Activity-dependent shedding of heparin-binding EGF-like growth factor in brain neurons

BBRC Biochemical and Biophysical Research Communications 348 (2006) 963–970 www.elsevier.com/locate/ybbrc

Activity-dependent shedding of heparin-binding EGF-like growth factor in brain neurons q Yuji Shishido a, Takayuki Tanaka a, Ying-shan Piao a, Kazuaki Araki a, Nobuyuki Takei a, Shigeki Higashiyama b, Hiroyuki Nawa a,* b

a Division of Molecular Neurobiology, Brain Research Institute, Niigata University, Niigata 951-8585, Japan PRESTO/JST, Department of Biochemistry and Molecular Genetics, Ehime University Graduate School of Medicine, Ehime 791-0295, Japan

Received 14 July 2006 Available online 31 July 2006

Abstract Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially produced as a membrane-anchored precursor (proHB-EGF) and subsequently liberated from the cell membrane through ectodomain shedding. Here, we characterized the molecular regulation of pro-HB-EGF shedding in the central nervous system. Cultured neocortical or hippocampal neurons were transfected with the alkaline-phosphatase-tagged pro-HB-EGF gene and stimulated with various neurotransmitters. Both kainate and N-methlyl-D-aspartate, but not agonists for metabotropic glutamate receptors, promoted pro-HB-EGF shedding and HB-EGF release, which were attenuated by an exocytosis blocker and metalloproteinase inhibitors. In the brain of transgenic mice over-expressing human pro-HB-EGF, kainateinduced seizure activity decreased content of pro-HB-EGF-like immunoreactivity and conversely increased levels of soluble HB-EGF. There was concomitant phosphorylation of EGF receptors (ErbB1) following seizures, suggesting that seizure activities liberated HBEGF and activated neighboring ErbB1 receptors. Therefore, we propose that glutamatergic neurotransmission in the central nervous system plays a crucial role in regulating ectodomain shedding of pro-HB-EGF.  2006 Elsevier Inc. All rights reserved. Keywords: HB-EGF; ErbB1; EGF; Shedding; TACE; Glutamate; NMDA

Heparin-binding epidermal growth factor (EGF)-like growth factor, HB-EGF, is a member of the EGF superfamily and binds to both homomeric and heteromeric ErbB receptors [1,2]. A secreted form of mature HB-EGF (referred to HB-EGF thereafter) influences cell proliferation, migration, and survival of various types of cells q

Abbreviations: AP, alkaline phosphatase; HB-EGF, heparin-binding epidermal growth factor-like growth factor; NMDA, N-methly-D-aspartate; ADAM, a disintegrin and metalloproteinase; GPCRs, G proteincoupled receptors; a-CaMK II, alpha-calmodulin-dependent protein kinase 2; AMPA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BoTX, botulinum toxin; ACPD, 1-aminocyclopentane-1,3-dicarboxylic acid; DHPG, (RS)-3,5-dihydroxyphenylglycine; TPA, 12-O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; CNS, central nervous system. * Corresponding author. Fax: +81 25 227 0815. E-mail address: [email protected] (H. Nawa). 0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.07.129

[3–5]. In the central nervous system (CNS), pro-HB-EGF and HB-EGF are highly enriched in the neocortex and cerebellum [6–8], and HB-EGF has been implicated in neuronal survival and glial/stem cell proliferation [9–11]. For example, administration of HB-EGF rescues neurons from neurodegeneration following ischemia and dopaminergic lesions with 6-hydroxydopamine [12,13]. Recent studies also indicate that this growth factor contributes to neurogenesis in the subventricular zone and adult hippocampus, thus maintaining multipotent neural progenitor cells expressing ErbB1 receptors [10,14]. HB-EGF is processed from its precursor protein, proHB-EGF, which is anchored in the plasma membrane. Pro-HB-EGF encodes a signal peptide, a heparin-binding domain, an EGF-like domain, an extracellular juxtamembrane, a transmembrane domain, and a cytoplasmic tail [15]. Pro-HB-EGF is susceptible to proteolytic cleavage,

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namely ectodomain shedding, and converted to the mature secreted factor, HB-EGF [16,17]. Pro-HB-EGF lacks proliferative activity and may inhibit mitosis of neighboring cells [18,19]. Accordingly, ectodomain shedding is essential for pro-HB-EGF to exert its biological effects, and the regulation of this proteolytic cleavage has been examined mainly in the peripheral tissues and cells [1,2]. Activation of metalloproteinases initiates ectodomain shedding but these enzymes also target the precursors for several proteins including pro-neuregulins and pro-tumor necrosis factor-alpha [16,20,21]. The enzymatic activities of a disintegrin and metalloproteinase (ADAM) and matrix metalloproteinase are induced by protein kinase C (PKC) activation and cleave pro-HB-EGF within the juxtamembrane domain [22–24]. Among various stimuli for protein kinase C, the activation of G protein-coupled receptors (GPCRs) is one of the most potent activators of ectodomain shedding. Ligand stimulation of GPCRs triggers the activation of ADAM proteases, leading to ectodomain shedding of pro-HB-EGF in cancer cells or peripheral organs [24–27]. However, the molecular nature of this process remains to be characterized in the nervous system. In the present study, first we examined if neural activity influences shedding of pro-HB-EGF or exocytosis of HBEGF in the brain. Second, we investigated the molecular nature of the trigger(s) for the ectodomain shedding of pro-HB-EGF from neurons. To overcome technical difficulties associated with monitoring HB-EGF in vivo, as the receptor traps most of HB-EGF following shedding, we utilized transgenic mice that over-express human proHB-EGF gene in the brain, and primary cultured neurons that express alkaline phosphatase-tagged pro-HB-EGF. Materials and methods Reagents. Glutamate, kainic acid, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), botulinum toxin (BoTX), 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), and (RS)-3,5-dihydroxyphenylglycine (DHPG) were all obtained from Wako Chemicals. 12-O-tetradecanoylphorbol-13-acetate (TPA) was purchased from Calbiochem Inc. (Tokyo, Japan). BAPTA and GM6001 were obtained from Chemicon Int. (CA, USA). KB-R7785 was obtained from Nippon Organon K.K. (Osaka, Japan). The goat anti-HB-EGF neutralizing antibody was obtained from R&D Systems (Minneapolis, MN, USA). Transgenic mice. The full-length human pro-HB-EGF gene was subcloned into a vector carrying the mouse a-CaMK II promoter [28]. After removal of most vector sequences, lineralized DNA was microinjected into fertilized mouse eggs at the Nihon SLC Co. (Aichi, Japan). Male transgenic mice carrying human HB-EGF gene were mated with female C57B6L/J mice to yield mice that were heterozygous for the human proHB-EGF transgene. Genotyping of founders was performed by PCR using genomic DNA from samples of tail with the primers: CGATCACT AGTAACGGCCGC and GTGGATACAGTGGGAGGGTC. All the animal experiments were approved by the Animal Use and Care Committee of Niigata University. Southern blot analysis. Southern blotting was performed to characterize the integrated HB-EGF transgene. Briefly, genomic DNA isolated from the mutant offspring was digested with PstI, separated on a 1% agarose gel, transferred to a nylon membrane (Amersham–Pharmacia), and fixed by cross-linking with UV irradiation and by baking at 80 C for

5 min. The filter was pre-hybridized for 2 h and hybridized overnight at 42 C in 50% formamide hybridization buffer with 0.5% SDS, 1% blocking reagent, and 10 ng/ml of probe. Following hybridization, the filter was washed twice for 20 h at 42 C in 50% formamide, 2 · sodium chloride/ sodium citrate buffer (SSC), 5· Denhardt’s solution, and 1% sodium dodecyl sulfate (SDS) followed by washing with 0.1· SSC, 0.1% SDS at 60 C. The probe was generated from a 0.5-kb DNA fragment (Fig. 4A) external to the targeting vector labeled with [32P]dCTP using the random primed DNA labeling kit (Boehringer–Mannheim). Signal was visualized with BAS2000 analyzer (Fuji Film, Tokyo, Japan). Human pro-HB-EGF protein levels were verified by immunoblotting for all mice used for analysis. Cell culture. Whole cerebral neocortices and hippocampi of embryonic rats (Sprague-Dawley, embryonic day 18–19) were mechanically dissociated and plated onto poly-D-lysine-coated dishes at a density of 1000– 1500 cells/mm2. Primary neurons were grown with Dulbecco’s modified Eagle’s medium containing 1 mM glutamine (Ultrapure, Ajinomoto, Tokyo, Japan) and 10% fetal bovine serum [29]. The following day, the medium was replaced with serum-free medium containing nutrient mixture N2 (100 lg/ml of transferrin, 5 lg/ml of bovine insulin, 16 lg/ml of putrescine, 20 nM progesterone, and 30 nM sodium selenite), and 10 mM Hepes (pH 7.3) (serum-free N2 medium). Primary cultures of glial cells were prepared from embryonic rats (Sprague–Dawley, embryonic day 18– 19). Briefly, whole neocortices of rats were mechanically dissociated and plated onto poly-D-lysine-coated dishes. Dissociated cells were first grown for 7 days with Dulbecco’s modified Eagle’s medium containing 1 mM glutamine and 10% fetal calf serum. Stimulation of cells was carried out as follows. Neuronal cultures are rinsed with Hank’s buffered saline (HBS) and challenged for 30 min with glutamate (100 lM), kainic acid (50 lM), NMDA (100 lM in either presence or absence of magnesium), AMPA (100 lM), ACPD (500 lM), DHPG (10 lM), dopamine (500 lM), serotonin (500 lM), and TPA (100 ng/ml). Some cultures were preincubated with BoTX (100 nM), BAPTA (3 mM), GM6001 (25 lM), or KB-R7785 (5 lM) 10 min before drug challenge [5]. KB-R7785 was selected as one of the most potent inhibitors of ErbB1 ligand shedding [25]. Western blot analysis. Protein samples for Western blotting were prepared from mouse tissues or from cultured neurons. These samples were homogenized in 2% SDS by sonication and denatured in the presence of 100 lM dithiothreitol (Wako) at 95 C for 5 min. Protein concentrations were determined by a micro-BCA kit (Protein Assay Reagent; Pierce). Twenty micrograms of protein was subjected to SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher and Schuell) by electrophoresis. The membrane was probed with anti-HBEGF neutralizing antibody (1:1000, Santa Crutz, CA). After extensive washing, the immunoreactivity on the membrane was detected with an anti-goat immunoglobulin conjugated to horseradish peroxidase, followed by a chemiluminescence reaction (ECL kit; Amersham, UK). The immunoreactivity of the bands was quantified by densitometric analysis. Enzyme immunoassay. Soluble HB-EGF levels were measured by sandwich enzyme immunoassay (EIA), as described previously [8]. To determine HB-EGF concentrations, brain tissues were homogenized in 20–50 vol. of homogenization buffer (phosphate-buffered saline, PBS) containing protease inhibitors (200 kallikrein U/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzethonium chloride, 1 mM benzamidine (Sigma Chemical Co, St. Louis, MO, USA), and 1 mM ethylenediaminetetraacetic acid). Brain homogenates were centrifuged at 14,000g for 30 min at 4 C and the supernatants were stored at 80 C until use. The protein concentrations in the samples were determined using a Micro BCA kit (Pierce, Rockland, IL, USA). Tissue extracts were loaded into wells of EIA titer plates that had been coated with anti-hHB-EGF (R&D Systems, Minneapolis, MN, USA) antibodies (as a primary antibody). The biotinylated secondary antibodies were detected using streptavidin–b-galactosidase (1: 10,000, Sigma, St. Louis, MO, USA). The bgalactosidase activity retained in each well was measured by incubation with 200 mM 4-methylumbelliferyl-b-D-galactosidase (Sigma). The amount of the resulting fluorescent product was monitored using a Fluorolite-1000 fluorometer (Dynatech Laboratories, Chantilly, VA, USA) with excitation at 364 nm and emission at 448 nm.

Y. Shishido et al. / Biochemical and Biophysical Research Communications 348 (2006) 963–970 Detection of phosphorylated EGF receptors. Protein extracts were prepared from the brains of transgenic mice or wild-type mice at 8 weeks of age. Kainic acid (20 mg/kg) was intraperitoneally injected to mice to induce seizures. Thirty minutes after seizures emerged, mice were sacrificed by cervical translocation and brain was dissected out. For immunoprecipitation, each brain was homogenized in 10 vol. of lysis buffer (50 mM Tris–HCl buffer (pH 7.4) with 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 lg/ml aprotinin, 1 lg/ml leupeptin, 1 lg/ml pepstatin, 1 mM Na3VO4, and 1 mM NaF). Alternatively, tissue was homogenized with 2· sodium dodecyl sulfate (SDS) sample buffer (100 mM Tris–HCl (pH 6.8) buffer, 4% SDS, 100 mM dithiothreitol, 20% glycerol, and 0.0001% bromophenol blue). After centrifugation at 14,000 rpm for 20 min, supernatants were recovered and the protein content was determined using a Micro BCA kit (Pierce Chemical, Rockland, IL, USA). Each brain lysate (1 mg protein) was incubated with 2 lg of anti-ErbB1 antibody (Upstate Biotechnology, Lake Placid, NY, USA) at 4 C overnight. Protein G–agarose beads (100 ll; Amersham–Pharmacia, Tokyo, Japan) were then added, and the mixture was incubated for 1 h. The beads carrying the bound immune complexes were pelleted at 5000 rpm for 5 min, washed three times with the lysis buffer, and resuspended with 100 ll of 2· SDS sample buffer. Protein extracts were subjected to SDS–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher and Schull, Germany) by electrophoresis. The membrane for immunoprecipitates was probed with an antiphosphotyrosine antibody (4G10, Upstate Biotechnology). After extensive washing, the immunoreactivity on the membrane was detected using antimouse immunoglobulin conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and chemiluminescence (ECL kit, Amersham–Pharmacia). Phosphatase assay for pro-HB-EGF shedding. The expression vector of HB-EGF fused with human placental alkaline phosphatase (AP) has been characterized previously [30]. Primary cultured neurons were prepared as described above and grown in 24-well plates at a density of 2 · 105 cells/ well. Neurons were transfected with the vector carrying the cDNA for APtagged pro-HB-EGF with the calcium phosphate method. After 2 days, cultured neurons were challenged with chemical reagents or neurotransmitters described above for 60 min. A 0.1 ml aliquot of culture supernatant containing AP-tagged HB-EGF was harvested and heated for 10 min at 65 C in order to inactivate endogenous alkaline phosphatase activity. Culture supernatant was incubated with an equal volume of substrate solution (1 M diethanolamine, 0.01% MgCl2, and 1 mg/mL p-nitrophenyl phosphate, pH 9.8) and optical absorbance of the enzyme products was measured at 405 nm using a microplate reader. Statistics. All the data are presented as means ± SEM. Statistical significance was evaluated with either Student’s t-test or two-way repeated measures analysis of variance (ANOVA).

Results Glutamate receptor stimulation triggers pro-HB-EGF shedding in culture To detect ectodomain shedding of pro-HB-EGF from cultured neurons, we tagged the extracellular domain of pro-HB-EGF gene with human placental alkaline phosphatase (AP) and transfected the cDNA for the AP-tagged pro-HB-EGF gene into neuron-enriched cultures prepared from rat embryos [30]. Two days after transfection, cortical or hippocampal neurons were challenged with various types of neurotransmitters or their analogues. The AP enzyme activity recovered in culture supernatants was measured (Fig. 1A). Application of glutamate, kainic acid, and NMDA (in the absence of Mg2+) increased the AP activity

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Fig. 1. Effects of neurotransmitter challenge on shedding of pro-HB-EGF in neuron-enriched cultures. Efficacy of pro-HB-EGF shedding was estimated in cortical (A) and hippocampal (B) cultures. Cultured neurons were prepared from embryonic neocortex and hippocampus, and transfected with an expression vector carrying AP-tagged pro-HB-EGF. Two days after transfection, cultures were treated with 100 lM glutamate (Glu), 50 lM kainic acid (KA), 100 lM AMPA, 100 lM NMDA, 500 lM ACPD, 10 lM DHPG, or 100 ng/ml TPA (positive control) for 60 min. AP activity in the culture supernatant was measured. Each bar represents mean ± SEM of six independent cultures. *P < 0.01 for the stimulus effect.

in the culture medium 2.9-, 3.5-, and 3.0-fold, respectively. In contrast, AMPA and metabotropic glutamate receptor agonists (ACPD, DHPG) failed to increase the AP activity in culture supernatants (Fig. 1A). Dopamine and serotonin (both 500 lM), both of which activate GPCRs, also did not influence the AP activity in supernatants (data not shown). In cultured hippocampal neurons, glutamate, NMDA, and kainate similarly increased AP activity in the culture medium (Fig. 1B). Thus, glutamate-related ligands for ionotropic glutamate receptors, but not for metabotropic receptors, activate neuronal pro-HB-EGF shedding or release of the resultant HB-EGF. Ectodomain shedding of pro-HB-EGF and exocytosis of HB-EGF from neurons The increase in release of AP-tagged HB-EGF following stimulation could involve exocytosis as well. It is uncertain whether ectodomain shedding occurs in vesicles destined for exocytosis, or if pro-HB-EGF shedding occurs on cell surfaces. Using various inhibitors for exocytosis or shedding, we examined the contribution of shedding to the AP activity recovered in culture supernatants. Application

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of kainate to culture liberated the AP activity from cortical and hippocampal neurons (Fig. 2). The amount of AP activity remaining in cortical cells was approximately less than 30% of total cellular AP activity before stimulation (data not shown). The metalloproteinase inhibitor, GM6001 [31], and the relatively specific inhibitor for ADAM17 (TACE), KB-R7785 [32], fully inhibited the kainate-induced release of AP-tagged HB-EGF into the medium of neocortical cultures (Fig. 2A). Therefore, we assume that release of AP-tagged HB-EGF from cortical neurons mainly represents ectodomain shedding of pro-HB-EGF. In contrast to the results with cortical cultures, these inhibitors were less effective at blocking the release of AP-tagged HB-EGF in hippocampal cultures. KB-R7785 and GM6001 partially attenuated the release of AP-tagged HB-EGF by half. We also estimated the contribution of vesicular exocytosis to the AP release. To inhibit exocytosis, we employed the Ca2+ chelator BAPTA [33] and the exocytosis blocker BoTX [34]. Extracellular depletion of Ca2+ with BAPTA reduced the amounts of AP-tagged HB-EGF in culture supernatant of glutamate-challenged cortical and hippocampal neurons to half, compared with that observed with Fig. 3. A Ca2+ chelator and an exocytosis inhibitor attenuate release of pro-HB-EGF following glutamate stimuli. Primary neuronal cortical (A) and hippocampal (B) cultures were prepared and transfected with an expression vector carrying AP-tagged pro-HB-EGF as described above (see Fig. 1). Cultures were pre-treated with or without 3 mM BAPTA or 100 nM BoTX for 30 min and then treated with 100 lM glutamate (Glu). AP activity in culture supernatant was measured. Each bar represents mean ± SEM of eight experiments. *P < 0.01 in comparison with untreated control cultures.  P < 0.05 in comparison with the cultures stimulated with glutamate alone.

glutamate stimulation alone (Fig. 3), although we cannot rule out the possibility that BAPTA also depleted other metal ions and inhibited shedding enzymes. BoTX is a more specific inhibitor for regulated vesicular release. BoTX significantly attenuated the HB-EGF release following glutamate challenge in hippocampal culture. In cultured neocortical neurons, however, the amount of AP release triggered by the stimulation of BoTX plus glutamate was not significantly different from that triggered by glutamate alone. Again, the regulation of HB-EGF release appeared to differ between neocortical and hippocampal neurons. Fig. 2. Metalloproteinase inhibitors attenuate shedding of pro-HB-EGF. Primary neuronal cultures from the neocortex (A) and hippocampus (B) were prepared and transfected with an expression vector carrying APtagged pro-HB-EGF as described above (See Fig. 1). Cultures were pretreated with or without 5 lM KB-R7785 (KBR) or 25 lM GM6001 (GM) and then treated with 50 lM kainate (KA) for 30 min. AP activity in culture supernatant was measured. Each bar represents mean ± SEM of seven experiments. *P < 0.01 in comparison with untreated control cultures.  P < 0.05 in comparison with the cultures stimulated with kainate alone.

Shedding of pro-HB-EGF triggered by seizure activity in vivo To monitor shedded HB-EGF in vivo and compensate for the absorption of this ligand by ErbB1, we generated transgenic mice that over-express the pro-HB-EGF gene in forebrain neurons. The transgene encoded human proHB-EGF driven by the mouse alpha-calmodulin-dependent protein kinase II (alpha-CaMK II) promoter (Fig. 4A). Among 32 mouse lines produced, Southern blots

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bands were markedly decreased in the neocortex compared to non-treated animals (Fig. 5A). In the hippocampus, there were similar reductions in pro-HB-EGF-like immunoreactivity (Fig. 5B). In parallel, we monitored mature HB-EGF levels in brain extracts with a sensitive two-site enzyme immunoassay. Kainate-induced seizures significantly elevated HB-EGF levels 3.7- and 3.4-fold in the neocortex and hippocampus, respectively (Fig. 5C). These results suggest that excitatory neural activity promotes the conversion of pro-HB-EGF to HB-EGF in the brain. We also examined whether the in vivo production of HBEGF leads to neighboring receptor activation. To examine ErbB1 activation we immunoprecipitated the receptor from brain tissue of seizure-induced animals and probed its immunoblot with anti-phosphotyrosine antibody (Fig. 6). There were marked increases in phospho-ErbB1 levels in

Fig. 4. Generation of pro-HB-EGF transgenic mice. Transgenic mice that express full-length human prepro-HB-EGF mRNA were generated with the vector carrying a promoter for mouse aCaMKII joined to a 5 0 -intron and a 3 0 -intron plus polyA signal from SV40. (A) DNA fragment of 10.3 kb was introduced into the pronucleus of mouse fertilized eggs by standard procedures. A box with slanted lines indicates the probe region for Southern blotting. (B) Tail DNA of transgenic mice was subjected to Southern blotting for human HB-EGF cDNA. (C) Protein extracts were prepared from the whole brains of transgenic mice (6 lines) at 8 weeks old, and subjected to Western blot analysis with a goat anti-human HB-EGF antibody (the left panel). Tissue distributions of pro-HB-EGF proteins in the mouse line #10–2 are shown in the right panel. CB, cerebellum; CT, neocortex; ST, striatum; HP, hippocampus; Ky, kidney; and Ht, heart. Note: Pro-HB-EGF protein was preferentially expressed in the hippocampus and prefrontal cortex.

revealed that seven lines carried the transgene(s). Western blots revealed that five lines expressed human pro-HBEGF protein in the brain (Fig. 4B). We focused on transgenic line #10–2, one line that properly expressed the pro-HB-EGF protein in the brain in an alpha-CaMK II promoter-dependent manner (Fig. 4C). The large molecular weights of HB-EGF-like immunoreactivities (18– 30 kD) on blots were consistent with the reported sizes of pro-HB-EGFs that are susceptible to ectodomain shedding [16,34]. Using this transgenic mouse line over-expressing proHB-EGFs, we examined whether neuronal activity can influence ectodomain shedding of pro-HB-EGF in vivo. Thirty minutes after chronic seizures initiated by kainate injection, the intensities of the putative pro-HB-EGF

Fig. 5. Kainate-induced seizures trigger shedding/release of pro-HB-EGF protein. Protein extracts were prepared from frontal cortex (A) or hippocampus (B) of transgenic mice. Western blots were probed with the anti-HB-EGF antibody, as shown inset. Pro-HB-EGF immunoreactivity was measured with the quantitative analysis software NIH image. Each bar represents mean ± SEM of five experiments. (C) Levels of HB-EGF protein were measured in the KA-treated and vehicle-treated transgenic mice (line #10–2). Tissue homogenates were cleared by ultracentrifugation and soluble fractions were subjected to EIA for HB-EGF. Solid bar, KAtreatment; open bar, controls. PFC, prefrontal cortex; HIP, hippocampus. Each bar represents the mean ± SEM of five experiments. *P < 0.01 for the stimulus effect.

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Fig. 6. Kainate administration enhances EGF receptor phosphorylation in the prefrontal cortex and hippocampus. Transgenic mice (line #10–2) were treated with 20 mg/kg of kainic acid to induce seizures for 30 min. Protein extracts were prepared from the frontal cortex (A) or hippocampus (B) of transgenic mice and subjected to immunoprecipitation with the anti-ErbB1 antibody. Western blots were probed with an anti-phosphotyrosine antibody as shown in the upper panel. The phospho-ErbB1 immunoreactivity was measured with the quantitative analysis software NIH image. Each bar represents mean ± SEM of five experiments. *P < 0.01 for the stimulus effect.

the neocortex and hippocampus following seizure induction in pro-HB-EGF-over-expressing transgenic mice. These results indicate that excitatory neurotransmission can trigger ectodomain shedding of pro-HB-EGF in the brain. Discussions Neurons express various members of the EGF superfamily such as TGFalpha and HB-EGF [6–8]. Although all members in the EGF family are initially produced as membrane-anchored precursors and require ectodomain shedding for their maturation, the shedding of their precursors in the CNS is poorly understood. The present study characterizes the regulation of pro-HB-EGF shedding in vitro and in vivo. We employed transgenic mice overexpressing pro-HB-EGF in in vivo experiments and a fusion protein construct for alkaline phosphatase-tagged pro-HB-EGF in culture examinations. With these tools we were able to monitor HB-EGF release in neuronal cultures and brain homogenates. We conclude that, among several neurotransmitters, glutamate is the most potent agent that triggers ectodomain shedding of pro-HB-EGF. This process involves the activation of NMDA-type or kainate-type glutamate receptors. In addition, seizure activity

induces ectodomain shedding of pro-HB-EGF in vivo. Accordingly, excitatory neurotransmission appears to play a crucial role in regulating ectodomain shedding of pro-HB-EGF in the CNS. In non-neuronal cells, ectodomain shedding of pro-HBEGF generally occurs on cell surfaces. However, it remains to be determined whether ectodomain shedding occurs in exocytosis vesicles and/or on cell surfaces in neurons. The ineffectiveness of the exocytosis blocker, BoTX, and the effectiveness of the shedding inhibitors for cortical HBEGF release suggest that most of pro-HB-EGF was located and subjected to ectodomain shedding on cell surfaces. In contrast to cortical cultures, HB-EGF release from hippocampal neurons exhibited different sensitivities to the inhibitors for exocytosis and shedding. The exocytosis blocker as well as the shedding inhibitors attenuated the release of the AP tag. The partial inhibition of HB-EGF release may reflect pre-existing vesicular pools of mature HB-EGF. These findings suggest that neuronal release of HB-EGF involves the activation of metalloproteinase-mediated shedding, although regulation of HB-EGF release considerably differs among types of neurons. Metalloproteinases in the ADAM family are potential candidates for the proteases involved in the ectodomain shedding of pro-HB-EGF. ADAM10 and ADAM17 (TACE) are expressed in the hippocampus, neocortex, and cerebellum [36]. These enzymes are suggested to function as an alpha-secretase and participate in the processing of beta-amyloid precursor protein. We detected significant levels of mRNAs for ADAM 10 and 17, but not for ADAM12, in cortical cultures (data not shown). From pharmacological results with ADAM inhibitors we speculate that TACE is activated by glutamate and promotes ectodomain shedding of pro-HB-EGF in brain neurons. Although ligands for GPCRs often activate these metalloproteinases [30,37], dopamine, serotonin, and metabotropic glutamate receptor agonists all failed to trigger ectodomain shedding of pro-HB-EGF. We did not expect these results in the present study. Future studies will be required to determine if neurons may have unique regulatory mechanism(s) or intracellular signals that control the activation of ADAM metalloproteinases following the activity of ionotropic glutamate receptors [35]. Pro-HB-EGF is expressed in the subventricular zone [9], suggesting that shedding of pro-HB-EGF might regulate the proliferation and migration of neural progenitors or their immediate decedents that contribute to post-injury repair in the brain [13,14]. In addition, HB-EGF has unique biological activities on developing neurons; it negatively influences the expression of AMPA receptors, potassium channels, and synaptic proteins [38–40], and stimulates dopaminergic development and survival [11]. Thus, the regulated ectodomain shedding of pro-HBEGF may contribute to activity-dependent brain development or organization as well. Ozaki et al. have reported that the neural activity required for the shedding of proneuregulin-1 also leads to synaptic plasticity [21]. Similarly,

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a tight regulatory link between shedding process of proHB-EGF and excitatory neurotransmission presumably indicates its contribution to synaptic or developmental plasticity of the central nervous system. Acknowledgments We are grateful to Nippon Organon K.K. and Dr. S. Kozaki for providing us with KB-R7785 and BoTX, respectively. We also thank Dr. E. Mekada for discussion and suggestions. This study was supported by Grant-inAid for Basic Scientific Research (B) from Japan Society for the Promotion of Science, Grants for Promotion of Niigata University Research Projects, and Core Research for Evolutional Science and Technology from Japan Science and Technology Corporation. References [1] R. Iwamoto, E. Mekada, ErbB and HB-EGF signaling in heart development and function, Cell Struct. Funct. 31 (2006) 1–14. [2] S. Higashiyama, D. Nanba, ADAM-mediated ectodomain shedding of HB-EGF in receptor cross-talk, Biochim. Biophys. Acta 1751 (2005) 110–117. [3] S. Yamazaki, R. Iwamoto, K. Saeki, M. Asakura, S. Takashima, A. Yamazaki, R. Kimura, H. Mizushima, H. Moribe, S. Higashiyama, M. Endoh, Y. Kaneda, S. Takagi, S. Itami, N. Takeda, G. Yamada, E. Mekada, Mice with defects in HB-EGF ectodomain shedding show severe developmental abnormalities, J. Cell Biol. 163 (2003) 469–475. [4] Y. Umeda, Y. Miyazaki, H. Shiinoki, S. Higashiyama, Y. Nakanishi, Y. Hieda, Involvement of heparin-binding EGF-like growth factor and its processing by metalloproteinases in early epithelial morphogenesis of the submandibular gland, Dev. Biol. 237 (2001) 202–211. [5] S. Tokumaru, S. Higashiyama, T. Endo, T. Nakagawa, J.I. Miyagawa, K. Yamamori, Y. Hanakawa, H. Ohmoto, K. Yoshino, Y. Shirakata, Y. Matsuzawa, K. Hashimoto, N. Taniguchi, Ectodomain shedding of epidermal growth factor receptor ligands is required for keratinocyte migration in cutaneous wound healing, J. Cell Biol. 151 (2000) 209–220. [6] K. Mishima, S. Higashiyama, Y. Nagashima, Y. Miyagi, A. Tamura, N. Kawahara, N. Taniguchi, A. Asai, Y. Kuchino, T. Kirino, Regional distribution of heparin-binding epidermal growth factorlike growth factor mRNA and protein in adult rat forebrain. 1, J. Neurosci. Lett. 213 (1996) 153–156. [7] Y. Hayase, S. Higashiyama, M. Sasahara, S. Amano, T. Nakagawa, N. Taniguchi, F. Hazama, Expression of heparin-binding epidermal growth factor-like growth factor in rat brain, Brain Res. 784 (1998) 163–178. [8] Y.S. Piao, Y. Iwakura, N. Takei, H. Nawa, Differential distributions of peptides in the epidermal growth factor family and phosphorylation of ErbB 1 receptor in adult rat brain, Neurosci. Lett. 390 (2005) 21–24. [9] T. Nakagawa, M. Sasahara, Y. Hayase, M. Haneda, H. Yasuda, R. Kikkawa, S. Higashiyama, F. Hazama, Neuronal and glial expression of heparin-binding EGF-like growth factor in central nervous system of prenatal and early-postnatal rat, Dev. Brain Res. 108 (1998) 263– 272. [10] H.I. Kornblum, S.D. Zurcher, Z. Werb, R. Derynck, K.B. Seroogy, Multiple trophic actions of heparin-binding epidermal growth factor (HB-EGF) in the central nervous system, Eur. J. Neurosci. 11 (1999) 3236–3246. [11] L.M. Farkas, K. Krieglstein, Heparin-binding epidermal growth factor-like growth factor (HB-EGF) regulates survival of midbrain dopaminergic neurons, J. Neural. Transm. 109 (2002) 267–277.

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