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The mitogen-activated protein kinase cascade mediates neurotrophic effect of epidermal growth factor in cultured rat hippocampal neurons
Neuroscience Letters 282 (2000) 89±92 www.elsevier.com/locate/neulet
The mitogen-activated protein kinase cascade mediates neurotrophic effect of epidermal growth factor in cultured rat hippocampal neurons Kazuho Abe*, Hiroshi Saito Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan Received 17 December 1999; received in revised form 27 January 2000; accepted 27 January 2000
Abstract Epidermal growth factor (EGF) has been reported to support the survival of cultured brain neurons. In the present study, we investigated whether the neurotrophic effect of EGF is mediated by the mitogen-activated protein kinase (MAPK) cascade in cultured rat hippocampal neurons. Recombinant human EGF (0.1±10 ng/ml) induced phosphorylation of p44/42 MAPK (ERK1/2) in a concentration-and time-dependent manner. EGF-induced ERK1/2 phosphorylation and promotion of neuronal survival were both blocked by U0126 and PD98059, inhibitors of the MAPK-activating enzyme MEK. These results suggest that the MEK/ERK signal transduction cascade is involved in the neurotrophic effect of EGF. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Epidermal growth factor; Tyrosine kinase; Mitogen-activated protein kinase; Neuronal survival; Rat hippocampal neurons; Culture
Epidermal growth factor (EGF) is a single-chain polypeptide composed of 53 amino acids and well-known as a potent mitogen for a variety of cell types [7]. In addition, it has been demonstrated that EGF promotes the survival of primary cultured neurons from various brain regions [2,3,14,15]. Since EGF and its receptors are present in the brain neurons [9,11,21], it is possible that EGF functions as a neurotrophic factor in the brain. However, very little is known about the molecular mechanism underlying the neurotrophic effect of EGF. Among the mitogen-activated protein kinases (MAPKs), p44 MAPK (44 kDa; extracellular signal-regulated kinase 1; ERK1) and p42 MAPK (42 kDa; ERK2) play a pivotal role in mediation of cellular responses to a variety of signaling molecules [13,17,18]. The EGF receptor tyrosine kinase is known to be linked to the G-protein Ras, which stimulates the MAPK signal transduction cascade. Ras activates MAPK kinase kinases, which phosphorylate and activate MAPK kinases (MAPK/ERK kinase; MEK), which in turn phosphorylate and activate ERK1/2. The activated ERK1/2 translocates into the nucleus and plays a role in the regulation of gene transcription. The present study was undertaken * Corresponding author. Tel.: 181-3-5841-4781; fax: 181-35841-4786. E-mail address: [email protected] (K. Abe)
to determine if this signal transduction cascade is required for the neurotrophic effect of EGF. Recombinant human EGF (a generous gift from Wakunaga Pharmaceutical Co., Ltd.) was dissolved to a concentration of 10 mg/ml in phosphate-buffered saline (PBS) supplemented with 1 mg/ml bovine serum albumin, and stored at 2208C until use. Primary cultures of hippocampal neurons were prepared from 18-day-old embryos of Wistar rats as described previously [4]. Brie¯y, dissociated hippocampal cells were suspended in a modi®ed Eagle's medium containing N1 supplements (5 mg/ml human transferrin, 5 mg/ml bovine insulin, 20 nM progesterone, 100 mM putrescine and 30 nM sodium selenite) and seeded on polylysine-coated 48-well culture plates at a density of 100 000 cells/cm 2. No mitotic inhibitors were required in these minimal conditions, and .95% of the cells were neuronal as de®ned by staining with microtubule-associated protein-2. The culture medium was changed to a fresh medium 2 days after the plating, and the cells were cultured for a further 4 days. During this period neurons acquired long and extensively branched neurites. Six days after the plating, the medium was changed again. Tucker et al. [20] previously reported that ERK1/2 was activated by treatment with EGF for 3 or 5 min in cultured rat hippocampal neurons. To further characterize EGF-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 86 7- 3
90
K. Abe, H. Saito / Neuroscience Letters 282 (2000) 89±92
induced phosphorylation of ERK1/2, we performed Western blot analysis with two different antibodies. Anti-ERK1/2 antibody (Promega Co., Madison, WI, USA) recognizes both inactive and active forms of ERK1/2, and indicates the expression level of total ERK1/2. On the other hand, anti-phospho ERK1/2 antibody (Promega Co., Madison, WI, USA) recognizes phosphorylated ERK1/2 only, and indicates the activation of ERK1/2. Since the neuronal survival was stable up to 6 days after the plating, the cells at day 6 in culture were exposed to 0.1±10 ng/ml EGF for 5 min±12 h. The cells were rinsed with ice-cold PBS and solubilized in sample buffer. The samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer onto a polyvinylidine ¯uoride membrane ®lter at 1 mA/cm 2 for 60 min at room temperature. The ®lters were incubated in PBS containing 0.5% Tween 20 (PBS-T) and 5% non-fat milk for 1 h at room temperature and then with anti-ERK1/2 antibody (1:5000 dilution in PBS-T) or anti-phospho ERK1/2 antibody (1:10 000 dilution in PBS-T) overnight at 48C. The
Fig. 2. In¯uences of signal transduction inhibitors on EGFinduced ERK1/2 phosphorylation in cultured rat hippocampal neurons. The cells were pretreated with 0.1±10 mM AG1478 (A;K, O,), 0.1±10 mM U0126 (B;A, B,) or 1±30 mM PD98059 (B;L, P,) for 20 min, and then exposed to 5 ng/ml EGF in the absence (W,X) or presence of the inhibitors for 30 min. The cell extract was subjected to Western blot analysis. The level of phosphorylated ERK1 (white symbols) or phosphorylated ERK2 (black symbols) was expressed by taking the control level (none) as 1. Data are means ^ SEM (n 4).
Fig. 1. Effect of EGF on total or phosphorylated ERK1/2 level in cultured rat hippocampal neurons. (A) Concentration-dependency. The cells were exposed to 0.1±10 ng/ml EGF for 30 min, and the cell extract was subjected to Western blot analysis with anti-ERK1/2 antibody or anti-phospho ERK1/2 (pERK1/2) antibody. Upper panel: representative blots. Lower panel: quantitative data. The level of phosphorylated ERK1 (W) or phosphorylated ERK2 (X) is expressed by taking the control level (none) as 1. (B) Time-course. The cells were exposed to 5 ng/ml EGF for 5, 15, 30 min, 1, 3, 5, 8 or 12 h. The level of phosphorylated ERK1 (W) or phosphorylated ERK2 (X) was expressed by taking the basal level (0 min) as 1. All data are means ^ SEM (n 4).
®lters were washed in PBS-T (3 £ 10 min) at room temperature and further incubated with horseradish peroxidaseconjugated anti-rabbit IgG (1:5000 dilution in PBS-T) for 1 h at room temperature. The blots were washed in PBS-T (3 £ 10 min), and immunoreactive proteins were visualized on a ®lm with an enhanced chemiluminescence kit (NEN Life Science Products, Inc., Boston, MA, USA). Optical density on the ®lm was measured with a computer imaging system (Imaging Technology Inc., Ontario, Canada), and protein level was estimated from the standard curve, which was constructed using several dilutions of samples in each analysis. As shown in Fig. 1A, addition of EGF induced phosphorylation of both ERK1 and ERK2, with no change in total ERK1/2. The effect of EGF was concentration dependent in the range of 0.1±10 ng/ml. The EGFinduced ERK1/2 phosphorylation rapidly occurred within 5 min and peaked 30 min±1 h after (Fig. 1B). The EGFinduced ERK1 phosphorylation and ERK2 phosphorylation were both blocked by the EGF receptor tyrosine kinase inhibitor AG1478 [16] and by the MEK inhibitors, U0126 [10] and PD98059 [8] (Fig. 2). To examine if these signals are required for the neurotrophic effect of EGF, we investigated the effects of
K. Abe, H. Saito / Neuroscience Letters 282 (2000) 89±92
AG1478, U0126 and PD98059 on neuronal survival. When the culture period was prolonged, neuronal survival was gradually decreased. The number of surviving neurons at day 9 in culture was decreased to approximately 40% of that at day 6. Thus, EGF and inhibitors were added at day 6 in culture, and the cultures were ®xed at day 9. The number of surviving neurons was counted under a microscope as described previously [1]. Consistent with our previous observation [2], addition of EGF (0.1±10 ng/ml) promoted the survival of hippocampal neurons in a concentration-dependent manner (data not shown, n 5). The EGF receptor tyrosine kinase inhibitor AG1478 at 1 mM had no effect on neuronal survival in the absence of EGF, but signi®cantly blocked the survival-promoting effect of EGF (Fig. 3A). A higher concentration (10 mM) of AG1478 showed toxicity by itself. The MEK inhibitors, U0126 (0.1±10 mM) and PD98059 (1±30 mM), blocked the survival-promoting effect of EGF, without affecting neuronal survival in the absence of EGF (Fig. 3A). It has previously been reported that phosphatidylinositol 3-kinase and protein kinase C are required for the anti-apoptotic effect of EGF in an epithelial tumor cell line [12]. Thus, we also investigated the effects of LY294002, a speci®c phosphatidylinositol 3-kinase inhibitor, and GF109203X, a speci®c protein kinase C inhibitor, on neuronal survival in our cultures. However, the survival-promoting effect of EGF was not affected by these inhibitors (Fig. 3B). We have previously con®rmed that GF-109203X at 0.1 mM completely blocks phorbol ester-induced morphological changes in cultured astrocytes [6]. Phosphatidylinositol 3kinase and protein kinase C are unlikely to be involved in the survival-promoting effect of EGF in cultured rat hippocampal neurons. Signal transduction mechanisms underly-
91
ing the effect of EGF on cell survival may be different with types of death or cells. EGF is known to stimulate the proliferation of astroglial cells. To test the possibility that EGF affects neuronal survival by increasing the number of astroglial cells, we performed immunostaining with monoclonal antibodies to glial ®brillary acidic protein (GFAP), a marker protein for astroglial cells, as described previously [5]. However, the percentage of GFAP-positive cells was less than 5% both in control cultures and in cultures treated with 5 ng/ml EGF for 3 days (data not shown, n 5). Furthermore, Western blot analysis with anti-GFAP antibody demonstrated that GFAP level was negligibly low in control and EGF-treated cultures (data not shown, n 4). For comparison, pure astrocyte cultures were prepared from the cerebral cortex of neonatal rats, as described previously [5]. GFAP level per mg protein in our neuronal cultures was estimated less than 2% of that in pure astrocyte cultures. Therefore, the effect of EGF observed in the present study is unlikely to be mediated by astroglial cells. The main ®ndings in the present study were: (1) EGF induced ERK1/2 phosphorylation without affecting ERK1/2 expression; (2) EGF-induced ERK1/2 phosphorylation was blocked by the tyrosine kinase inhibitor AG1478 and MEK inhibitors, U0126 and PD98059; and (3) these inhibitors blocked survival-promoting effect of EGF. Since ERK1/2 phosphorylation rapidly occurred within 5 min after addition of EGF, the response is probably due to a direct action of EGF on neurons. Furthermore, the effective concentration of EGF in inducing ERK1/2 phosphorylation (Fig. 1) was consistent with that in promoting neuronal survival [2]. The effective concentrations of AG1478, U0126 and PD98059 in blocking the survival-promoting effect of EGF (Fig. 3A) were very
Fig. 3. In¯uences of signal transduction inhibitors on survival-promoting effect of EGF in cultured rat hippocampal neurons. The cells were pretreated with 0.1±10 mM AG1478 (A), 0.1±10 mM U0126 (A), 1±30 mM PD98059 (A), 0.1±10 mM LY294002 (B) or 0.01±1 mM GF109203X (B) for 20 min, and then exposed to 5 ng/ml EGF in the absence or presence of the inhibitors for 3 days. The number of surviving neurons was counted under a microscope. Data are means ^ SEM (n 5). **P , 0:01 vs. cont/-EGF; #P , 0:05, ##P , 0:01 vs. EGF alone; Duncan's multiple range test.
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K. Abe, H. Saito / Neuroscience Letters 282 (2000) 89±92
consistent with those in blocking EGF-induced ERK1/2 phosphorylation (Fig. 2A,B). These results suggest that EGF promotes neuronal survival through ERK1/2 phosphorylation, which is regulated by the EGF receptor tyrosine kinase and MEK. Phosphorylated ERK1/2 is known to translocate into the nucleus and elicit changes in patterns of gene expression, including the induction of the immediate-early genes such as c-fos [19]. Further investigations to identify targets of phosphorylated ERK1/2 will give clues for understanding molecular mechanisms underlying neurotrophic effect of EGF. [1] Abe, K., Takayanagi, M. and Saito, H., Effects of recombinant human basic ®broblast growth factor and its modi®ed protein CS23 on survival of primary cultured neurons from various regions of fetal rat brain. Jpn. J. Pharmacol., 53 (1990) 221±227. [2] Abe, K., Takayanagi, M. and Saito, H., A comparison of neurotrophic effects of epidermal growth factor and basic ®broblast growth factor in primary cultured neurons from various regions of fetal rat brain. Jpn. J. Pharmacol., 54 (1990) 45±51. [3] Abe, K., Takayanagi, M. and Saito, H., Basic ®broblast growth factor and epidermal growth factor promote survival of primary cultured cerebellar neurons from neonatal rats. Jpn. J. Pharmacol., 56 (1990) 113±116. [4] Abe, K. and Kimura, H., Amyloid b toxicity consists of a Ca 21-independent early phase and a Ca 21-dependent late phase. J. Neurochem., 67 (1996) 2074±2078. [5] Abe, K. and Saito, H., Adenosine stimulates stellation of cultured rat cortical astrocytes. Brain Res., 804 (1998) 63±71. [6] Abe, K. and Saito, H., The p44/42 mitogen-activated protein kinase cascade is involved in the induction and maintenance of astrocyte stellation mediated by protein kinase C. Neurosci. Res., 36 (2000) 251±257. [7] Carpenter, G. and Cohen, S., Epidermal growth factor. Annu. Rev. Biochem., 48 (1979) 193±216. [8] Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. and Saltiel, A.R., A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA, 92 (1995) 7686± 7689. [9] Fallon, J.H., Seroogy, K.B., Loughlin, S.E., Morrison, R.S., Bradshaw, R.A., Knauer, D.J. and Cunningham, D.D., Epidermal growth factor immunoreactive material in the
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[21]
central nervous system: location and development. Science, 224 (1984) 1107±1109. Favata, M.F., Horiuchi, K.Y., Manos, E.J., Daulerio, A.J., Stradley, D.A., Feeser, W.S., Can Dyk, D.E., Pitts, W., Earl, R.A., Hobbs, F., Copeland, R.A., Magolda, R.L., Scherle, P.A. and Trzakos, J.M., Identi®cation of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem., 273 (1998) 18623±18632. Gomez-Pinilla, F., Knauer, D.J. and Nieto-Sampedoro, M., Epidermal growth factor immunoreactivity in rat brain: development and cellular localization. Brain Res., 438 (1988) 385±390. Lan, L. and Wong, N.S., Phosphatidylinositol 3-kinase and protein kinase C are required for the inhibition of caspase activity by epidermal growth factor. FEBS Lett., 444 (1999) 90±96. Lewis, T.S., Shapiro, P.S. and Ahn, N.G., Signal transduction through MAP kinase cascades. Adv. Cancer Res., 74 (1998) 49±139. Morrison, R.S., Kornblum, H.I., Leslie, F.M. and Bradshaw, R.A., Trophic stimulation of cultured neurons from neonatal rat brain by epidermal growth factor. Science, 238 (1987) 72±75. Morrison, R.S., Keating, R.F. and Moskal, J.R., Basic ®broblast growth factor and epidermal growth factor exert differential trophic effects on CNS neurons. J. Neurosci., 21 (1988) 71±79. Osherov, N. and Levitzki, A., Epidermal-growth-factordependent activation of the src-family kinases. Eur. J. Biochem., 225 (1994) 1047±1053. Seger, R. and Krebs, E.G., The MAPK signaling cascade. FASEB J., 9 (1995) 726±735. Sugden, P.H. and Clerk, A., Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors. Cell. Signal., 9 (1997) 337±351. Thomson, S., Mahadevan, L.C. and Clayton, A.L., MAP kinase-mediated signalling to nucleosomes and immediate-early genes. Semin. Cell Dev. Biol., 10 (1999) 205± 214. Tucker, M.S., Eves, E.M., Wainer, B.H. and Rosner, M.R., Activation of mitogen-activated protein kinase by epidermal growth factor in hippocampal neurons and neuronal cell lines. J. Neurochem., 61 (1993) 1376±1387. Werner, M.H., Nanney, L.B., Stoscheck, C.M. and King, L.E., Localization of immunoreactive epidermal growth factor receptor in human nervous system. J. Histochem. Cytochem., 36 (1988) 81±86.
The mitogen-activated protein kinase cascade mediates neurotrophic effect of epidermal growth factor in cultured rat hippocampal neurons Kazuho Abe*, Hiroshi Saito Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan Received 17 December 1999; received in revised form 27 January 2000; accepted 27 January 2000
Abstract Epidermal growth factor (EGF) has been reported to support the survival of cultured brain neurons. In the present study, we investigated whether the neurotrophic effect of EGF is mediated by the mitogen-activated protein kinase (MAPK) cascade in cultured rat hippocampal neurons. Recombinant human EGF (0.1±10 ng/ml) induced phosphorylation of p44/42 MAPK (ERK1/2) in a concentration-and time-dependent manner. EGF-induced ERK1/2 phosphorylation and promotion of neuronal survival were both blocked by U0126 and PD98059, inhibitors of the MAPK-activating enzyme MEK. These results suggest that the MEK/ERK signal transduction cascade is involved in the neurotrophic effect of EGF. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Epidermal growth factor; Tyrosine kinase; Mitogen-activated protein kinase; Neuronal survival; Rat hippocampal neurons; Culture
Epidermal growth factor (EGF) is a single-chain polypeptide composed of 53 amino acids and well-known as a potent mitogen for a variety of cell types [7]. In addition, it has been demonstrated that EGF promotes the survival of primary cultured neurons from various brain regions [2,3,14,15]. Since EGF and its receptors are present in the brain neurons [9,11,21], it is possible that EGF functions as a neurotrophic factor in the brain. However, very little is known about the molecular mechanism underlying the neurotrophic effect of EGF. Among the mitogen-activated protein kinases (MAPKs), p44 MAPK (44 kDa; extracellular signal-regulated kinase 1; ERK1) and p42 MAPK (42 kDa; ERK2) play a pivotal role in mediation of cellular responses to a variety of signaling molecules [13,17,18]. The EGF receptor tyrosine kinase is known to be linked to the G-protein Ras, which stimulates the MAPK signal transduction cascade. Ras activates MAPK kinase kinases, which phosphorylate and activate MAPK kinases (MAPK/ERK kinase; MEK), which in turn phosphorylate and activate ERK1/2. The activated ERK1/2 translocates into the nucleus and plays a role in the regulation of gene transcription. The present study was undertaken * Corresponding author. Tel.: 181-3-5841-4781; fax: 181-35841-4786. E-mail address: [email protected] (K. Abe)
to determine if this signal transduction cascade is required for the neurotrophic effect of EGF. Recombinant human EGF (a generous gift from Wakunaga Pharmaceutical Co., Ltd.) was dissolved to a concentration of 10 mg/ml in phosphate-buffered saline (PBS) supplemented with 1 mg/ml bovine serum albumin, and stored at 2208C until use. Primary cultures of hippocampal neurons were prepared from 18-day-old embryos of Wistar rats as described previously [4]. Brie¯y, dissociated hippocampal cells were suspended in a modi®ed Eagle's medium containing N1 supplements (5 mg/ml human transferrin, 5 mg/ml bovine insulin, 20 nM progesterone, 100 mM putrescine and 30 nM sodium selenite) and seeded on polylysine-coated 48-well culture plates at a density of 100 000 cells/cm 2. No mitotic inhibitors were required in these minimal conditions, and .95% of the cells were neuronal as de®ned by staining with microtubule-associated protein-2. The culture medium was changed to a fresh medium 2 days after the plating, and the cells were cultured for a further 4 days. During this period neurons acquired long and extensively branched neurites. Six days after the plating, the medium was changed again. Tucker et al. [20] previously reported that ERK1/2 was activated by treatment with EGF for 3 or 5 min in cultured rat hippocampal neurons. To further characterize EGF-
0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 0) 00 86 7- 3
90
K. Abe, H. Saito / Neuroscience Letters 282 (2000) 89±92
induced phosphorylation of ERK1/2, we performed Western blot analysis with two different antibodies. Anti-ERK1/2 antibody (Promega Co., Madison, WI, USA) recognizes both inactive and active forms of ERK1/2, and indicates the expression level of total ERK1/2. On the other hand, anti-phospho ERK1/2 antibody (Promega Co., Madison, WI, USA) recognizes phosphorylated ERK1/2 only, and indicates the activation of ERK1/2. Since the neuronal survival was stable up to 6 days after the plating, the cells at day 6 in culture were exposed to 0.1±10 ng/ml EGF for 5 min±12 h. The cells were rinsed with ice-cold PBS and solubilized in sample buffer. The samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer onto a polyvinylidine ¯uoride membrane ®lter at 1 mA/cm 2 for 60 min at room temperature. The ®lters were incubated in PBS containing 0.5% Tween 20 (PBS-T) and 5% non-fat milk for 1 h at room temperature and then with anti-ERK1/2 antibody (1:5000 dilution in PBS-T) or anti-phospho ERK1/2 antibody (1:10 000 dilution in PBS-T) overnight at 48C. The
Fig. 2. In¯uences of signal transduction inhibitors on EGFinduced ERK1/2 phosphorylation in cultured rat hippocampal neurons. The cells were pretreated with 0.1±10 mM AG1478 (A;K, O,), 0.1±10 mM U0126 (B;A, B,) or 1±30 mM PD98059 (B;L, P,) for 20 min, and then exposed to 5 ng/ml EGF in the absence (W,X) or presence of the inhibitors for 30 min. The cell extract was subjected to Western blot analysis. The level of phosphorylated ERK1 (white symbols) or phosphorylated ERK2 (black symbols) was expressed by taking the control level (none) as 1. Data are means ^ SEM (n 4).
Fig. 1. Effect of EGF on total or phosphorylated ERK1/2 level in cultured rat hippocampal neurons. (A) Concentration-dependency. The cells were exposed to 0.1±10 ng/ml EGF for 30 min, and the cell extract was subjected to Western blot analysis with anti-ERK1/2 antibody or anti-phospho ERK1/2 (pERK1/2) antibody. Upper panel: representative blots. Lower panel: quantitative data. The level of phosphorylated ERK1 (W) or phosphorylated ERK2 (X) is expressed by taking the control level (none) as 1. (B) Time-course. The cells were exposed to 5 ng/ml EGF for 5, 15, 30 min, 1, 3, 5, 8 or 12 h. The level of phosphorylated ERK1 (W) or phosphorylated ERK2 (X) was expressed by taking the basal level (0 min) as 1. All data are means ^ SEM (n 4).
®lters were washed in PBS-T (3 £ 10 min) at room temperature and further incubated with horseradish peroxidaseconjugated anti-rabbit IgG (1:5000 dilution in PBS-T) for 1 h at room temperature. The blots were washed in PBS-T (3 £ 10 min), and immunoreactive proteins were visualized on a ®lm with an enhanced chemiluminescence kit (NEN Life Science Products, Inc., Boston, MA, USA). Optical density on the ®lm was measured with a computer imaging system (Imaging Technology Inc., Ontario, Canada), and protein level was estimated from the standard curve, which was constructed using several dilutions of samples in each analysis. As shown in Fig. 1A, addition of EGF induced phosphorylation of both ERK1 and ERK2, with no change in total ERK1/2. The effect of EGF was concentration dependent in the range of 0.1±10 ng/ml. The EGFinduced ERK1/2 phosphorylation rapidly occurred within 5 min and peaked 30 min±1 h after (Fig. 1B). The EGFinduced ERK1 phosphorylation and ERK2 phosphorylation were both blocked by the EGF receptor tyrosine kinase inhibitor AG1478 [16] and by the MEK inhibitors, U0126 [10] and PD98059 [8] (Fig. 2). To examine if these signals are required for the neurotrophic effect of EGF, we investigated the effects of
K. Abe, H. Saito / Neuroscience Letters 282 (2000) 89±92
AG1478, U0126 and PD98059 on neuronal survival. When the culture period was prolonged, neuronal survival was gradually decreased. The number of surviving neurons at day 9 in culture was decreased to approximately 40% of that at day 6. Thus, EGF and inhibitors were added at day 6 in culture, and the cultures were ®xed at day 9. The number of surviving neurons was counted under a microscope as described previously [1]. Consistent with our previous observation [2], addition of EGF (0.1±10 ng/ml) promoted the survival of hippocampal neurons in a concentration-dependent manner (data not shown, n 5). The EGF receptor tyrosine kinase inhibitor AG1478 at 1 mM had no effect on neuronal survival in the absence of EGF, but signi®cantly blocked the survival-promoting effect of EGF (Fig. 3A). A higher concentration (10 mM) of AG1478 showed toxicity by itself. The MEK inhibitors, U0126 (0.1±10 mM) and PD98059 (1±30 mM), blocked the survival-promoting effect of EGF, without affecting neuronal survival in the absence of EGF (Fig. 3A). It has previously been reported that phosphatidylinositol 3-kinase and protein kinase C are required for the anti-apoptotic effect of EGF in an epithelial tumor cell line [12]. Thus, we also investigated the effects of LY294002, a speci®c phosphatidylinositol 3-kinase inhibitor, and GF109203X, a speci®c protein kinase C inhibitor, on neuronal survival in our cultures. However, the survival-promoting effect of EGF was not affected by these inhibitors (Fig. 3B). We have previously con®rmed that GF-109203X at 0.1 mM completely blocks phorbol ester-induced morphological changes in cultured astrocytes [6]. Phosphatidylinositol 3kinase and protein kinase C are unlikely to be involved in the survival-promoting effect of EGF in cultured rat hippocampal neurons. Signal transduction mechanisms underly-
91
ing the effect of EGF on cell survival may be different with types of death or cells. EGF is known to stimulate the proliferation of astroglial cells. To test the possibility that EGF affects neuronal survival by increasing the number of astroglial cells, we performed immunostaining with monoclonal antibodies to glial ®brillary acidic protein (GFAP), a marker protein for astroglial cells, as described previously [5]. However, the percentage of GFAP-positive cells was less than 5% both in control cultures and in cultures treated with 5 ng/ml EGF for 3 days (data not shown, n 5). Furthermore, Western blot analysis with anti-GFAP antibody demonstrated that GFAP level was negligibly low in control and EGF-treated cultures (data not shown, n 4). For comparison, pure astrocyte cultures were prepared from the cerebral cortex of neonatal rats, as described previously [5]. GFAP level per mg protein in our neuronal cultures was estimated less than 2% of that in pure astrocyte cultures. Therefore, the effect of EGF observed in the present study is unlikely to be mediated by astroglial cells. The main ®ndings in the present study were: (1) EGF induced ERK1/2 phosphorylation without affecting ERK1/2 expression; (2) EGF-induced ERK1/2 phosphorylation was blocked by the tyrosine kinase inhibitor AG1478 and MEK inhibitors, U0126 and PD98059; and (3) these inhibitors blocked survival-promoting effect of EGF. Since ERK1/2 phosphorylation rapidly occurred within 5 min after addition of EGF, the response is probably due to a direct action of EGF on neurons. Furthermore, the effective concentration of EGF in inducing ERK1/2 phosphorylation (Fig. 1) was consistent with that in promoting neuronal survival [2]. The effective concentrations of AG1478, U0126 and PD98059 in blocking the survival-promoting effect of EGF (Fig. 3A) were very
Fig. 3. In¯uences of signal transduction inhibitors on survival-promoting effect of EGF in cultured rat hippocampal neurons. The cells were pretreated with 0.1±10 mM AG1478 (A), 0.1±10 mM U0126 (A), 1±30 mM PD98059 (A), 0.1±10 mM LY294002 (B) or 0.01±1 mM GF109203X (B) for 20 min, and then exposed to 5 ng/ml EGF in the absence or presence of the inhibitors for 3 days. The number of surviving neurons was counted under a microscope. Data are means ^ SEM (n 5). **P , 0:01 vs. cont/-EGF; #P , 0:05, ##P , 0:01 vs. EGF alone; Duncan's multiple range test.
92
K. Abe, H. Saito / Neuroscience Letters 282 (2000) 89±92
consistent with those in blocking EGF-induced ERK1/2 phosphorylation (Fig. 2A,B). These results suggest that EGF promotes neuronal survival through ERK1/2 phosphorylation, which is regulated by the EGF receptor tyrosine kinase and MEK. Phosphorylated ERK1/2 is known to translocate into the nucleus and elicit changes in patterns of gene expression, including the induction of the immediate-early genes such as c-fos [19]. Further investigations to identify targets of phosphorylated ERK1/2 will give clues for understanding molecular mechanisms underlying neurotrophic effect of EGF. [1] Abe, K., Takayanagi, M. and Saito, H., Effects of recombinant human basic ®broblast growth factor and its modi®ed protein CS23 on survival of primary cultured neurons from various regions of fetal rat brain. Jpn. J. Pharmacol., 53 (1990) 221±227. [2] Abe, K., Takayanagi, M. and Saito, H., A comparison of neurotrophic effects of epidermal growth factor and basic ®broblast growth factor in primary cultured neurons from various regions of fetal rat brain. Jpn. J. Pharmacol., 54 (1990) 45±51. [3] Abe, K., Takayanagi, M. and Saito, H., Basic ®broblast growth factor and epidermal growth factor promote survival of primary cultured cerebellar neurons from neonatal rats. Jpn. J. Pharmacol., 56 (1990) 113±116. [4] Abe, K. and Kimura, H., Amyloid b toxicity consists of a Ca 21-independent early phase and a Ca 21-dependent late phase. J. Neurochem., 67 (1996) 2074±2078. [5] Abe, K. and Saito, H., Adenosine stimulates stellation of cultured rat cortical astrocytes. Brain Res., 804 (1998) 63±71. [6] Abe, K. and Saito, H., The p44/42 mitogen-activated protein kinase cascade is involved in the induction and maintenance of astrocyte stellation mediated by protein kinase C. Neurosci. Res., 36 (2000) 251±257. [7] Carpenter, G. and Cohen, S., Epidermal growth factor. Annu. Rev. Biochem., 48 (1979) 193±216. [8] Dudley, D.T., Pang, L., Decker, S.J., Bridges, A.J. and Saltiel, A.R., A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA, 92 (1995) 7686± 7689. [9] Fallon, J.H., Seroogy, K.B., Loughlin, S.E., Morrison, R.S., Bradshaw, R.A., Knauer, D.J. and Cunningham, D.D., Epidermal growth factor immunoreactive material in the
[10]
[11]
[12]
[13] [14]
[15]
[16] [17] [18] [19]
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