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2, thereby inducing neurite outgrowth in PC12 cells
Molecular Brain Research 123 (2004) 18 – 26 www.elsevier.com/locate/molbrainres
Research report
PACAP activates Rac1 and synergizes with NGF to activate ERK1/2, thereby inducing neurite outgrowth in PC12 cells Yoshiyuki Sakai a, Hitoshi Hashimoto a, Norihito Shintani a, Hironori Katoh b, Manabu Negishi b, Chihiro Kawaguchi a, Atsushi Kasai a, Akemichi Baba a,c,* a
Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan b Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto 606-8502, Japan c Laboratory of Molecular Pharmacology, Osaka University Medical School, Suita, Osaka 565-0871, Japan Accepted 23 December 2003
Abstract The mechanisms linked to the neuritogenic effect of PACAP acting in synergy with NGF were analyzed in PC12 cells. Recently, we have shown that PACAP synergizes with NGF to stimulate PACAP gene transcription and neurite outgrowth, differentially dependent on both the ERK1/2 and p38 MAP kinase pathways in PC12 cells. This suggests that PACAP modulates mitogen signaling pathways governing cell differentiation, in part through MAP kinase activation and an autocrine mechanism. Here, we studied the mechanism of the underlying neuritogenic actions of PACAP. PACAP induced transient activation of Rac1, a small GTPase involved in neurite outgrowth, in a PI3-kinaseindependent manner, and stimulated accumulation of active Rac1 at filamentous actin-rich protrusions on the cell surface to induce subsequent neurite formation. PACAP had no additional effect on the activity of Rac1 beyond the effect of NGF and failed to activate Ras or Cdc42. By contrast, simultaneous treatment with PACAP and NGF acts in synergy to induce prolonged activation of ERK1/2. These results indicate for the first time that PACAP induces activation of Rac1 associated with neurite outgrowth and suggest that the synergistic effect of PACAP and NGF on neurite extension is due to enhanced activation of ERK1/2. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotrophic factors: biological effects Keywords: Pituitary adenylate cyclase-activating polypeptide; Nerve growth factor; Neurite outgrowth; Rac1; Extracellular signal-regulated kinase; Mitogenactivated protein kinase
1. Introduction Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamic tissues and a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of peptides [3,20,41]. PACAP exists in two amidated forms, PACAP38 and PACAP27, which share an identical 27-amino-acid Nterminus, and are alternatively processed from the precursor preproPACAP. PACAP is found in neuronal elements of the * Corresponding author. Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan Tel: +81-6-6879-8180; fax: +81-6-6879-8184. E-mail address: [email protected] (A. Baba). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2003.12.013
brain and peripheral tissues, where it acts as a pleiotropic neuropeptide via three heptahelical G-protein-linked receptors, one PACAP-specific (PAC1) receptor and two receptors that are shared with VIP (VPAC1 and VPAC2) [12]. Although VPAC receptors are mainly coupled to the adenylate cyclase pathway, PAC1 receptor is coupled to several transduction systems that activate adenylate cyclase, phospholipase C, and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and mobilize calcium [3,4,41]. Recently, we have also shown the involvement of p38 MAP kinase in PAC1 receptor signaling in PC12 cells [34,35]. Several lines of evidence suggest that PACAP exerts neurotrophic and neuroprotective effects in PC12 cells that largely overlap with those induced by NGF, including stimulation of tyrosine hydroxylase gene expression [8],
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dopamine release [2], neurite outgrowth [4,9,24], and prevention of cell death induced by a variety of insults [13,39]. However, the mechanisms underlying these actions of PACAP, and how the roles are shared with NGF remain largely unknown. Recently, we demonstrated that PACAP alone stimulates PACAP gene transcription, acts synergistically with NGF to stimulate transcription, and potentiates NGF-induced neurite outgrowth in PC12 cells [17,34]. The enhancement of PACAP mRNA expression by PACAP and NGF was effectively blocked by the p38 MAP kinase inhibitor SB203580, and less effectively blocked by the ERK kinase 1/2 (MEK1/2) inhibitor PD98059. In contrast, neurite outgrowth induced by PACAP plus NGF was reduced by PD98059, but was not significantly affected by SB203580 [34]. These results suggest that PACAP modulates mitogen signaling pathways governing cell differentiation, in part through activation of MAP kinase. Recent studies have shown a mechanistic role for ERK MAP kinases in the regulation of neurite elongation: the duration of ERK activation determines the switch from proliferation to differentiation [11,26,29], although sustained activation of ERK alone is insufficient to induce neurite outgrowth [40]. The Rho family of small GTPases, consisting of Cdc42, Rac, and Rho, has been implicated in the reorganization of the actin cytoskeleton and subsequent morphological changes in various cellular functions [10,33]. Among them, Rac1 has been shown to play crucial roles in neuronal network formation including axonal development and spine formation [10,23,30]. NGF activates Rac1 in a Ras- and PI3-kinase-dependent manner, and induces the rapid formation of filamentous actin (F-actin)-rich protrusions where active Rac1 accumulates, and subsequent extension of neuritic processes [29,31,32,43,45]. Formation of actin-rich protrusions is blocked by inhibiting PI3-kinase, but is insensitive to the ERK kinase inhibitor PD98059, while neurite extension requires activation of the ERK pathway. In a previous study of ours, PACAP or NGF induced a flat-shaped morphology with few short processes in neurites, while combined together they induced a few long and straight neurites from a single PC12h cell [34]. However, the signaling mechanism underlying both the neuritogenic effect of PACAP and the potentiating effect of PACAP on the actions of NGF remain largely unknown. In this study, we investigated the signal transduction mechanisms linked to the neuritogenic effect of PACAP acting in synergy with NGF.
2. Materials and methods 2.1. Cell culture Rat pheochromocytoma PC12 cells were grown on polystyrene tissue culture plates or dishes, coated with
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poly-D-lysine in Dulbecco’s modified Eagle’s culture medium (DMEM) containing 10% fetal calf serum and 5% horse serum. Cultures were maintained at 37jC in a humidified atmosphere of 5% CO2 and 95% air. 2.2. Pull down assay Pull-down assays to measure Rac1, Cdc42, and Ras activities were performed as previously described [23,45] with several modifications. PC12 cells were seeded in 100mm culture dishes coated with poly-D-lysine at a density of 8 –10 106 cells/dish, cultured for 24 h, and serum-starved in serum-free DMEM for 12 h. Cells were preincubated with or without inhibitors for 30 min. Cells were then stimulated with PACAP38 (Peptide Institute, Osaka, Japan) and/or NGF (2.5S NGF; Alomone Labs) for the indicated times, washed twice in ice-cold phosphate-buffered saline (PBS) containing 1 mM Na3VO4, and lysed for 5 min with ice-cold cell lysis buffer (50 mM Tris –HCl, pH 7.4, 100 mM NaCl,
Fig. 1. Time-dependent induction of Rac1 activity by PACAP38 in PC12 cells. A: PC12 cells were stimulated with 10 8 M PACAP38 for the indicated time periods. The cell lysates were incubated with GST-CRIB, and the amount of GTP-bound Rac1 was determined by immunoblotting using a monoclonal anti-Rac1 antibody (upper panel). The total amount of Rac1 in cell lysates is also shown (lower panel). B: Quantification was performed by densitometric analysis. Rac1 activity is indicated by the amount of GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole cell lysates. Rac1 activity is expressed as the fold increase over the value of serum-starved cells at time 0 min. Data are the mean F S.E. of four to seven independent experiments. *P < 0.05, ***P < 0.001 versus the control cells, post hoc Student’s t-test.
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2 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 Ag/ml aprotinin, and 1 Ag/ml leupeptin) containing 8 Ag of the glutathione S-transferase (GST)-fused Cdc42/Rac-interactive binding domain of rat aPAK (GST-CRIB) for GTPbound Rac1 and Cdc42, or 50 Ag of the GST-fused Rasbinding domain of human c-Raf1 (GST-Raf) for GTP-bound Ras. Cell lysates were then centrifuged for 5 min at 10,000 g at 4 jC, and the supernatant was incubated with glutathione-Sepharose 4B beads for 45 min at 4 jC. After the beads were washed with the cell lysis buffer without protease inhibitors, the bound proteins were resolved by 12.5% SDS-polyacrylamide gel electrophoresis and immunoblotted using either a mouse monoclonal anti-Rac1 antibody (1:500 dilution; Transduction Laboratories), a rabbit polyclonal anti-Cdc42 antibody (1:100 dilution; Santa Cruz Biotechnology) or a mouse monoclonal anti-Ras antibody (1:1000 dilution; Sigma-Aldrich). Densitometric analysis was performed using NIH Image software, and the amounts
of GTP-bound Rac1, Cdc42, and Ras were normalized to the total amounts of Rac1, Cdc42, and Ras in cell lysates, respectively. 2.3. Immunofluorescence staining Immunofluorescence staining was performed as previously described [45] with several modifications. PC12 cells cultured on poly-D-lysine-coated glass coverslips in 24well plates were fixed with a 3.7% formaldehyde/PBS solution for 20 min, permeabilized in 0.2% Triton X-100 for 10 min, and incubated with 10% fetal calf serum in PBS for 30 min to block nonspecific antibody binding. Rac1 was stained with an anti-Rac1 monoclonal antibody (1:500 dilution) for 1 h, and incubated with an FITCconjugated donkey anti-mouse IgG (1:500 dilution; Molecular Probes) for 1 h. F-actin was stained with rhodamine-conjugated phalloidin (Molecular Probes) in PBS (0.5 units/ml) for 1 h.
Fig. 2. Subcellular distribution of Rac1 and F-actin in PACAP38- or NGF-stimulated PC12 cells. After stimulation with 10 8 M PACAP38 (A) or 50 ng/ml NGF (B) for the indicated times, cells were fixed and stained with an anti-Rac1 monoclonal antibody followed by a FITC-conjugated secondary antibody (green) and rhodamine-conjugated phalloidin (red). Note that the red – green overlap leads to yellow in the Merged image. The results shown are representative of three independent experiments. The bars represent 10 Am.
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Fig. 2 (continued).
2.4. Immunoblot analysis Immunoblot analysis to measure ERK phosphorylation was performed as described previously [35]. PC12 cells were serum-starved, incubated with PACAP38 and/or NGF for the indicated times, and then subjected to SDS-PAGE and immunoblotting using a rabbit anti-phospho-ERK antibody (1:1000 dilution; New England Biolabs).
3. Results
dependent increase in the amount of cellular GTP-bound Rac1, reaching a maximum at 4– 5 min. The level decreased gradually and returned to basal levels within 30 min of the stimulation. Cdc42, a small GTP-binding protein related to Rac and Ras, has been implicated in neurite outgrowth [6]. We therefore measured the amount of activated Cdc42: however, in contrast to Rac1, cellular GTP-bound Cdc42 was not changed after PACAP38 stimulation (data not shown). 3.2. Subcellular distribution of Rac1 and F-actin in PACAP38-stimulated PC12 cells
3.1. Effect of PACAP38 on Rac1 activity in PC12 cells It has been shown that both PACAP and NGF induce neurite outgrowth in PC12 cells through the ERK signal transduction pathway [24,34], and that Rac is involved in the NGF effect [43,45]. To examine whether Rac1 is involved in PACAP-induced PC12 cell neurite outgrowth, we measured the amount of cellular GTP-bound active form of Rac1 using the GST-fused CRIB domain of PAK (GSTCRIB). As shown in Fig. 1, PACAP38 induced a time-
NGF activates and accumulates Rac1 at the F-actin-rich protrusions on the cell surface, and results in subsequent neurite extension in PC12 cells [45]. We next examined the subcellular distribution of Rac1 and F-actin after stimulation with PACAP38. Immunofluorescence staining of Rac1 using an anti-Rac1 antibody revealed that Rac1 was mainly present in the cytoplasm of unstimulated PC12 cells (0 time in Fig. 2). One minute after the addition of PACAP38, PC12 cells produced a few protrusions from the cell membrane,
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Table 1 Quantification of Rac1 or F-actin distribution in PACAP38- or NGFstimulated PC12 cells Time (min) 0 1 3 15
10
8
M PACAP38
%
50 ng/ml NGF
5.6 F 1.7 10.2 F 1.5* 25.1 F 2.3*** 16.8 F 5.0*
19.9 F 3.2** 50.3 F 2.8*** 46.7 F 6.9***
Cells exhibiting a redistribution of Rac1 and F-actin were defined as cells that possessed at least one protrusion positively stained with anti-Rac1 antibody and phalloidin, and were scored as percentages of the total cells. At least 50 cells were assessed in one experiment, and data are the mean F S.E. of four independent experiments. * P < 0.05 versus control, post hoc Student’s t-test. ** P < 0.01 versus control, post hoc Student’s t-test. *** P < 0.001 versus control, post hoc Student’s t-test.
and Rac1 and F-actin were accumulated and co-localized at these protrusions (Fig. 2A; Table 1). Within 15 min, these Factin-rich protrusions had begun to extend and form short processes where Rac1 was still accumulated. Consistent with a previous report, NGF induced a rapid accumulation of Rac1 at the F-actin-rich protrusions, over a time course similar to that seen with PACAP38 (Fig. 2B). As shown in Table 1, the number of cells exhibiting redistribution of Rac1 and F-actin, with at least one protrusion positively stained with anti-Rac1 antibody, increased significantly in both PACAP38- and NGF-stimulated PC12 cells, where the increase in NGF-stimulated cells was approximately twice that in PACAP38-stimulated cells.
Fig. 4. Combined effects of PACAP38 and NGF on Rac1 activity in PC12 cells. PC12 cells were stimulated for 3 min with PACAP38 (10 8 or 10 7 M) in the absence or presence of NGF (5 or 50 ng/ml). The cell lysates were incubated with GST-CRIB, and the amount of GTP-bound Rac1 was determined by immunoblotting using a monoclonal anti-Rac1 antibody. The Rac1 activity is indicated by the amount of GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole cell lysates, and the value of Rac1 activity is expressed as the fold increase over the value of the control cells. Data are the mean F S.E. of four independent experiments. Statistically significant differences were assessed by Student’s t-test. N.S., not significant.
3.3. Effect of kinase inhibitors on PACAP-induced Rac1 activation NGF induces a PI3-kinase dependent transient activation of Rac1 and accumulation of active Rac1 at protrusion sites on the cell surface [45]. To determine whether PI3-kinase is involved in PACAP-induced Rac1 activation, cells were
Fig. 3. Effects of inhibitors of PI3-kinase (wortmannin), ERK kinase 1/2 (MEK1/2) (PD98059), and p38 MAP kinase (SB203580) on PACAP38 (10 8 M)-induced Rac1 activation after 3 min in PC12 cells. Three inhibitors, wortmannin (1 AM), PD98059 (50 AM), and SB203580 (20 AM), all produced no significant effect on PACAP38-induced Rac1 activity, while wortmannin significantly inhibited NGF (50 ng/ml)-induced activation of Rac1. The cell lysates were incubated with GST-CRIB, and the amount of GTP-bound Rac1 was determined by immunoblotting using a monoclonal anti-Rac1 antibody. Rac1 activity is indicated by the amount of GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole cell lysates, and the value of Rac1 activity is expressed as the fold increase over the value of the control cells. Data are the mean F S.E. of four to seven independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control cells; #P < 0.05 versus the cells treated with 50 ng/ml NGF alone, Student’s t-test.
Fig. 5. Prolonged effects of PACAP38 and/or NGF on ERK1/2 phosphorylation in PC12 cells. PC12 cells were incubated for 5 min, 120 min, or 24 h with the indicated concentrations of PACAP38 and/or NGF. Phosphorylated ERK (pERK) 1/2 were detected by immunoblot analysis using a rabbit anti-phospho-ERK antibody. The results shown are representative of three to six independent experiments.
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treated with a PI3-kinase inhibitor, wortmannin. As shown in Fig. 3, wortmannin (1 AM) had no effect on PACAPinduced activation of Rac1, while it significantly inhibited NGF-induced activation of Rac1. The inhibitors of MEK1/2 (50 AM PD98059) and p38 MAPK (20 AM SB203580) have been shown to effectively inhibit the PACAP mRNA expression induced by PACAP38 in PC12 cells [34]. We therefore examined the effect of these inhibitors on Rac1 activity. However, neither PD98059 (50 AM) nor SB203580 (20 AM) had any effect on PACAP-induced activation of Rac1. 3.4. Effect of combined treatment with PACAP and NGF on Rac1 activity and ERK1/2 activity PACAP potentiates NGF-induced neurite outgrowth in PC12 cells [34]. We next determined the effect of combined treatment with PACAP and NGF on Rac1 activity. Although either PACAP or NGF induced activation of Rac1, the effect
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of them combined was not additive but equal to that of NGF alone (Fig. 4). The sustained activation of ERK is known to be required for neuronal differentiation of PC12 cells [11,26]. We then examined the effect of combined treatment with PACAP and NGF on the levels of phosphorylated ERK1/2 (Fig. 5). In agreement with a previous report [4], either PACAP or NGF induced ERK1/2 phosphorylation after 5 min of stimulation. These increases persisted for at least 120 min. Treatment with PACAP and NGF combined had more than an additive effect: PACAP (10 9 M) markedly potentiated NGF (50 ng/ml)-induced ERK1/2 phosphorylation, even after 120 min. 3.5. Ras activity in PC12 cells treated with PACAP38 and/ or NGF It has also been proposed that NGF-induced activation of ERK involves Ras-dependent signaling [36]. Ras induces activation of Rac, and thus this signaling pathway likely plays an important role in the initiation of NGF-induced neurite outgrowth [45]. To determine whether this is also true for PACAP-induced neurite formation, we measured Ras activity in PC12 cells stimulated with PACAP38 either alone, or together with NGF (Fig. 6). Although NGF increased the amount of cellular GTP-bound Ras, PACAP38 had no effect on the amount of GTP-bound Ras both in unstimulated and NGF-stimulated PC12 cells.
4. Discussion
Fig. 6. Effects of PACAP38 and/or NGF on Ras activity in PC12 cells. A: PC12 cells were stimulated for 1 min with PACAP38 (10 nM), NGF (50 ng/ ml), or a combination of PACAP38 and NGF. The cell lysates were incubated with GST-Raf, and the amount of GTP-bound Ras was determined by immunoblotting using a monoclonal anti-Ras antibody (upper panel). The total amount of Ras in cell lysates (lower panel) are also shown. B: Quantification was performed by densitometric analysis. Ras activity is indicated by the amount of GST-Raf-bound Ras normalized to the amount of Ras in whole cell lysates, and the value of Ras activity is expressed as the fold increase over the value of the control cells. Data are the mean F S.E. of four to seven independent experiments. ***P < 0.001 versus the control cells; #P < 0.05 versus the cells treated with 10 nM PACAP38 alone, Student’s t-test.
PACAP induces neurite outgrowth and potentiates NGFinduced neurite outgrowth in PC12 cells; however, the underlying mechanism has been poorly understood. In the present study, we show that (1) PACAP induces transient activation of Rac1, but not Cdc42 or Ras, in a PI3-kinaseindependent manner; (2) PACAP stimulates the accumulation of active Rac1 at protrusion sites on the cell surface, inducing F-actin-rich protrusions and sites of subsequent neurite formation; (3) PACAP does not potentiate NGFinduced activation of Rac1 and Ras; however, (4) simultaneous treatment with PACAP and NGF acts in synergy to induce prolonged activation of ERK1/2. These results indicate that PACAP-induced neurite outgrowth involves neuritogenic action via activation of Rac1 and subsequent neurite extension by ERK activation, and that although PACAP and NGF share common intracellular events leading to neurite outgrowth, synergism between them arises, at least in part, via activation of ERKs. In PC12 cells, PACAP, like NGF, induces differentiation of sympathetic neuron-like cells and eventual cessation of cell proliferation [4]. NGF induces a sustained activation of ERK that is critical for the neuronal differentiation of PC12 cells [11,26]. However, prolonged receptor-mediated activation of MAPK alone is insufficient to induce neurite
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outgrowth [40]. Activation of Rac1 and accumulation of the activated Rac1 at F-actin-rich protrusions are also required for NGF-induced neurite outgrowth in PC12 cells [45]. In the present study, we analyzed the role of Rac1 in PACAPinduced neurite outgrowth, and show that PACAP induces a temporal activation and spatial localization of Rac1, similar to that observed in cells stimulated by NGF. However, in contrast to NGF, PACAP induced Rac1 activation through a mechanism independent of PI3-kinase signaling pathway. The failure of PACAP to potentiate NGF-induced activation of Rac1 may be explained by a ceiling effect. Recently, we have shown that PACAP38 induces sustained activation of ERK lasting at least 60 min at concentrations as low as 10 12 M in cultured astrocytes and that this effect is completely abolished by the cyclic AMP antagonist Rp-cAMP but not by the protein kinase A inhibitor H89 [21]. These results suggest the involvement of a cyclic AMP-dependent but protein kinase A-independent pathway in PACAP receptor signaling. Maillet et al. [25] has recently shown that Rac activation results from activation of Rap1 through the cAMP guanine nucleotideexchange factor Epac1 but not protein kinase A. Based on these observations, we speculate that cyclic AMP/Epac/ Rap1 may be involved in PACAP-induced activations of Rac1 and ERK1/2 in PC12 cells. It has been shown that through activation of the ERK pathway, NGF induces sustained and strong expression of p35, the neuron-specific activator of Cdk5, in PC12 cells, and prolongs the activation of Cdk5, which is required for NGF-induced neurite outgrowth [11]. In addition, sustained activation of ERK (>80 min) is needed for the strong induction of p35 in PC12 cells [11]. In the present study, PACAP can either act alone or synergistically with NGF to induce ERK activation for at least 120 min. Furthermore, a previous study of ours [34] showed that neurite outgrowth induced by PACAP and NGF combined was partly inhibited by PD98059. These results suggest that the synergistic effects between PACAP and NGF on neurite outgrowth arises, at least in part, via prolonged ERK activation. Rac is involved in both the regulation of cell morphology and activation of p38 [28]. We have previously shown that PACAP, either alone or combined with NGF, stimulates PACAP gene transcription [17] and that p38 plays a critical role in this PACAP effect [34]. p38 is known to be a prominent activator of the tumor suppressor p53 [5]. Interestingly, PACAP-selective PAC1 receptor expression is induced by p53 and its transcriptional coactivator Zac1 [7,22]. In the developing central nervous system in vivo, PACAP restrains neuronal precursor proliferation, possibly via autocrine and/or paracrine pathways [38]. Taken together, these results imply that PACAP signaling modulates the MAP kinase signaling pathways governing cell growth and differentiation, in part through the Rac/p38 pathway and an autocrine/paracrine mechanism. We have previously cloned the cDNA for the PAC1 receptor [14] and determined the structural organization of
the mouse genes for the PAC1 receptor [1], VPAC1 receptor [16], and PACAP ligand [44], and localized the PAC1 receptor mRNA in the rat brain [15]. In order to understand the function of PACAP-dependent signaling in vivo, we generated mice deficient in PACAP [19] or PAC1 receptor exon 2 [18]. PACAP-deficient mice had a high early mortality rate and displayed central nervous system phenotypes including behavioral and psychological abnormalities [19] and impairment of hippocampal long-term potentiation [27]. In addition, brain levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) decreased slightly [19,37], and manifests itself at the earliest age tested (four weeks of age) (unpublished observation). PACAP as well as the PAC1 receptor are expressed in the embryonic neural tube, where they may regulate neurogenesis and patterning [42]. Although it has not yet been determined whether the phenotypes of PACAP-deficient mice resulted from developmental defects, we speculate that Rac1 and MAP kinases are hierarchically and/or coordinately involved in the multiple roles of PACAP in neuronal function and development. However, it is evident that future studies are required to provide direct evidence that Rac1 is implicated in the control of neurite outgrowth and activation of the p38.
Acknowledgements This research was supported, in part, by Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-inAid for Scientific Research (A) and (B) from Japan Society for the Promotion of Science, and by grants from Taisho Pharmaceutical, AstraZeneca, and The Naito Foundation.
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Research report
PACAP activates Rac1 and synergizes with NGF to activate ERK1/2, thereby inducing neurite outgrowth in PC12 cells Yoshiyuki Sakai a, Hitoshi Hashimoto a, Norihito Shintani a, Hironori Katoh b, Manabu Negishi b, Chihiro Kawaguchi a, Atsushi Kasai a, Akemichi Baba a,c,* a
Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan b Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto 606-8502, Japan c Laboratory of Molecular Pharmacology, Osaka University Medical School, Suita, Osaka 565-0871, Japan Accepted 23 December 2003
Abstract The mechanisms linked to the neuritogenic effect of PACAP acting in synergy with NGF were analyzed in PC12 cells. Recently, we have shown that PACAP synergizes with NGF to stimulate PACAP gene transcription and neurite outgrowth, differentially dependent on both the ERK1/2 and p38 MAP kinase pathways in PC12 cells. This suggests that PACAP modulates mitogen signaling pathways governing cell differentiation, in part through MAP kinase activation and an autocrine mechanism. Here, we studied the mechanism of the underlying neuritogenic actions of PACAP. PACAP induced transient activation of Rac1, a small GTPase involved in neurite outgrowth, in a PI3-kinaseindependent manner, and stimulated accumulation of active Rac1 at filamentous actin-rich protrusions on the cell surface to induce subsequent neurite formation. PACAP had no additional effect on the activity of Rac1 beyond the effect of NGF and failed to activate Ras or Cdc42. By contrast, simultaneous treatment with PACAP and NGF acts in synergy to induce prolonged activation of ERK1/2. These results indicate for the first time that PACAP induces activation of Rac1 associated with neurite outgrowth and suggest that the synergistic effect of PACAP and NGF on neurite extension is due to enhanced activation of ERK1/2. D 2004 Elsevier B.V. All rights reserved. Theme: Development and regeneration Topic: Neurotrophic factors: biological effects Keywords: Pituitary adenylate cyclase-activating polypeptide; Nerve growth factor; Neurite outgrowth; Rac1; Extracellular signal-regulated kinase; Mitogenactivated protein kinase
1. Introduction Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamic tissues and a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon family of peptides [3,20,41]. PACAP exists in two amidated forms, PACAP38 and PACAP27, which share an identical 27-amino-acid Nterminus, and are alternatively processed from the precursor preproPACAP. PACAP is found in neuronal elements of the * Corresponding author. Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan Tel: +81-6-6879-8180; fax: +81-6-6879-8184. E-mail address: [email protected] (A. Baba). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2003.12.013
brain and peripheral tissues, where it acts as a pleiotropic neuropeptide via three heptahelical G-protein-linked receptors, one PACAP-specific (PAC1) receptor and two receptors that are shared with VIP (VPAC1 and VPAC2) [12]. Although VPAC receptors are mainly coupled to the adenylate cyclase pathway, PAC1 receptor is coupled to several transduction systems that activate adenylate cyclase, phospholipase C, and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase and mobilize calcium [3,4,41]. Recently, we have also shown the involvement of p38 MAP kinase in PAC1 receptor signaling in PC12 cells [34,35]. Several lines of evidence suggest that PACAP exerts neurotrophic and neuroprotective effects in PC12 cells that largely overlap with those induced by NGF, including stimulation of tyrosine hydroxylase gene expression [8],
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dopamine release [2], neurite outgrowth [4,9,24], and prevention of cell death induced by a variety of insults [13,39]. However, the mechanisms underlying these actions of PACAP, and how the roles are shared with NGF remain largely unknown. Recently, we demonstrated that PACAP alone stimulates PACAP gene transcription, acts synergistically with NGF to stimulate transcription, and potentiates NGF-induced neurite outgrowth in PC12 cells [17,34]. The enhancement of PACAP mRNA expression by PACAP and NGF was effectively blocked by the p38 MAP kinase inhibitor SB203580, and less effectively blocked by the ERK kinase 1/2 (MEK1/2) inhibitor PD98059. In contrast, neurite outgrowth induced by PACAP plus NGF was reduced by PD98059, but was not significantly affected by SB203580 [34]. These results suggest that PACAP modulates mitogen signaling pathways governing cell differentiation, in part through activation of MAP kinase. Recent studies have shown a mechanistic role for ERK MAP kinases in the regulation of neurite elongation: the duration of ERK activation determines the switch from proliferation to differentiation [11,26,29], although sustained activation of ERK alone is insufficient to induce neurite outgrowth [40]. The Rho family of small GTPases, consisting of Cdc42, Rac, and Rho, has been implicated in the reorganization of the actin cytoskeleton and subsequent morphological changes in various cellular functions [10,33]. Among them, Rac1 has been shown to play crucial roles in neuronal network formation including axonal development and spine formation [10,23,30]. NGF activates Rac1 in a Ras- and PI3-kinase-dependent manner, and induces the rapid formation of filamentous actin (F-actin)-rich protrusions where active Rac1 accumulates, and subsequent extension of neuritic processes [29,31,32,43,45]. Formation of actin-rich protrusions is blocked by inhibiting PI3-kinase, but is insensitive to the ERK kinase inhibitor PD98059, while neurite extension requires activation of the ERK pathway. In a previous study of ours, PACAP or NGF induced a flat-shaped morphology with few short processes in neurites, while combined together they induced a few long and straight neurites from a single PC12h cell [34]. However, the signaling mechanism underlying both the neuritogenic effect of PACAP and the potentiating effect of PACAP on the actions of NGF remain largely unknown. In this study, we investigated the signal transduction mechanisms linked to the neuritogenic effect of PACAP acting in synergy with NGF.
2. Materials and methods 2.1. Cell culture Rat pheochromocytoma PC12 cells were grown on polystyrene tissue culture plates or dishes, coated with
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poly-D-lysine in Dulbecco’s modified Eagle’s culture medium (DMEM) containing 10% fetal calf serum and 5% horse serum. Cultures were maintained at 37jC in a humidified atmosphere of 5% CO2 and 95% air. 2.2. Pull down assay Pull-down assays to measure Rac1, Cdc42, and Ras activities were performed as previously described [23,45] with several modifications. PC12 cells were seeded in 100mm culture dishes coated with poly-D-lysine at a density of 8 –10 106 cells/dish, cultured for 24 h, and serum-starved in serum-free DMEM for 12 h. Cells were preincubated with or without inhibitors for 30 min. Cells were then stimulated with PACAP38 (Peptide Institute, Osaka, Japan) and/or NGF (2.5S NGF; Alomone Labs) for the indicated times, washed twice in ice-cold phosphate-buffered saline (PBS) containing 1 mM Na3VO4, and lysed for 5 min with ice-cold cell lysis buffer (50 mM Tris –HCl, pH 7.4, 100 mM NaCl,
Fig. 1. Time-dependent induction of Rac1 activity by PACAP38 in PC12 cells. A: PC12 cells were stimulated with 10 8 M PACAP38 for the indicated time periods. The cell lysates were incubated with GST-CRIB, and the amount of GTP-bound Rac1 was determined by immunoblotting using a monoclonal anti-Rac1 antibody (upper panel). The total amount of Rac1 in cell lysates is also shown (lower panel). B: Quantification was performed by densitometric analysis. Rac1 activity is indicated by the amount of GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole cell lysates. Rac1 activity is expressed as the fold increase over the value of serum-starved cells at time 0 min. Data are the mean F S.E. of four to seven independent experiments. *P < 0.05, ***P < 0.001 versus the control cells, post hoc Student’s t-test.
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2 mM MgCl2, 1% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 Ag/ml aprotinin, and 1 Ag/ml leupeptin) containing 8 Ag of the glutathione S-transferase (GST)-fused Cdc42/Rac-interactive binding domain of rat aPAK (GST-CRIB) for GTPbound Rac1 and Cdc42, or 50 Ag of the GST-fused Rasbinding domain of human c-Raf1 (GST-Raf) for GTP-bound Ras. Cell lysates were then centrifuged for 5 min at 10,000 g at 4 jC, and the supernatant was incubated with glutathione-Sepharose 4B beads for 45 min at 4 jC. After the beads were washed with the cell lysis buffer without protease inhibitors, the bound proteins were resolved by 12.5% SDS-polyacrylamide gel electrophoresis and immunoblotted using either a mouse monoclonal anti-Rac1 antibody (1:500 dilution; Transduction Laboratories), a rabbit polyclonal anti-Cdc42 antibody (1:100 dilution; Santa Cruz Biotechnology) or a mouse monoclonal anti-Ras antibody (1:1000 dilution; Sigma-Aldrich). Densitometric analysis was performed using NIH Image software, and the amounts
of GTP-bound Rac1, Cdc42, and Ras were normalized to the total amounts of Rac1, Cdc42, and Ras in cell lysates, respectively. 2.3. Immunofluorescence staining Immunofluorescence staining was performed as previously described [45] with several modifications. PC12 cells cultured on poly-D-lysine-coated glass coverslips in 24well plates were fixed with a 3.7% formaldehyde/PBS solution for 20 min, permeabilized in 0.2% Triton X-100 for 10 min, and incubated with 10% fetal calf serum in PBS for 30 min to block nonspecific antibody binding. Rac1 was stained with an anti-Rac1 monoclonal antibody (1:500 dilution) for 1 h, and incubated with an FITCconjugated donkey anti-mouse IgG (1:500 dilution; Molecular Probes) for 1 h. F-actin was stained with rhodamine-conjugated phalloidin (Molecular Probes) in PBS (0.5 units/ml) for 1 h.
Fig. 2. Subcellular distribution of Rac1 and F-actin in PACAP38- or NGF-stimulated PC12 cells. After stimulation with 10 8 M PACAP38 (A) or 50 ng/ml NGF (B) for the indicated times, cells were fixed and stained with an anti-Rac1 monoclonal antibody followed by a FITC-conjugated secondary antibody (green) and rhodamine-conjugated phalloidin (red). Note that the red – green overlap leads to yellow in the Merged image. The results shown are representative of three independent experiments. The bars represent 10 Am.
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Fig. 2 (continued).
2.4. Immunoblot analysis Immunoblot analysis to measure ERK phosphorylation was performed as described previously [35]. PC12 cells were serum-starved, incubated with PACAP38 and/or NGF for the indicated times, and then subjected to SDS-PAGE and immunoblotting using a rabbit anti-phospho-ERK antibody (1:1000 dilution; New England Biolabs).
3. Results
dependent increase in the amount of cellular GTP-bound Rac1, reaching a maximum at 4– 5 min. The level decreased gradually and returned to basal levels within 30 min of the stimulation. Cdc42, a small GTP-binding protein related to Rac and Ras, has been implicated in neurite outgrowth [6]. We therefore measured the amount of activated Cdc42: however, in contrast to Rac1, cellular GTP-bound Cdc42 was not changed after PACAP38 stimulation (data not shown). 3.2. Subcellular distribution of Rac1 and F-actin in PACAP38-stimulated PC12 cells
3.1. Effect of PACAP38 on Rac1 activity in PC12 cells It has been shown that both PACAP and NGF induce neurite outgrowth in PC12 cells through the ERK signal transduction pathway [24,34], and that Rac is involved in the NGF effect [43,45]. To examine whether Rac1 is involved in PACAP-induced PC12 cell neurite outgrowth, we measured the amount of cellular GTP-bound active form of Rac1 using the GST-fused CRIB domain of PAK (GSTCRIB). As shown in Fig. 1, PACAP38 induced a time-
NGF activates and accumulates Rac1 at the F-actin-rich protrusions on the cell surface, and results in subsequent neurite extension in PC12 cells [45]. We next examined the subcellular distribution of Rac1 and F-actin after stimulation with PACAP38. Immunofluorescence staining of Rac1 using an anti-Rac1 antibody revealed that Rac1 was mainly present in the cytoplasm of unstimulated PC12 cells (0 time in Fig. 2). One minute after the addition of PACAP38, PC12 cells produced a few protrusions from the cell membrane,
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Table 1 Quantification of Rac1 or F-actin distribution in PACAP38- or NGFstimulated PC12 cells Time (min) 0 1 3 15
10
8
M PACAP38
%
50 ng/ml NGF
5.6 F 1.7 10.2 F 1.5* 25.1 F 2.3*** 16.8 F 5.0*
19.9 F 3.2** 50.3 F 2.8*** 46.7 F 6.9***
Cells exhibiting a redistribution of Rac1 and F-actin were defined as cells that possessed at least one protrusion positively stained with anti-Rac1 antibody and phalloidin, and were scored as percentages of the total cells. At least 50 cells were assessed in one experiment, and data are the mean F S.E. of four independent experiments. * P < 0.05 versus control, post hoc Student’s t-test. ** P < 0.01 versus control, post hoc Student’s t-test. *** P < 0.001 versus control, post hoc Student’s t-test.
and Rac1 and F-actin were accumulated and co-localized at these protrusions (Fig. 2A; Table 1). Within 15 min, these Factin-rich protrusions had begun to extend and form short processes where Rac1 was still accumulated. Consistent with a previous report, NGF induced a rapid accumulation of Rac1 at the F-actin-rich protrusions, over a time course similar to that seen with PACAP38 (Fig. 2B). As shown in Table 1, the number of cells exhibiting redistribution of Rac1 and F-actin, with at least one protrusion positively stained with anti-Rac1 antibody, increased significantly in both PACAP38- and NGF-stimulated PC12 cells, where the increase in NGF-stimulated cells was approximately twice that in PACAP38-stimulated cells.
Fig. 4. Combined effects of PACAP38 and NGF on Rac1 activity in PC12 cells. PC12 cells were stimulated for 3 min with PACAP38 (10 8 or 10 7 M) in the absence or presence of NGF (5 or 50 ng/ml). The cell lysates were incubated with GST-CRIB, and the amount of GTP-bound Rac1 was determined by immunoblotting using a monoclonal anti-Rac1 antibody. The Rac1 activity is indicated by the amount of GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole cell lysates, and the value of Rac1 activity is expressed as the fold increase over the value of the control cells. Data are the mean F S.E. of four independent experiments. Statistically significant differences were assessed by Student’s t-test. N.S., not significant.
3.3. Effect of kinase inhibitors on PACAP-induced Rac1 activation NGF induces a PI3-kinase dependent transient activation of Rac1 and accumulation of active Rac1 at protrusion sites on the cell surface [45]. To determine whether PI3-kinase is involved in PACAP-induced Rac1 activation, cells were
Fig. 3. Effects of inhibitors of PI3-kinase (wortmannin), ERK kinase 1/2 (MEK1/2) (PD98059), and p38 MAP kinase (SB203580) on PACAP38 (10 8 M)-induced Rac1 activation after 3 min in PC12 cells. Three inhibitors, wortmannin (1 AM), PD98059 (50 AM), and SB203580 (20 AM), all produced no significant effect on PACAP38-induced Rac1 activity, while wortmannin significantly inhibited NGF (50 ng/ml)-induced activation of Rac1. The cell lysates were incubated with GST-CRIB, and the amount of GTP-bound Rac1 was determined by immunoblotting using a monoclonal anti-Rac1 antibody. Rac1 activity is indicated by the amount of GST-CRIB-bound Rac1 normalized to the amount of Rac1 in whole cell lysates, and the value of Rac1 activity is expressed as the fold increase over the value of the control cells. Data are the mean F S.E. of four to seven independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control cells; #P < 0.05 versus the cells treated with 50 ng/ml NGF alone, Student’s t-test.
Fig. 5. Prolonged effects of PACAP38 and/or NGF on ERK1/2 phosphorylation in PC12 cells. PC12 cells were incubated for 5 min, 120 min, or 24 h with the indicated concentrations of PACAP38 and/or NGF. Phosphorylated ERK (pERK) 1/2 were detected by immunoblot analysis using a rabbit anti-phospho-ERK antibody. The results shown are representative of three to six independent experiments.
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treated with a PI3-kinase inhibitor, wortmannin. As shown in Fig. 3, wortmannin (1 AM) had no effect on PACAPinduced activation of Rac1, while it significantly inhibited NGF-induced activation of Rac1. The inhibitors of MEK1/2 (50 AM PD98059) and p38 MAPK (20 AM SB203580) have been shown to effectively inhibit the PACAP mRNA expression induced by PACAP38 in PC12 cells [34]. We therefore examined the effect of these inhibitors on Rac1 activity. However, neither PD98059 (50 AM) nor SB203580 (20 AM) had any effect on PACAP-induced activation of Rac1. 3.4. Effect of combined treatment with PACAP and NGF on Rac1 activity and ERK1/2 activity PACAP potentiates NGF-induced neurite outgrowth in PC12 cells [34]. We next determined the effect of combined treatment with PACAP and NGF on Rac1 activity. Although either PACAP or NGF induced activation of Rac1, the effect
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of them combined was not additive but equal to that of NGF alone (Fig. 4). The sustained activation of ERK is known to be required for neuronal differentiation of PC12 cells [11,26]. We then examined the effect of combined treatment with PACAP and NGF on the levels of phosphorylated ERK1/2 (Fig. 5). In agreement with a previous report [4], either PACAP or NGF induced ERK1/2 phosphorylation after 5 min of stimulation. These increases persisted for at least 120 min. Treatment with PACAP and NGF combined had more than an additive effect: PACAP (10 9 M) markedly potentiated NGF (50 ng/ml)-induced ERK1/2 phosphorylation, even after 120 min. 3.5. Ras activity in PC12 cells treated with PACAP38 and/ or NGF It has also been proposed that NGF-induced activation of ERK involves Ras-dependent signaling [36]. Ras induces activation of Rac, and thus this signaling pathway likely plays an important role in the initiation of NGF-induced neurite outgrowth [45]. To determine whether this is also true for PACAP-induced neurite formation, we measured Ras activity in PC12 cells stimulated with PACAP38 either alone, or together with NGF (Fig. 6). Although NGF increased the amount of cellular GTP-bound Ras, PACAP38 had no effect on the amount of GTP-bound Ras both in unstimulated and NGF-stimulated PC12 cells.
4. Discussion
Fig. 6. Effects of PACAP38 and/or NGF on Ras activity in PC12 cells. A: PC12 cells were stimulated for 1 min with PACAP38 (10 nM), NGF (50 ng/ ml), or a combination of PACAP38 and NGF. The cell lysates were incubated with GST-Raf, and the amount of GTP-bound Ras was determined by immunoblotting using a monoclonal anti-Ras antibody (upper panel). The total amount of Ras in cell lysates (lower panel) are also shown. B: Quantification was performed by densitometric analysis. Ras activity is indicated by the amount of GST-Raf-bound Ras normalized to the amount of Ras in whole cell lysates, and the value of Ras activity is expressed as the fold increase over the value of the control cells. Data are the mean F S.E. of four to seven independent experiments. ***P < 0.001 versus the control cells; #P < 0.05 versus the cells treated with 10 nM PACAP38 alone, Student’s t-test.
PACAP induces neurite outgrowth and potentiates NGFinduced neurite outgrowth in PC12 cells; however, the underlying mechanism has been poorly understood. In the present study, we show that (1) PACAP induces transient activation of Rac1, but not Cdc42 or Ras, in a PI3-kinaseindependent manner; (2) PACAP stimulates the accumulation of active Rac1 at protrusion sites on the cell surface, inducing F-actin-rich protrusions and sites of subsequent neurite formation; (3) PACAP does not potentiate NGFinduced activation of Rac1 and Ras; however, (4) simultaneous treatment with PACAP and NGF acts in synergy to induce prolonged activation of ERK1/2. These results indicate that PACAP-induced neurite outgrowth involves neuritogenic action via activation of Rac1 and subsequent neurite extension by ERK activation, and that although PACAP and NGF share common intracellular events leading to neurite outgrowth, synergism between them arises, at least in part, via activation of ERKs. In PC12 cells, PACAP, like NGF, induces differentiation of sympathetic neuron-like cells and eventual cessation of cell proliferation [4]. NGF induces a sustained activation of ERK that is critical for the neuronal differentiation of PC12 cells [11,26]. However, prolonged receptor-mediated activation of MAPK alone is insufficient to induce neurite
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outgrowth [40]. Activation of Rac1 and accumulation of the activated Rac1 at F-actin-rich protrusions are also required for NGF-induced neurite outgrowth in PC12 cells [45]. In the present study, we analyzed the role of Rac1 in PACAPinduced neurite outgrowth, and show that PACAP induces a temporal activation and spatial localization of Rac1, similar to that observed in cells stimulated by NGF. However, in contrast to NGF, PACAP induced Rac1 activation through a mechanism independent of PI3-kinase signaling pathway. The failure of PACAP to potentiate NGF-induced activation of Rac1 may be explained by a ceiling effect. Recently, we have shown that PACAP38 induces sustained activation of ERK lasting at least 60 min at concentrations as low as 10 12 M in cultured astrocytes and that this effect is completely abolished by the cyclic AMP antagonist Rp-cAMP but not by the protein kinase A inhibitor H89 [21]. These results suggest the involvement of a cyclic AMP-dependent but protein kinase A-independent pathway in PACAP receptor signaling. Maillet et al. [25] has recently shown that Rac activation results from activation of Rap1 through the cAMP guanine nucleotideexchange factor Epac1 but not protein kinase A. Based on these observations, we speculate that cyclic AMP/Epac/ Rap1 may be involved in PACAP-induced activations of Rac1 and ERK1/2 in PC12 cells. It has been shown that through activation of the ERK pathway, NGF induces sustained and strong expression of p35, the neuron-specific activator of Cdk5, in PC12 cells, and prolongs the activation of Cdk5, which is required for NGF-induced neurite outgrowth [11]. In addition, sustained activation of ERK (>80 min) is needed for the strong induction of p35 in PC12 cells [11]. In the present study, PACAP can either act alone or synergistically with NGF to induce ERK activation for at least 120 min. Furthermore, a previous study of ours [34] showed that neurite outgrowth induced by PACAP and NGF combined was partly inhibited by PD98059. These results suggest that the synergistic effects between PACAP and NGF on neurite outgrowth arises, at least in part, via prolonged ERK activation. Rac is involved in both the regulation of cell morphology and activation of p38 [28]. We have previously shown that PACAP, either alone or combined with NGF, stimulates PACAP gene transcription [17] and that p38 plays a critical role in this PACAP effect [34]. p38 is known to be a prominent activator of the tumor suppressor p53 [5]. Interestingly, PACAP-selective PAC1 receptor expression is induced by p53 and its transcriptional coactivator Zac1 [7,22]. In the developing central nervous system in vivo, PACAP restrains neuronal precursor proliferation, possibly via autocrine and/or paracrine pathways [38]. Taken together, these results imply that PACAP signaling modulates the MAP kinase signaling pathways governing cell growth and differentiation, in part through the Rac/p38 pathway and an autocrine/paracrine mechanism. We have previously cloned the cDNA for the PAC1 receptor [14] and determined the structural organization of
the mouse genes for the PAC1 receptor [1], VPAC1 receptor [16], and PACAP ligand [44], and localized the PAC1 receptor mRNA in the rat brain [15]. In order to understand the function of PACAP-dependent signaling in vivo, we generated mice deficient in PACAP [19] or PAC1 receptor exon 2 [18]. PACAP-deficient mice had a high early mortality rate and displayed central nervous system phenotypes including behavioral and psychological abnormalities [19] and impairment of hippocampal long-term potentiation [27]. In addition, brain levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) decreased slightly [19,37], and manifests itself at the earliest age tested (four weeks of age) (unpublished observation). PACAP as well as the PAC1 receptor are expressed in the embryonic neural tube, where they may regulate neurogenesis and patterning [42]. Although it has not yet been determined whether the phenotypes of PACAP-deficient mice resulted from developmental defects, we speculate that Rac1 and MAP kinases are hierarchically and/or coordinately involved in the multiple roles of PACAP in neuronal function and development. However, it is evident that future studies are required to provide direct evidence that Rac1 is implicated in the control of neurite outgrowth and activation of the p38.
Acknowledgements This research was supported, in part, by Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grant-inAid for Scientific Research (A) and (B) from Japan Society for the Promotion of Science, and by grants from Taisho Pharmaceutical, AstraZeneca, and The Naito Foundation.
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