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• W2159362912 abstract "We found that Grifola frondosa extracts induced the activation of mitogen-activated protein kinase (MAPK) in cultured PC12 cells, a line of rat pheochromocytoma cells. The active substance was isolated by a few chromatographic steps, including high-performance liquid chromatography, and was identified to be lysophosphatidylethanolamine (LPE) from various structural analyses. LPE from G. frondosa (GLPE) was confirmed to induce the activation of MAPK of cultured PC12 cells and was found to suppress cell condensation and DNA ladder generation evoked by serum deprivation, suggesting that the GLPE had antiapoptotic effects. Moreover, GLPE caused morphological changes in and upregulation of neurofilament M expression of PC12 cells, demonstrating that the GLPE could induce neuronal differentiation of these cells. The activation of MAPK by GLPE was suppressed by AG1478, an antagonist of epidermal growth factor receptor (EGFR), and by U0126, an inhibitor of MAPK kinase (MEK1/2), but not by K252a, an inhibitor of TrkA, or by pertussis toxin. These results demonstrate that GLPE induced the MAPK cascade [EGFR-MEK1/2-extracellular signal-regulated protein kinases (ERK1/2)] of PC12 cells, the activation of which induced neuronal differentiation and suppressed serum deprivation-induced apoptosis. This study has clarified for the first time the involvement of the MAPK signal cascade in LPE actions. We found that Grifola frondosa extracts induced the activation of mitogen-activated protein kinase (MAPK) in cultured PC12 cells, a line of rat pheochromocytoma cells. The active substance was isolated by a few chromatographic steps, including high-performance liquid chromatography, and was identified to be lysophosphatidylethanolamine (LPE) from various structural analyses. LPE from G. frondosa (GLPE) was confirmed to induce the activation of MAPK of cultured PC12 cells and was found to suppress cell condensation and DNA ladder generation evoked by serum deprivation, suggesting that the GLPE had antiapoptotic effects. Moreover, GLPE caused morphological changes in and upregulation of neurofilament M expression of PC12 cells, demonstrating that the GLPE could induce neuronal differentiation of these cells. The activation of MAPK by GLPE was suppressed by AG1478, an antagonist of epidermal growth factor receptor (EGFR), and by U0126, an inhibitor of MAPK kinase (MEK1/2), but not by K252a, an inhibitor of TrkA, or by pertussis toxin. These results demonstrate that GLPE induced the MAPK cascade [EGFR-MEK1/2-extracellular signal-regulated protein kinases (ERK1/2)] of PC12 cells, the activation of which induced neuronal differentiation and suppressed serum deprivation-induced apoptosis. This study has clarified for the first time the involvement of the MAPK signal cascade in LPE actions. Natural compounds with antioxidant activity, such as curcumin (1Yang F. Lim G.P. Begum A.N. Ubeda O.J. Simmons M.R. Ambegaokar S.S. Chen P. Curcumin inhibits formation of amyloid β oligomers and fibrils, binds plaques, and reduces amyloid in vivo.J. Biol. Chem. 2005; 280: 5892-5901Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar), phenolic yellow curry pigment, and naringenin, a major flavonone constituent isolated from Citrus junos, have been found to reduce the neurotoxicity generated by amyloid β protein and to result in reduced amyloid deposition and the amelioration of drug-induced amnesia in animal models of Alzheimer’s disease. Soy isoflavones have similarly been shown to influence the brain cholinergic system, reducing age-related neuron loss and the spatial cognition decline that occurs in elderly rats (2Heo H.J. Kim D.O. Effect of antioxidant flavanone, naringenin, from Citrus junos on neuroprotection.J. Agric. Food Chem. 2004; 52: 1520-1525Crossref PubMed Scopus (149) Google Scholar). These observations prompted us to search for natural compounds with neurotrophic and/or neuroprotective activities, because the population of the Western world is progressively aging, and this increase in the elderly population will mean an increase in age-related cognitive decline disorders such as Alzheimer’s disease. Recent investigations suggest that neurotrophic factors are involved in the cause and/or development of these diseases and that the development of compounds with activities like those of particular neurotrophic factors may be a promising avenue for protection against such diseases (3Angelucci F. Mathe A.A. Aloe L. Neurotrophic factors and CNS disorders: findings in odent models of depression and schizophrenia.Prog. Brain Res. 2004; 146: 151-165Crossref PubMed Scopus (113) Google Scholar, 4Duman R.S. Role of neurotrophic factors in the etiology and treatment of mood disorders.Neuromolecular Med. 2004; 5: 11-24Crossref PubMed Google Scholar, 5Tuszynski M.H. Blesch A. Nerve growth factor: from animal models of cholinergic neuronal degeneration to gene therapy in Alzheimer's disease.Prog. Brain Res. 2004; 146: 441-449PubMed Google Scholar). Although many edible fungi are cultivated all over the world, we focused on mushrooms, because they are marketed as healthful foods and their pharmacological activities have been studied. Tricholomalides from Tricholoma species (6Tsukamoto S. Macabalang A.D. Ohta T. Tricholomalides A-C, new neurotrophic diterpenes from the mushroom Tricholoma sp.J. Nat. Prod. 2003; 66: 1578-1581Crossref PubMed Scopus (43) Google Scholar), termitomycesphins from Termitomyces albuminosus (7Qi J. Ojika M. Sakagami Y. Termitomycesphins A-D, novel neuritogenic cerebrosides from the edible Chinese mushroom Termitomyces albuminosus.Tetrahedron. 2000; 56: 5835-5841Crossref Google Scholar), and dictyoquinazol and dictyophorines from Dictyophora indusiata (8Lee I.K. Yun B.S. Han G. Yoo I.D. Dictyoquinazols A, B, and C, new neuroprotective compounds from the mushroom Dictyophora indusiata.J. Nat. Prod. 2002; 65: 1769-1772Crossref PubMed Scopus (64) Google Scholar, 9Kawagishi H. Ishiyama D. Dictyophorines A and B, two stimulators of NGF-synthesis from the mushroom Dictyophora indusiata.Phytochemistry. 1997; 45: 1203-1205Crossref PubMed Scopus (36) Google Scholar) have been found to be active toward neurons. The biological activities of various mushroom components have been studied. For example, the exopolysaccharide from the culture broth of Hericium erinaceus was shown to enhance the growth and neuronal differentiation of PC12 cells (10Park Y.S. Lee H.S. Lee J.H. Lee S.Y. Lee H.Y. Effect of an exo-polysaccharide from the culture broth of Hericium erinaceus on enhancement of growth and differentiation of rat adrenal nerve cells.Cytotechnology. 2002; 39: 155-162Crossref PubMed Scopus (48) Google Scholar). The apoptosis of PC12 cells was evoked by the culture broth of Lentinula edodes (11Han B. Shinozawa T. Induction of apoptosis in cultured cells by extracts from shiitake (Lentinula edodes) mycelial culture broth.Mycoscience. 2000; 41: 623-631Crossref Scopus (2) Google Scholar), and the apoptosis of U937 cells was evoked by a lectin isolated from the mushroom Boletopsis leucomelas (12Koyama Y. Katsuno Y. Miyoshi N. Hayakawa S. Mita T. Isemura M. Apoptosis induction by lectin isolated from the mushroom Boletopsis leucomelas in U937 cells.Biosci. Biotechnol. Biochem. 2002; 66: 784-789Crossref PubMed Google Scholar). Grifola frondosa (Maitake mushroom), one of the most widely grown mushrooms in Gunma Prefecture of Japan, was selected as the source for isolation of active substances in this study because we were particularly interested in the activity of natural components toward neuronal cells. We evaluated the activity of mushroom extracts for the induction of phosphorylation of mitogen-activated protein kinase (MAPK) in cultured rat pheochromocytoma PC12 cells, because the Raf (MAPK kinase kinase)/MEK1/2 (MAPK kinases)/extracellular signal-regulated protein kinases (ERK1/2) (MAPKs, extracellular signal-regulated protein kinases) pathway has been well studied, and ERK1/2 activity serves as one of the checkpoints controlling cellular differentiation and proliferation. As a result, we identified lysophosphatidylethanolamine (LPE) as an active substance that induced the phosphorylation of ERK1/2, the activation of which was followed by inhibition of serum deprivation-induced apoptosis and induction of neuronal differentiation in cultured PC12 cells. G. frondosa (Maitake mushroom) powder was a gift from Yukiguni Maitake Co., Ltd. (Niigata, Japan). Ethyl acetate, n-hexane, chloroform, methanol, acetic acid, NaF, NaCl, and sodium deoxycholate were obtained from Kanto Chemical Co., Inc. (Tokyo, Japan). PMSF, Nonidet P-40, aprotinin, and agarose (type I, low electroendsosis) were purchased from Sigma (St. Louis, MO). BSA was obtained from Serologicals Corp. (Norcross, GA). SDS, Na3VO4, leupeptin, and an inhibitor for the Trk-type tyrosine kinase (K252a) were purchased from Wako Pure Chemical Industries (Osaka, Japan). RNase and a DNA size marker (100 bp ladder) came from MoBiTec (Goettingen, Germany) and Promega Corp. (Madison, WI), respectively. MEK inhibitors (PD98059 and U0126) were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Specific inhibitors for epidermal growth factor receptor (EGFR) tyrosine kinase (AG1478) and a Gi/o protein pertussis toxin (PTX) were from Calbiochem (San Diego, CA) and List Biological Laboratories, Inc. (Campbell, CA), respectively. Powdered G. frondosa (100 g) was immersed in 500 ml of a chloroform-methanol mixture (2:1, v/v) for 24 h at room temperature. The solvent containing the extracts were filtered through a filter paper (5C; Whatman, Blentford, UK), and the filtrate was evaporated to dryness. The solid extract (2.0 g) was then stirred in 10 ml of n-hexane at room temperature for 1 h, after which the mixture was centrifuged at 1,500 g at room temperature for 10 min. The supernatant was collected and evaporated to dryness. The sample was suspended in 10 ml of ethyl acetate, and the precipitate was then sequentially extracted, first with chloroform and then with methanol. The material that was obtained after the methanol extract had been evaporated to dryness was called the residue. The fraction that remained insoluble after each extraction was dissolved in chloroform-methanol (2:1, v/v) at a concentration of 100 mg/ml. An aliquot of the chloroform-methanol solution was injected into a HPLC apparatus equipped with a Develosil 60-10 (for normal phase) or Develosil ODS 60-10 (for reverse phase) column (20 × 250 mm, 10 μm particle size; Nomura Chemical Co., Ltd., Aichi, Japan), a PU-800 pump (JASCO, Tokyo, Japan), and an evaporative light-scattering detector (SEDEX model 75; Sedere, Inc., Cranbury, NJ). The column was run with hexane-ethyl acetate (80:20, v/v) for the normal phase or methanol for the reverse phase at a flow rate of 10 ml/min. Eluted substances were manually collected by referring to the signal detected by the evaporative light-scattering detector and subjected to TLC analysis using silica-gel 60 (Merck KgaA) with a developing solvent of chloroform-methanol-acetic acid-water (25:15:4:2, v/v/v/v). Detection was performed using sulfuric acid for total organisms, anthrone reagent for glycolipids, iron chloride reagent for sterols, Ditter’s reagent for phosphate, and ninhydrin reagent for amino bases. Components of each detection reagent were reported previously (13Daveloose D. Viret J. Simultaneous changes in lipid composition, fluidity and enzyme activity in piglet intestinal brush border membrane as affected by dietary polyunsaturated fatty acid deficiency.Biochim. Biophys. Acta. 1993; 1166: 229-237Crossref PubMed Scopus (37) Google Scholar, 14Ludmir J. Alvarez J.G. Increased levels of granulocyte-specific glycosphingolipids in preterm labor amniotic fluid.J. Liq. Chromatogr. 1993; 16: 1685-1693Crossref Scopus (4) Google Scholar, 15Entezami A.A. Venables B.J. Daugherty K.E. Analysis of lipids by one-dimensional thin-layer chromatography.J. Chromatogr. 1987; 387: 323-331Crossref PubMed Scopus (11) Google Scholar). PC12 cells were cultured as described previously (16Ito H. Nomoto H. Furukawa S. Role of low-affinity p75 receptor in nerve growth factor-inducible growth arrest of PC12 cells.J. Neurosci. Res. 2002; 69: 653-661Crossref PubMed Scopus (12) Google Scholar). In brief, the cells were maintained in DMEM (Sigma) supplemented with 10% heat-inactivated horse serum (Gibco BRL, Grand Island, NY) and 5% heat-inactivated FBS (Sanko Junyaku, Co., Ltd., Tokyo, Japan) or in serum-free medium (DMEM supplemented with 1% BSA), unless stated otherwise. All G. frondosa samples, such as the chloroform-methanol extract, fractions obtained by HPLC, and reagents, were prepared in serum-free DMEM and sonicated until fully emulsified. PC12 cells were seeded at a cell density of 2 × 106 cells/well into collagen-coated six-well plates containing medium with serum and precultured for 2 days at 37°C in an atmosphere of 95% air and 5% CO2. The cells were then washed with PBS and incubated with the above-mentioned culture medium containing a given sample from G. frondosa or various agents for 10 min at 37°C. Then, the culture plates were placed on ice, and each well was washed with 3 ml of 2 mM TBS containing 0.33 M NaF and 6.25 M Na3VO4 and subsequently lysed with 150 μl of 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40 (w/v), 1% sodium deoxycholate (w/v), 0.1% SDS (w/v), 50 mM NaF, 0.1% aprotinin (w/v), 0.1% leupeptin (w/v), 1 mM Na3VO4, and 1 mM PMSF. Cell lysates were collected using a cell scraper, transferred to 1.5 ml microcentrifuge tubes, and centrifuged at 15,000 g for 30 min at 4°C. The supernatant was collected and transferred to another tube, and the overall protein concentration was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL) with BSA as the standard. HPLC-time of flight MS was performed using a micro-time of flight focus (Bruker Daltonik GmbH, Bremen, Germany) equipped with a Develosil ODS 60-5 column (4.5 × 250 mm, 5 μm particle size; Nomura Chemical), and the active components were eluted by methanol. Infrared spectra were recorded with a Nicolet Magna-500 infrared spectrometer. 1H-NMR spectra were recorded with a JEOL AL-300 spectrometer, with chemical shifts being reported on the δ scale in ppm relative to Me4Si. 13C-NMR, 1H-1H total correlation spectroscopy, 1H-13C heteronuclear single quantum correlation, and 1H-13C 1H-detected heteronuclear multiple bond connectivity spectra were recorded with a Bruker AV-400 spectrometer. Proteins (20 μg) in each supernatant were mixed with SDS sample buffer and incubated for 5 min at 80°C. Protein samples were separated on SDS-polyacrylamide gels and electroblotted onto polyvinylidene difluoride filters (Fluorotrans membrane W, 0.2 μm; Nihon Genetics, Tokyo, Japan). Immunoblotting analysis was performed using monoclonal antibodies against p44/42 ERK, phospho p44/42 ERK (Cell Signaling Technology, Lake Placid, NY), and neurofilament M (NF-M; Sigma) as primary antibodies, followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega) as the secondary antibody. The blots were developed by the enhanced chemiluminescence method (Hyperfilm-ECL plus; Amersham Biosciences Corp., Piscataway, NJ). Each inhibitor was added to serum-containing medium to result in a final concentration of 1 μM (K252a, PTX, and AG1478), 50 μM (U0126), or 200 μM (PD98059). Cells were preincubated with each inhibitor for 20 h (PTX) or 4 h (all others) and cultured in the serum-containing medium supplemented with each extract or reagent for the appropriate times. The cells were then collected and subjected to analysis of phosphorylation or expression of proteins as described above. Cytotoxicity of U0126 or PD98059 was measured by MTT assay (17Nakamura Y. Yasuda M. Fujimori H. Kiyono M. Pan-Hou H. Cytotoxic effect of sodium nitroprusside on PC12 cells.Chemosphere. 1997; 34: 317-324Crossref PubMed Scopus (20) Google Scholar). PC12 cells maintained for 2 days on six-well plates (2 × 106 cells/well) at 37°C in an atmosphere containing 5% CO2 were washed with PBS and incubated in serum-free medium for 0, 1, 2, or 8 days. Then, the cells were collected and centrifuged at 500 g for 5 min. DNA was extracted from the cells using a QIAamp DNA mini kit (Qiagen, Valencia, CA). Fragmentation of DNA was confirmed on 3% agarose gels after electrophoresis and staining with ethidium bromide for visualization under ultraviolet transillumination. PC12 cells plated on poly-d-lysine-coated cover glasses (13 × 13 mm; Matsunami Glass Industries, Ltd., Osaka, Japan) were precultured in serum-containing medium for 24 h, washed with PBS, and incubated with the same medium containing mushroom extract or agent for the appropriate period. Cells were fixed with paraformaldehyde solution (4%, w/v) and incubated for 20 min in PBS containing Triton X-100 (0.3%, v/v). Nonspecific binding was blocked with Block Ace solution (2%, v/v; Dainippon Pharmaceutical Co., Ltd., Osaka, Japan). Cells were treated with anti-NF-M monoclonal antibody (Sigma) and then reacted with Alexa Fluor 488-conjugated anti-mouse IgG antibody (Molecular Probes, Eugene, OR). The stained samples were mounted on slide glasses by use of Tissue-Tek (Sakura Finetechnical Co., Ltd., Tokyo, Japan) and observed with a confocal laser scanning microscope (Radiance 2100; Bio-Rad, Hercules, CA). Activation of ERK1/2 is one of the checkpoints to assess the activation of the classical Ras/MAPK cascade (18Sturgill T.W. Ray L.B. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II.Nature. 1988; 334: 715-718Crossref PubMed Google Scholar), which is triggered by an engaged tyrosine kinase receptor or G protein-coupled receptor and results in proliferation and/or differentiation. Therefore, we tested whether the G. frondosa contained something that could activate this signal pathway. The whole extract obtained from 100 g of dried G. frondosa was fractionated with various solvents, and the extracts obtained by use of n-hexane (1.39 g), ethyl acetate (0.04 g), chloroform (0.02 g), and methanol (0.34 g) as well as the residue (0.11 g) were evaluated for their ability to induce the activation of ERK1/2 (Fig. 1A). The extracts obtained with low-polarity solvents showed stronger activity, as the amounts of the n-hexane and ethyl acetate extracts were 70 and 35 times, respectively, greater than those in the chloroform extract. Therefore, we chose the n-hexane extract as the starting material for the isolation of the putative active component. At first, the n-hexane extract was fractionated by preparative normal-phase HPLC, and the fraction with potent activity (indicated by the arrow in Fig. 1B) was subjected to reverse-phase HPLC (resulting in fraction E in Fig. 1C). Fraction E of the second HPLC with the activity was further analyzed by TLC. After development, the TLC plate was stained with sulfuric acid (for detection of all organic compounds), Dittmer’s reagent (for detection of phosphorus compounds), or ninhydrin (for detection of amino-containing compounds). The results showed that the active component was an amino group-containing phospholipid. The relative mobility value of the active components was in good agreement with that of LPE (data not shown). Infrared spectra of the active component showed the characteristic peak at 1,730.5 cm−1 for carbonyl stretching of the ester as well as a broad band at ∼3,000 cm−1 attributable to the primary amine salt (N-H stretching) along with C-H stretching peaks of alkanes. 1H-NMR spectra of the active component showed signals at δ = 3.08 ppm (2H) and 3.98 ppm (2H; protons of two methylenes of ethanolamine moiety) and δ = 3.82 ppm (2H), 3.90 ppm (1H), and 4.06 ppm (2H; protons of the glycerol moiety). 13C-NMR peaks of four carbons (both of the ethanolamine carbons and two of the three glycerol carbons) were split in two as a result of the spin coupling with 31P. 1H-NMR, infrared analysis, and mass spectra of synthetic 1-myristoyl-LPE, 1-palmitoyl-LPE, and 1-oleoyl-LPE were similar to those of the active component from G. frondosa (data not shown). As shown in Fig. 2, the tentative LPE concentrations calculated from the area of the total ion chromatogram were 0.8% for 1-myristoyl-LPE, 1.9% for 1-margaroyl-LPE, 4.1% for 1-palmitoyl-LPE, 3.2% for 1-palmitoleoyl-LPE, 39.1% for 1-oleoyl-LPE, 49.3% for 1-linoleoyl-LPE, and 1.6% for 1-linolenoyl-LPE. These results confirmed that the compound from G. frondosa that induced ERK1/2 activation in PC12 cells was LPE. Moreover, synthetic 1-myristoyl-LPE, 1-palmitoyl-LPE, and 1-oleoyl-LPE activated ERK1/2 of PC12 cells equally to GLPE (Fig. 3).Fig. 2Confirmation of fatty acids bound to lysophosphatidylethanolamine (LPE) obtained from G. frondosa. A: Pattern of total ion chromatography of fraction E shown in Fig. 1C. B: Exact mass measurements of peaks obtained by total ion chromatography. MW, molecular weight. C: Composition of fatty acids bound to LPE of G. frondosa (GLPE) calculated from peak areas on the total ion chromatogram (TIC).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Concentration-dependent phosphorylation of ERK1/2 of PC12 cells by GLPE, synthetic LPE, and lysophosphatidic acid (LPA). The concentrations indicated for each sample are final concentrations in the culture medium. Cells were treated with each sample for 30 min, and the amounts of phosphorylated ERK1/2 were visualized by Western immunoblotting. Data are normalized to the level of total ERK and expressed as means ± SEM of three separate experiments. Significant differences from the value of the corresponding control group were determined by Student’s t-test and are indicated at bottom as follows: a, P < 0.05; b, P < 0.01; c, P < 0.005.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GLPE was further fractionated by reverse-phase HPLC, and 90.8% of 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine obtained was used for the characterization of GLPE. Next, the effect of GLPE on the phosphorylation of ERK1/2 was examined, and we found that GLPE induced the phosphorylation of ERK1/2 in a dose-dependent manner from 200 to 2,000 μM (Fig. 3). It is known that nerve growth factor (NGF) induces the activation of MEK1/2, a MAPK kinase, and phosphorylates ERK1/2 (19Liu Y.Z. Latchman D.S. Activation of the Bcl-2 promoter by nerve growth factor is mediated by the p42/p44 MAPK cascade.Nucleic Acids Res. 1999; 27: 2086-2090Crossref PubMed Scopus (97) Google Scholar). Therefore, the effects of MEK inhibitors (U0126 and PD98059) on the induction of phosphorylation of ERK1/2 by NGF or GLPE were evaluated (Fig. 4). The induction of activation of ERK1/2 by both NGF and GLPE was similarly inhibited by both inhibitors; that is, it was suppressed greatly by U0126 and slightly by PD98059. Therefore, it was obvious that GLPE, as well as NGF, induces the activation of MEK1/2, resulting in the activation/phosphorylation of ERK1/2. The cytotoxicity of PD98059 or U0126 for PC12 cells was checked by MTT assay, because they were used at relatively high concentrations in this study. No toxicity was detected, even at the highest concentration shown in Fig. 4 (data not shown). The effects of K252a (an inhibitor of the tyrosine kinase of the high-affinity NGF receptor Trk) and AG1478 (an inhibitor of the tyrosine kinase of the EGFR) on GLPE-, NGF-, and EGF-induced phosphorylation of ERK1/2 of PC12 cells were examined next, as was the effect of PTX (one of the Gi/o inhibitors) on GLPE-induced phosphorylation (Fig. 5). K252a and AG1478 suppressed the induction of phosphorylation by NGF and EGF, respectively. The phosphorylation of ERK1/2 induced by GLPE was suppressed by AG1478, but not by K252a or PTX, suggesting that GLPE induced the phosphorylation of ERK1/2 via activation of the EGFR tyrosine kinase but not via the NGF receptor or PTX-sensitive G protein-coupled receptor (GPCR) (Gi/o). As NGF is known to prevent PC12 cells from undergoing apoptosis induced by serum deprivation (20Lindenboim L. Stein R. Apoptosis induced by serum deprivation of PC12 cells is not preceded by growth arrest and can occur at each phase of the cell cycle.Cancer Res. 1995; 55: 1242-1247PubMed Google Scholar), such activity of GLPE was investigated. PC12 cells were plated on collagen-coated 12-multiwell plates at a cell density of 104/well and cultured for 2 days in medium supplemented with 10% horse serum and 5% FBS. The medium was then exchanged for serum-free medium containing 1% BSA and various concentrations of GLPE or NGF, and the cells were cultured for another 2 days. A substantial number of cells underwent apoptosis when the cells were cultured in control medium containing neither NGF nor GLPE, as judged from the cell body condensation and DNA ladder formation (Fig. 6). However, most PC12 cells survived and generated neurites in the presence of NGF or GLPE. These results demonstrate that GLPE suppressed the apoptosis of PC12 cells induced by serum deprivation as efficiently as NGF. PC12 cells were plated on poly-d-lysine-coated 12-multiwell plates at a cell density of 2 × 106 or 1 × 104 cells/well for immunoblot analysis or morphological observation, respectively, and cultured for 2 days in the presence of NGF or EGF at 50 ng/ml or GLPE at 200–1,000 μM. Expression of NF-M protein of each culture was analyzed by immunostaining of the cells (Fig. 7) and by Western immunoblotting (Fig. 8). From the results of the immuostaining, the expression intensity of NF-M protein and the percentage of NF-M-positive cells increased when the cells vigorously extended neurites 5 days after the addition of NGF. However, neither EGF nor vehicle treatment (control) had any effects. GLPE significantly increased NF-M expression in PC12 cells at 2 days, but the expression became reduced by 5 days. Neurite outgrowth by GLPE was weaker than that by NGF.Fig. 8Expression of NF-M in PC12 cells in serum-containing medium supplemented with U0126, GLPE, EGF, or NGF. PC12 cells were plated on poly-d-lysine-coated six-multiwell plates at 104 cells/well and cultured in serum-containing medium for 2 days. The medium was then changed to serum-containing medium supplemented with (B) or without (A, C) U0126 (50 nM) and NGF (50 ng/ml), EGF (50 ng/ml), or GLPE (200–1,000 μM), and the cells were cultured for another 2 or 5 days. The cells were collected, and the amount of NF-M expression was measured by immunoblotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The results of the immunoblot analysis (Fig. 8) revealed that the addition of NGF increased the expression level of NF-M protein but that EGF or vehicle (control) treatment was not efficient. On the other hand, different from the immunostaining results, GLPE at 1,000 μM was necessary for detectable expression of NF-M. Comparing the results of Figs. 7, 8, we suspect that cell density might affect the efficient dose of GLPE for NF-M expression. NF-M expression induced by NGF (Fig. 8B) and neurite outgrowth (Fig. 9) were markedly inhibited by U0126. Similarly, the neurite outgrowth and NF-M expression induced by GLPE were obstructed by U0126. From these results, we propose that GLPE induced neurotrophic effects on PC12 cells via induction of MEK1/2 activation, because phosphorylation of ERK1/2 in PC12 cells is known to be caused predominantly by the activation of MEK1/2 (21Kleijn M. Proud C.G. The activation of eukaryotic initiation factor (eIF)2B by growth factors in PC12 cells requires MEK/ERK signalling.FEBS Lett. 2000; 476: 262-265Crossref PubMed Scopus (18) Google Scholar). The results described above show that GLPE elicited the phosphorylation of ERK1/2 via the EGFR (Fig. 5) but weakly affected the proliferation of PC12 cells in a way different from EGF. Additionally, GLPE had neurotrophic effects (i.e., it induced neuronal differentiation of PC12 cells like NGF). However, the induction of NF-M expression and neurite outgrowth by GLPE was weaker than that by NGF. A well-known neurotrophic factor, NGF, acts on cultured PC12 cells and induces many processes, including neurite outgrowth for their neuronal differentiation into sympathetic neuron-like cells. These NGF actions require both phosphorylation of the NGF receptor, TrkA, expressed on the cell surface, as a trigger and subsequent activation of the MAPK cascade (22Kimmelman A.C. Rodriguez N.N. Chan A.M.L. R-Ras3/M-Ras induces neuronal differentiation of PC12 cells through cell-type-specific activation of the mitogen-activated protein kinase cascade.Mol. Cell. Biol. 2002; 22: 5946-5961Crossref PubMed Scopus (46) Google Scholar). Based on these facts, extracts of G. frondosa were tested and found to induce ERK1/2 phosphorylation of PC12 cells as efficiently as NGF. An active component was isolated from the extracts and identified as LPE. To date, the pharmacological effects of LPE are unclear; although phosphatidylethanolamine is a common cell membrane component, and LPE is easily derived from phosphatidylethanolamine by deacylation by phospholipase A2. The LPE concentration necessary to induce the activation of ERK1/2 of PC12 cells was almost 200 μM in our assay system. On the other hand, Howe and Marshall (23Howe L.R. Marshall C.J. Lysophosphatidic acid stimulates mitogen-activated protein kinase activation vi" @default.
• W2159362912 created "2016-06-24" @default.
• W2159362912 creator A5011759251 @default.
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• W2159362912 date "2006-07-01" @default.
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• W2159362912 title "Lysophosphatidylethanolamine in Grifola frondosa as a neurotrophic activator via activation of MAPK" @default.
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