY5Y cells. Five results sustain this finding: 1) the observed heterologous desensitization found for LPE- and LPA-induced [Ca2+]i increases, 2) the abrogation of LPE-induced response by the LPA1 and LPA3 antagonist Ki16425 supported the involvements of LPA1 and/or LPA3, 3) the complete inhibition of LPE-induced response by the LPA1 antagonist, AM-095, 4) the observation that LPA1 was expressed in SH-SY5Y cells, and 5) the Gi/o-coupling character of LPA1 and the PTX-sensitivity of LPE-induced [Ca2+]i increase. LPE-induced [Ca2+]i increases have been previously observed in ovarian and breast cancer cells and in pheochromocytoma cells (Park
In ovarian cancer cells, LPE-induced [Ca2+]i increase was not found to be mediated through Ki16425, VPC32183, or AM-095-sensitive receptors (Park
In the present study, LPE-induced Ca2+ responses of synthetic LPE analogues were cell type dependent. In particular, ether-linked 18:0 LPE and ester-linked 18:0 LPE produced more than 50% of the response elicited by ester-linked 18:1 LPE in SK-OV3, SH-SY5Y, and PC-12 cells, but did not produce any response in MDA-MB-231 cells (Park
Therefore, in SH-SY5Y cells, LPE was found to act on LPA1 to induce [Ca2+]i increase via Gi/o proteins, phospholipase C, and IP3R, and LPA was found to use LPA1 and LPA2 to mobilize Ca2+ (Fig. 7). Significance of this study is not only LPE action on LPA1 in SH-SY5Y cells but also involvement of Gi/o proteins and phospholipase C in LPA Ca2+ signaling. In previous studies using SH-SY5Y cells, LPA-induced Ca2+ mobilization was shown to be independent on phosphoinositide signaling and not mediated through pertussis toxin-sensitive Gi/o proteins (Young
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant no. NRF-2011-0021158) and by the Korean National Research Foundation funded by the Korean government (MSIP) (Grant no. 2009-0083538).