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J. Biol. Chem., Vol. 279, Issue 47, 48808-48816, November 19, 2004
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1-induced Migration*
¶¶
From the
Department of Human and Animal Biology, University of Torino, Torino 10123, Italy, the ||Department of Medical Sciences, University of Piemonte Orientale, Novara 28100, Italy, and the **Department of Pharmacological Sciences, University of Milan, Milan 20133, Italy
Received for publication, July 23, 2004 , and in revised form, August 24, 2004.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 (NRG11) stimulus induced ErbB4 tyrosine phosphorylation and phosphatidylinositol 3-kinase (PI3K) recruitment and activation (as demonstrated by Akt phosphorylation) either directly (ErbB4 cyt1 isoform) or indirectly (ErbB4 cyt2 isoform). We examined the ability of the four ErbB4 isoforms to induce chemotaxis and cell proliferation in response to NRG11 stimulation. Using migration assays, we observed that only ErbB4-expressing cells stimulated with NRG11 showed a significant increase in migration, whereas the growth rate remained unchanged. Additional assays showed that inhibition of PI3K (but not of phospholipase C
) dramatically reduced migratory activity. Our data show that ErbB4 signaling via PI3K activation plays a fundamental role in controlling NRG11-induced migration. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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An interesting ErbB4 feature (recently reviewed in Ref. 3) is the existence of isoforms generated by alternative splicing (4). One isoform pair (5) is characterized by alternative splicing of exons located in the extracellular juxtamembrane region conferring (JMa), or not (JMb), susceptibility to proteolytic cleavage (6) by a member of the ADAM (a disintegrin and metalloprotease) family, the tumor necrosis factor-
-converting enzyme (7). ErbB4 proteolytic cleavage produces a membrane-associated 80-kDa fragment that can be degraded by proteasome activity following polyubiquitination (8) or that can be the substrate for subsequent -secretase cleavage, which releases the cytoplasmic domain from the membrane and allows, intriguingly, nuclear translocation of a fragment (9, 10) that can act as cotranscriptional activator of the Yes-associated protein (11, 12).
The other ErbB4 isoform pair (13) is characterized by the presence (cyt1) or absence (cyt2) of a cytoplasmic exon containing a docking site for phosphatidylinositol 3-kinase (PI3K). The existence of these isoforms suggests that potentially four isoforms may exist and evoke functional differences. Actually, in stable NIH3T3 transfectants, it has been shown that the ErbB4 isoform that does not activate PI3K (cyt2) mediates proliferation, but not survival or chemotaxis, as does cyt1 (13).
ErbB4 ligands belong to two groups: the neuregulins (NRG1–4), also termed heregulins, and some members of the EGF family (betacellulin, epiregulin, and HB-EGF). NRG1 and NRG2 exist in a number of splicing isoforms and recognize also ErbB3 as a binding site, whereas NRG3 and NRG4 interact, with lower affinity, only with ErbB4 (1). In most assays, NRG1 with a -type EGF-like domain is 10–100 times more potent than NRG1 with an -type EGF-like domain. Moreover, experiments performed with mice with targeted mutations showed that NRG1 is involved in nervous system and cardiac development, whereas NRG1 is involved in breast development (reviewed in Ref. 14).
In the central nervous system, NRG1 is expressed by both neurons and glia (15–17), and ErbB4 is expressed in various part of the brain and nervous system (18–22), including the retina (23), the hippocampus (24, 25) and, as we (26, 27) and others (22) have previously shown, the olfactory bulb. Interestingly, cells coming out of the rostral migratory stream of the subependymal layer as well as isolated cells in the granule cell layer, possibly migrating cells, strongly express ErbB4, and ErbB4 immunoreactivity is found in all the periglomerular and mitral/tufted cells of the olfactory bulb (26, 28). Furthermore, ErbB4 is preferentially expressed by interneurons that migrate tangentially from the ventral to the dorsal rat telencephalon (29). ErbB4 knockout mice exhibit defects in cranial neural crest migration and cranial nerve segregation in the developing hindbrain, but die at embryonic day 10.5 because of abnormal heart development (30, 31). Recently, heart defects were rescued in mutant ErbB4 mice by expressing ErbB4 under the control of a cardiac-specific myosin promoter (32). Rescued mice display aberrant cranial nerve architecture and an increased number of large interneurons within the cerebellum. Expression of a dominant-negative ErbB4 construct in primary cultures of neuronal cells showed an additional ErbB4 requirement for cell migration in the developing cerebellum: the phenotype described includes defects in the migration of cerebellar granule cells (expressing NRGs) along radial glial fibers (usually expressing ErbB4) (33).
All of this evidence concerning a role for ErbB4 in cell migration in the nervous system prompted us to find a suitable in vitro model in which to investigate more deeply the involvement of ErbB4 in migration. For this purpose, we chose ST14A, an ErbB1/ErbB2/ErbB3-expressing cell line derived from embryonic day 14 rat striatal primordia by retroviral transduction of a temperature-sensitive allele of the SV40 large T antigen (34). In this model, cell division and expression of nestin persist at 33 °C, the permissive temperature, whereas at the non-permissive temperature of 39 °C, cell division ceases, nestin expression decreases, and microtubule-associated protein-2 expression increases (35). Unlike cells derived from somatic fusion (36), they contain a normal complement of chromosomes without contamination from a neuroblastoma cell line. Following intracerebral grafting procedures, ST14A cells develop into a high percentage of microtubule-associated protein-2- and glial fibrillary acidic protein-positive cells, suggesting that they behave as multipotent neural progenitors (37). These properties, together with the stability of their phenotype over several years since derivation, associated with the feasibility of their engineering and subcloning, identify them as an appropriate model for molecular and biochemical investigations of gene function (38–40).
In this study, we engineered ST14A cells to express ErbB4 and demonstrate that ErbB4 expression is necessary to confer on neural progenitor cells the ability to respond to NRG11 through migration. This response is shown to be mediated by PI3K activation either directly or indirectly through ErbB3 heterodimerization.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The ST14A cell line was derived from primary cells dissociated from embryonic day 14 rat striatal primordia and conditionally immortalized by retroviral transduction of the temperature-sensitive variant tsA58/U19 of the SV40 large T antigen (34). Cells were cultured on dishes (BD Biosciences) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100 units/ml penicillin, 0.1 mg/ml streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, and 10% heat-inactivated fetal bovine serum (FBS; Invitrogen). Stable transfectants were grown in medium containing 5 µg/ml puromycin (Sigma). Cells were grown as monolayers at the permissive temperature of 33 °C in a 5% CO2 atmosphere saturated with H2O. Cells were allowed to grow to near confluence, and adherent cells were harvested by the trypsin/EDTA method.
The EGF-like domain of mouse NRG11 was produced in our laboratory as a His-tag fusion protein in Escherichia coli (41). The PI3K inhibitor LY294002 was dissolved in dimethyl sulfoxide (20 mM stock solution) and stored at –20 °C. The phospholipase C inhibitor U73122
[GenBank]
was dissolved in chloroform according to the manufacturer's instructions to a final concentration of 2 mM, divided in aliquots, lyophilized, stored at –20 °C, and resuspended in dimethyl sulfoxide just before use to obtain a 2 mM stock solution. In all experiments, control samples received the same concentration of dimethyl sulfoxide or other vehicle.
Polyclonal anti-ErbB4 (C-18, sc-283) and monoclonal anti-phosphotyrosine (PY99) primary antibodies were from Santa Cruz Biotechnology. Polyclonal anti-PI3K p85 antibody was from Upstate Biotechnology, Inc. Polyclonal anti-phospho-Ser473 Akt and anti-Akt antibodies were from Cell Signaling. Horseradish peroxidase-linked donkey anti-rabbit and sheep anti-mouse secondary antibodies were from Amersham Biosciences.
Proliferation Assay—The proliferation assay was performed according to the protocol described by Kueng et al. (42). Briefly, cells were plated in 200 µl of DMEM containing 10% FBS at a density of 1000 cells/well in 96-well plates. The following day (t = 0), the medium was replaced with 2% FBS-containing DMEM with or without 5 nM NRG11 (100 µl/well, 8 wells/treatment). For each plate, an 8-well line without cells was treated as the entire plate and used as a blank in the following microplate reader analysis. At t = 0, a 96-well plate was fixed and used as a cell growth starting point. Cell growth was subsequently calculated at 2 (t = 48 h), 4 (t = 96 h), and 6 (t = 144 h) days after the initiation of treatments. Briefly, the medium was removed, and cells were fixed by addition of 100 µl of 2% glutaraldehyde in phosphate-buffered saline (PBS). After being shaken (200 cycles/min) for 20 min at room temperature, plates were washed five times by submersion in deionized water and air-dried for at least 24 h. Plates were then stained by addition of a solution (100 µl/well) containing 0.1% crystal violet dissolved in freshly prepared 200 mM boric acid (pH 9.0). After being shaken (200 cycles/min) for 20 min at room temperature, plates were washed five times by submersion in deionized water and air-dried for at least 24 h. Bound dye was solubilized by addition of 10% acetic acid (100 µl/well) and 5 min of shaking at room temperature. The absorbance of dye extracts was measured directly in plates using a Microplate Reader (Bio-Rad) at a wavelength of 590 nm.
Reverse Transcription (RT)-PCR and Cloning of Rat ErbB4 cDNAs—Total RNA was prepared from olfactory bulbs obtained from one adult Wistar rat (Charles River Laboratories) using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Total RNA (1 µg) was subsequently reverse-transcribed to cDNA in a total volume of 25 µl using 200 units of reverse transcriptase (U. S. Biochemical Corp.) in 1x buffer (U. S. Biochemical Corp.) with 7.5 µM random exanucleotide primers (Amersham Biosciences), 0.05% Triton X-100, 0.5 mM dNTPs (Amersham Biosciences), 0.1 µg/µl acetylated bovine serum albumin (Amersham Biosciences), and 33 units of RNasin (Amersham Biosciences). As a control, an enzyme-less reaction was performed for each sample. PCRs were carried out in a total volume of 50 µl containing 5 µl of cDNA, enzyme-less reaction sample, or water (negative controls), 250 nM each 5'- and 3'-primers (see below), 2.5 units of PfuTurbo® DNA polymerase (Stratagene), 100 µM dNTPs, and 5% glycerol. The primers used (produced by Sigma) were designed according to the GenBankTM/EBI rat ErbB4 sequence (accession number NM_021687 [GenBank] ; corresponding to the JMa-cyt1 isoform, herein numbered considering the start site as +1): primer ErbB4-11 (5'-TGCTAGCCAA-AAATGAAGCTGGCG-3', with the artificial NheI site underlined and the ATG codon in boldface) and primer ErbB4-4 (5'-GTTTCTCCTTCA-GGTACCCAGATTCC-3') to obtain the amplicons corresponding to nucleotides –7 to +2228 and primer ErbB4-5 (5'-GCACAGCACCCAAT-CAAGCTC-3') and primer ErbB4-6 (5'-TACTGGCCTTGGGGTAGAA-GGAAG-3') to obtain the amplicons corresponding to nucleotides +2105 to +4025. Samples were amplified using a simplified touchdown PCR protocol: denaturation at 95 °C for 5 min; followed by 10 cycles of denaturation at 95 °C for 30 s, annealing at 66 °C for 30 s, and extension at 72 °C for 3 min; followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 3 min. The extension step of the last cycle was increased to 30 min. PCR products were separated by electrophoresis on a 0.8% agarose gel, stained with ethidium bromide, and visualized with UV light. A 1-kb DNA ladder (Invitrogen) or BenchTop pGEM DNA markers (Promega) was used as a size marker. PCR products were purified from agarose gel using the GenEluteTM gel purification kit (Sigma). To obtain the full-length ErbB4 cDNAs, the previously obtained amplicons (nucleotides –7 to +2228 and nucleotides +2105 to +4025) were used as templates in the presence of primers ErbB4-11 and ErbB4-6 in recombinant PCR according to the following protocol: denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 63 °C for 30 s, and extension at 72 °C for 8 min. The extension step of the last cycle was increased to 30 min. PCR products were separated by electrophoresis on a 0.8% agarose gel, purified from agarose gel using the GenEluteTM gel purification kit, and cloned into the pCR®-BluntII-TOPO® vector (Invitrogen) using the Zero Blunt® TOPO® PCR cloning kit (Invitrogen) following the manufacturer's instructions.
The DNA of 18 single colonies was obtained using the GenEluteTM plasmid miniprep kit (Sigma). For each clone, the orientation of the cDNA in the vector (sense or antisense) was detected by restriction enzyme digestions using EcoRI and BamHI (Amersham Biosciences). To identify the different ErbB4 isoforms (cyt1/cyt2 and JMa/JMb), individual sense clones were analyzed by PCR; the juxtamembrane domain (fragment +1773 to +2019) was amplified with primer ErbB-4-12 (5'-GAAATGTCCAGATGGCCTACAG-3') and primer ErbB4-13 (5'-AACGGCAAATGTCAGAGCCATG-3'), and the cytoplasmic domain (fragment +3054 to +3267) was amplified with primer ErbB4-1 (5'-T-GCTGAGGAATATTTGGTCCCCCA-3') and primer ErbB4-2 (5'-TCTG-GTATGGTGCTGGTTGTGGCT-3'). PCRs were carried out in a total volume of 50 µl containing 5 µl of DNA or water (negative controls), 250 nM each specific primer, 1 unit of RED TaqTM DNA polymerase (Sigma), 100 µM dNTPs, and 5% glycerol. Samples were amplified by denaturation at 95 °C for 3 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 63 °C (JMa/JMb) or 60 °C (cyt1/cyt2) for 30 s, and extension at 72 °C for 1 min. The extension step of the last cycle was increased to 20 min. To amplify the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment corresponding to nucleotides 544–732 (GenBankTM/EBI accession number M32599 [GenBank] ), PCRs were carried out using primer GAPDH1 (5'-TGGCATTGTGGAAGGGCTCA-TTGAC-3') and primer GAPDH2 (5'-ATGCCAGTGAGCTTCCCGTTC-AGC-3'); samples were amplified by denaturation at 95 °C for 3 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min. PCR products were separated by electrophoresis on 2% agarose gels. For all PCRs, the Thermal Cycler GeneAmp® PCR System 2400 (PerkinElmer Life Sciences) was used.
Sequencing of ErbB4 cDNAs—Two cDNA clones (3A and 3G) corresponding to the full-length JMa-cyt1 and JMa-cyt2 isoforms were completely sequenced on both strands (Genelab Laboratories, Rome, Italy). A cDNA clone (3E) corresponding to the JMb isoform was sequenced on both strands in the region between nucleotides –7 and +2196. The primers used for sequencing were designed according to the GenBankTM/EBI rat ErbB4 sequence (accession number NM_021687 [GenBank] ).
Expression Vectors—To generate stable cell lines expressing ErbB4 JMa-cyt1 and JMa-cyt2, the cDNAs corresponding to the two sequenced isoforms were subcloned into the pIRESpuro2 vector (Clontech); NheI-NotI fragments obtained from pCR®-BluntII-TOPO®-ErbB4 clones (containing the entire rat ErbB4 cDNAs) were subcloned into the NheI and NotI sites of pIRESpuro2. To generate stable cell lines expressing ErbB4 JMb-cyt1 and JMb-cyt2, a Csp45I-HpaI fragment containing the entire juxtamembrane region was obtained from the JMb-sequenced clone and used to replace the JMa region in clones pIRESpuro2-ErbB4-JMa-cyt1 and pIRESpuro2-ErbB4-JMa-cyt2, yielding pIRESpuro2-ErbB4-JMb-cyt1 and pIRESpuro2-ErbB4-JMb-cyt2, respectively. The rat ErbB3 expression vectors pcDNA3-B3 (43) and pcDNA3-B3-6F (44), in which codons corresponding to Tyr1051, Tyr1194, Tyr1219, Tyr1257, Tyr1273, and Tyr1286 were substituted with phenylalanine codons, were a generous gift of Dr. John G. Koland (Department of Pharmacology, University of Iowa, College of Medicine, Iowa City, IA).
Transfections—For stable transfections, growing ST14A cells were transfected in triplicate with 10 µg of DNA for each construct using 20 µl of LipofectAMINE and Opti-MEM I (Invitrogen) according to the manufacturer's recommendations. DNA was prepared using the GenEluteTM endotoxin-free plasmid maxiprep kit (Sigma). Cells transfected with the pIRESpuro2 vector alone were generated to be used as controls. Cells were cultured in DMEM as described above; and 48 h after transfection, three different doses of puromycin (5, 10, and 20 µg/ml) for each construct were added to the three plates. Individual clones were recovered and screened for their ErbB4 expression level by Western blotting as described below.
For transient transfections, growing COS-7 cells were transfected using specific combinations of expression plasmids as indicated. Briefly, 5 µg of each plasmid were mixed in 1 ml of PBS and added to 1 ml of PBS containing 800 µg/ml DEAE-dextran. COS-7 cells were washed twice with 10 ml of PBS, covered with the DNA/DEAE-dextran/PBS solution, and incubated for 40 min at 37 °C. Then, 10 ml of DMEM containing 10% FBS and 0.1 mM chloroquine were added to the cells, and the cells, spontaneously detached after severe resuspension, were split in two and incubated at 37 °C for 3 h. After removing the medium, cells were treated for 3 min with DMEM containing 10% FBS and 10% Me2SO, washed once with DMEM, and finally covered with DMEM containing 10% FBS. Twenty-four hours after plating, the cells were washed with PBS and serum-starved in DMEM for an additional 24 h before NRG11 stimulation.
Immunoprecipitation and Western Blotting—For immunoprecipitation experiments, confluent cells grown on 10-cm dishes were serumstarved overnight. The following day, 1 mM sodium orthovanadate was added to both the untreated and treated cells for at least 30 min prior to addition of 5 nM NRG11. After 10 min of NRG11 stimulation, cells were rinsed twice with ice-cold PBS containing 1 mM sodium orthovanadate and lysed for 20 min on ice with 500 µl of cold extraction buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 2 mM EGTA, 1 mM ZnCl2, 10% glycerol, and 1% Triton X-100 supplemented just prior to use with 50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Roche Applied Science)). For immunoprecipitations to be assayed for PI3K activity, cells were lysed with 500 µl of cold lysis buffer (25 mM Hepes (pH 8), 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, 2 mM EGTA, 1 mM ZnCl2, and 10% glycerol supplemented just prior to use with 50 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and the mixture of protease inhibitors) (45). Lysates were collected with a cell scraper, placed in microcentrifuge tubes, rocked at 4 °C for 10 min, and spun at 4 °C for 20 min at 11,000 x g to discard cell debris. Protein concentration was determined using the BCA kit for protein determination, and lysates (1.5–2 mg) were diluted to equal concentrations using extraction buffer (1 mg/ml). Agarose-conjugated protein A beads (50 µl; Santa Cruz Biotechnology) pre-washed three times with extraction buffer were preincubated for 45 min with primary antibody (1 µg/mg of protein), and lysates were added and incubated overnight with constant rotation at 4 °C. As a control, representative samples (NRG11-treated or not) were incubated with protein A beads without primary antibody. The next day, immunoprecipitates were spun at 4 °C for 1 min at 900 x g and rinsed four times with ice-cold extraction buffer. To release immunoprecipitates from the beads, 2x sample buffer (62.5 mM Tris (pH 6.8), 4% SDS, 480 mM 2-mercaptoethanol, and 40% glycerol) was added, and the proteins were denatured at 100 °C for 5 min. Proteins were resolved by 8% SDS-PAGE, transferred to a HybondTM C Extra membrane (Amersham Biosciences) following the manufacturer's instruction, and blocked for 1 h at 37 °C in 1x TBST (150 mM NaCl, 10 mM Tris-HCl (pH 7.4), and 0.1% Tween) plus 5% nonfat milk. The membranes were incubated overnight at 4 °C in primary antibodies diluted 1:500 in TBST plus 1% nonfat milk. The next day, they were rinsed four times with TBST for 5 min each at room temperature and incubated for 1 h at room temperature with horseradish peroxidase-linked anti-rabbit (1:3000) or anti-mouse (1:10,000) secondary antibody (diluted in TBST plus 1% nonfat milk). Membranes were rinsed four times with TBST for 5 min each at room temperature, and specific binding was detected by the enhanced chemiluminescence ECL system (Amersham Biosciences) using HyperfilmTM (Amersham Biosciences). In some cases, the membrane was stripped for 30 min at 50 °C in 0.05 M phosphate buffer (pH 6.5), 10 M urea, and 0.1 M 2-mercaptoethanol, blocked, tested with secondary antibody to verify accurate cleaning, and reprobed with the indicated primary antibodies for further analysis. The molecular masses of proteins were estimated relative to the electrophoretic mobility of cotransferred prestained molecular mass markers (Invitrogen and Bio-Rad).
Total Protein Extraction—Total proteins were extracted by lysing cells with extraction buffer as described above or by solubilizing cells in boiling Laemmli buffer (2.5% SDS and 0.125 M Tris-HCl (pH 6.8)), followed by 5 min of denaturation at 100 °C in 240 mM 2-mercaptoethanol and 18% glycerol. Protein concentration was determined by the BCA method, and equal amounts of proteins were loaded onto each lane, separated by SDS-PAGE, transferred to a HybondTM C Extra membrane, and analyzed as described above. Bands were quantified by densitometry using the Gel Doc image documentation system (Bio-Rad) with Quantity One software. Conclusions were drawn after experiments were repeated a minimum of three times.
PI3K Assays—PI3K activity was assayed in the ErbB4 immunoprecipitates in the presence of [-32P]ATP (Amersham Biosciences) and phosphoinositides (Sigma) as described previously (45, 46).
Motility Assay—The wound healing motility assay was used to measure two-dimensional movement. Cells were grown to confluence in 12-well plates and serum-starved for 24 h, and then a cross-shaped wound was made on the monolayers using a sterile 200-µl pipette tip. The cells were rinsed three times with PBS and placed in serum-free DMEM with or without 5 nM NRG11. To obtain good reproducibility, in all experiments, the left arm of the cross was photographed. Photos were taken at the initiation of the experiment (t = 0) and 24 h later (t = 24 h) using an Olympus IX50 inverted microscope equipped with a Cool SNAP-Pro CCD camera (Media Cybernetics, Silver Spring, MD); images were edited with Image Pro-Plus software (Media Cybernetics). The areas were measured using the measurement program included. The two-dimensional movement of the cells was quantified by measuring the surface area of the wound at t = 0 and comparing it with the surface area at t = 24 h: healing percentage = (1 – t24/t0) x 100.
Migration Assay—The Transwell migration assay was used to measure three-dimensional movement. Cells (105) resuspended in 200 µl of DMEM containing 2% FBS were seeded in the upper chamber of a Transwell (cell culture insert, no. 353097, BD Biosciences) on a porous transparent polyethylene terephthalate membrane (8.0-µm pore size, 1 x 105 pores/cm2). The lower chamber (a 24-well plate well) was filled with DMEM containing 2% FBS with or without 5 nM recombinant NRG11. The 24-well plates containing cell culture inserts were incubated at 33 °C in a 5% CO2 atmosphere saturated with H2O. After 18 h of incubation, cells attached to the upper side of the membrane were mechanically removed using a cotton-tipped applicator. Cells that migrated to the lower side of the membrane were rinsed with PBS, fixed with 2% glutaraldehyde in PBS for 15 min at room temperature, washed five times with water, stained with 0.1% crystal violet and 20% methanol for 20 min at room temperature, washed five times with water, air-dried, and photographed using an Olympus IX50 inverted microscope equipped with a Cool SNAP-Pro CCD camera; images were edited with Image Pro-Plus software. Migrated cells (2 mm2 of microscopic field/sample) were counted using the enclosed measurement program. Inhibitors (LY294002, U73122
[GenBank]
, or the same concentration of dimethyl sulfoxide as a control) were added to both the upper and lower chambers 30 min prior to addition of NRG11. In each case, the final concentration represented a 1:200 dilution of stock solutions in Me2SO, and control cultures were treated with Me2SO at a 1:200 dilution. We ruled out the possibility that the effect of migration inhibition was due to decreased viability caused by inhibitor toxicity by treating 1 x 105 cells (plated in a 24-well plate) for 18 h with the same amounts of inhibitors used for migration assays.
Statistical Analysis—Results from different experiments (performed at least three times in duplicate) were averaged and expressed as means ± S.D. One-way analysis of variance was used to compare data as indicated in the figure legends. Data were graphed using Microsoft Excel.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 (data not shown). At t = 18 h, the time used for motility and migration assays, we did not observe significant differences between different clones and between treated and untreated cells; however, comparing all clones at t2 = 48 h and t4 = 96 h, we observed that they can be divided into two groups: a lower rate group, including wild-type ST14A cells and cells expressing ErbB4 JMb-cyt1 isoforms (b1-7 and b1-15), and a higher rate group, including the remaining three isoforms.
NRG11 Activates the ErbB4 Receptor—To establish whether the different isoforms can be activated by treatment with exogenous NRG11, we performed immunoprecipitation experiments. Proteins from whole cell lysates of selected serum-starved clones treated or not with recombinant NRG11 were immunoprecipitated using anti-ErbB4 antibody. The immunoprecipitates were separated by SDS-PAGE, transferred to membranes, and probed for phosphotyrosines (Fig. 4B); after stripping, the membranes were reprobed for ErbB4 (Fig. 4A). Bands of phosphoproteins at the appropriate molecular masses were detected: an 
180-kDa band (present in all the isoforms) corresponding to the entire receptor and an 80-kDa band (present only in the JMa isoforms) derived by proteolytic cleavage attributable to tumor necrosis factor--converting enzyme, a member of the transmembrane ADAM protease family, also designated ADAM17 (7), which recognizes the JMa isoform, but not the JMb isoform. Shedding activity releases the ErbB4 ectodomain fragment into the medium and leaves an 80-kDa fragment, representing the transmembrane and cytoplasmic domains associated with the cell (48), which is an active tyrosine kinase at least in vitro (8). In the b1-7 clone, we observed an unusual 90-kDa band that did not correspond to the known proteolytic intracellular fragment and, moreover, did not seem to be tyrosine-phosphorylated. Our data show that the amount of phosphorylated ErbB4 (180 kDa) increased when the receptors were stimulated by NRG11, exhibiting different intensities among clones, being higher for clones b1, b2, and a2 and lower for clone a1.
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1 and, intriguingly, that all the ErbB4 isoforms co-immunoprecipitated with PI3K. Such interaction with PI3K has been described downstream from NRG1 in NIH3T3 cells transfected with ErbB4 cyt1, but not in NIH3T3 transfected with ErbB4 cyt2 (13). The two isoforms differ by 16 amino acids present only in the cytoplasmic tail of ErbB4 cyt1, including a sequence that matches the consensus binding sequence for the p85 subunit of PI3K (49). NIH3T3 cells analyzed by Kainulainen et al. (13) expressed one transfected ErbB4 isoform (a1 or a2) and moderate levels of endogenous ErbB2 (47), but not ErbB1 and ErbB3. In contrast, ST14A cells transfected with one of the four ErbB4 isoforms (a1, a2, b1, or b2) expressed not only ErbB2, but also the EGF receptor and ErbB3. ErbB3 is the only member of the ErbB family that incorporates multiple PI3K-binding sites and has been shown to be a major recruiter of PI3K (50–52). Each of the six YXXM motifs has the potential to individually bind the PI3K holoenzyme, albeit to a significantly lower extent than the six YXXM motifs together (44).
As a consequence of NRG11-induced heterodimerization with ErbB4, ErbB3 could account for ErbB4 cyt2 and PI3K co-immunoprecipitation. To test this hypothesis, we transiently expressed, in COS-7 cells endogenously expressing ErbB2, but not ErbB3 (44), ErbB4 JMb-cyt1 or JMb-cyt2 together with wild-type ErbB3 or ErbB3-6F, a mutant ErbB3 protein unable to bind PI3K (44), or the parent vector as a control and examined the ability of these receptors to associate with PI3K upon stimulation by NRG11. Immunoprecipitation analysis confirmed that, whereas ErbB4 cyt1 could bind PI3K independently of ErbB3, ErbB4 cyt2 needed wild-type ErbB3 to co-immunoprecipitate with PI3K (Fig. 5).
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1 stimulation. To prove PI3K activation, as a preliminary test, PI3K activity was analyzed in ErbB4 immunoprecipitates by in vitro kinase assays in which the phosphatidylinositol 3-phosphate product was resolved by thin layer chromatography (data not shown). PI3K activity co-immunoprecipitated with both the ErbB4 cyt1 and cyt2 isoforms, although with lower intensity with the cyt2 isoform.
Phosphorylation of Akt provides an excellent indication of PI3K activation in the cell; phosphorylation at Thr308 and Ser473 is critical for activation of the protein Ser/Thr kinase activity of Akt (53). Therefore, to confirm PI3K activation, we investigated Akt phosphorylation at Ser473 in ST14A cells expressing either the ErbB4 cyt1 isoform (clones b1-15 and a1-2) or the ErbB4 cyt2 isoform (clones b2-16 and a2-6) following NRG11stimulation (Fig. 6). We observed a very strong NRG11-dependent increase in phosphorylation of Akt at Ser473 in cells expressing ErbB4 cyt1 and cyt2. The very slight phosphorylation observed in control cells (vector) could be the consequence of ErbB2-ErbB3 heterodimer activation (44) and demonstrates that the strong phosphorylation observed in our samples can be ascribed to ErbB4 activation.
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1 treatment can modulate motility, defined here as two-dimensional movement or chemokinesis. We selected as a model the wound healing assay, in which the repopulation of cells in a cell-free region (wound) can be examined quantitatively. Stable ErbB4 clones and control cells were grown until they progressively reached confluence and then serum-starved for 24 h. A cross-shaped wound was created in the monolayers, and cells were placed in medium with or without NRG11 and photographed. Twenty-four hours later, the cross-shaped wound was photographed in the same region, and the healing percentage was quantified comparing the wound area at t = 0 and t = 24 h. Fig. 7 (upper) shows representative digital images of the wounded region before and after the incubation period; the healing percentage was quantified and is displayed graphically (Fig. 7, lower). Our results show that NRG11 treatment induced a statistically significant motility increase only in clones expressing ErbB4 JMb-cyt1 and JMb-cyt2. We suppose that the wound (and the consequent absence of contact inhibition) stimulates motility and proliferation so strongly that the differences between clones and between treated and untreated samples cannot be appreciated significantly. Only clone a2-6 showed a significant increase in motility compared with control cells; however, it displayed high basal activity observed also in the Transwell assays discussed below.
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1 at 10 pM to 10 nM. Cells were plated in the upper chamber of a Transwell filter and allowed to migrate for 18 h in response to NRG11 added to the lower chamber before being fixed, stained, and counted. Fig. 8 shows that the number of migrating cells was augmented significantly in the presence of increasing amounts of NRG11 from 1 nM, with a peak at 5 nM.
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1 added to the lower chamber. Fig. 9 shows that, for each ErbB4 isoform, NRG11 treatment significantly increased the number of cells that migrated through the filter for each treated clone compared with the respective untreated clone and with the treated negative control (empty vector).
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) Activity—Data highlighting the need for PI3K in ErbB4 (cyt1 isoform)-mediated migration (13) and our results concerning ErbB4 and PI3K co-immunoprecipitation prompted us to investigate a possible role of this signal transduction protein in cell migration. We performed Transwell assays in the presence of 5 nM NRG11 and increasing amounts of the PI3K inhibitor LY294002 (1–100 µM) (Fig. 10). To exclude a vehicle inhibitory effect, all samples received the same final concentration of dimethyl sulfoxide. Our data show that, in ErbB4-expressing cells, increasing amounts of the PI3K inhibitor resulted in greater reduction in NRG11-induced migration. In contrast, when we performed Transwell assays in the presence of 5 nM NRG11 and increasing amounts of the phospholipase C inhibitor U73122
[GenBank]
(0.1–5 µM) (data not shown), we did not observe a significant decrease in NRG11-induced migration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1-induced migration and that this phenomenon occurs via a PI3K-mediated mechanism. We began our research prompted by several intriguing studies suggesting that ErbB4 may be involved in cell migration and, specially, in neural cell migration. In fact, interneurons tangentially migrating from the ganglionic eminences express ErbB4 (29). When the glial ErbB4 receptor is blocked, neurons fail to induce radial glial formation, and their migration along radial glial fibers is impaired (33). ErbB4 knockout mice (genetically rescued from embryonic lethality) display defects in cranial neural crest migration and increased numbers of large interneurons within the cerebellum (32). In our laboratory, long-distance migration and differentiation of neuronal precursors in adult mammalian brain have been extensively investigated in the paradigm represented by the subependymal layer-olfactory bulb system (reviewed in Ref. 54). The olfactory bulb, together with the dentate gyrus of the hippocampus, belongs to those few specific regions of the brain undergoing constant neurogenesis during adulthood (55). Newly formed cells of the olfactory bulb are generated from multipotent stem cells that reside in the subependymal layer of the lateral ventricles (see Ref. 56 for a review). The progeny of these stem cells undergo long-distance tangential migration along the rostral extension (rostral migratory stream) of the subependymal layer, reaching the main (57) and accessory (58) olfactory bulb, where they differentiate into the two principal classes of interneurons, the granule and periglomerular cells. Intriguingly, cells coming out of the rostral migratory stream of the subependymal layer, as well as isolated cells in the granule cell layer, possibly migrating cells, strongly express ErbB4, and ErbB4 immunoreactivity is found in all the periglomerular and mitral/tufted cells of the olfactory bulb (26, 28).
Many efforts have been made to solve the ErbB1 signal transduction mechanism, whereas little is known about ErbB4 receptor signaling. Neuregulin-stimulated ErbB4 receptor (cyt1 isoform) has been shown to interact with the adaptor protein Shc (Src homology and collagen domain) and with the p85 subunit of PI3K (59). In addition, it has been demonstrated that, in NIH3T3 cells overexpressing ErbB4 (cyt1 isoform), HB-EGF induces association of PI3K activity with ErbB4 and is a potent chemotactic factor (60). Moreover, chemotaxis is inhibited by wortmannin, a PI3K inhibitor, suggesting a possible role for PI3K in mediating HB-EGF-stimulated chemotaxis.
A number of studies in several types of cell lines (reviewed in Ref. 61) showed that, in response to external stimuli, PI3K activates protein kinase B/Akt, which phosphorylates Raf-1 at Ser259, inhibiting Raf-1 and the subsequent extracellular signal-regulated kinase activity (62, 63). This pathway has been recently confirmed for neuregulin-induced ErbB4 signal transduction (64).
Previous studies suggest that neuregulin (and other growth factors)-activated PI3K may be involved in actin reorganization, formation of lamellipodia, membrane ruffling, and cell migration (65–68). PI3K cooperates with the small GTPases Rac and Cdc42 and with p21-activated kinase-1 in the formation of actin-rich extensions via stimulation of actin polymerization (66, 69–71). There is also evidence for the association of PI3K with microtubules (72) and for the involvement of the microtubule system in the outgrowth of cell processes induced by PI3K (73, 74).
The aim of our research was to study the role of ErbB4 in neural cell migration. We began our analysis by seeking a suitable in vitro model, and we identified an embryonic neuroepithelial striatal immortalized cell line, ST14A (34), which lacks ErbB4, but expresses all other members of the ErbB family. Starting from data describing two different isoform pairs (5, 13), we cloned the full-length cDNAs encoding the four potential ErbB4 isoforms (4) and expressed them in our cell model. We found that NRG11 induced ErbB4 phosphorylation and co-immunoprecipitation of PI3K with all ErbB4 isoforms. In addition, we demonstrated that, following NRG11 stimulation, all ErbB4 isoforms activated PI3K and induced Akt phosphorylation at Ser473, which provides an excellent indication of PI3K activation in the cell (53).
However, according to a previous study (13), only the ErbB4 cyt1 isoform should interact with PI3K because the cyt2 isoform lacks the consensus sequence (49) for the binding of this signal transduction protein. Nevertheless, the previous model is different: fibroblasts expressing moderate levels of ErbB2 (47) and stably transfected with an ErbB4 isoform were studied, whereas we studied a neural progenitor cell line endogenously expressing ErbB1, ErbB2, and ErbB3 and one transfected ErbB4 isoform. Therefore, in NIH3T3 fibroblasts, ErbB4 can homodimerize or heterodimerize only with ErbB2. ErbB2 has been described to associate with Src (75, 76), a potential mediator of PI3K activation (77). Nevertheless, in NIH3T3 cells, it has been shown that cyt2, the ErbB4 isoform that does not activate PI3K, mediates proliferation, but not survival and chemotaxis (13), which are mediated by PI3K, suggesting that ErbB2 cannot account for the PI3K recruitment mediated by NRG11-stimulated ErbB4.
Intriguingly, there are six PI3K-binding motifs in the C-terminal domain of ErbB3 (50–52), and one simple explanation may include direct recruitment of PI3K by the activated ErbB3 receptor. We tested this hypothesis by expressing ErbB4 cyt2 together with wild-type ErbB3 (43) or ErbB3 mutated at the six tyrosines included in the PI3K-binding motifs (44), and we demonstrated that the ErbB4 cyt2 isoform could co-immunoprecipitate with PI3K only when coexpressed with the wild-type ErbB3 receptor.
Finally, to test our preliminary hypothesis, we performed migration assays and showed that ErbB4 confers to ST14A cells the ability to respond to NRG11 through migration. This ligand is not ErbB4-specific and could activate the ErbB3 receptor; nevertheless, control cells expressing ErbB3, but lacking ErbB4, failed to migrate, indicating that, in our cell model, ErbB4 is necessary for this activity. Our data show that every ErbB4 isoform (endowed or not with the PI3K-binding motif) mediates NRG11-induced migration, although with different intensity, and we have demonstrated that PI3K activation is necessary for ErbB4-mediated migration either directly or indirectly through ErbB3 heterodimerization.
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FOOTNOTES |
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The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY375306
[GenBank]
, AY375307
[GenBank]
, and AY375308
[GenBank]
. 
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: Dipt. di Biologia Animale e dell'Uomo, Università degli Studi di Torino, Via Accademia Albertina 13, 10123 Torino, Italia. Tel.: 39-11-670-4688; Fax: 39-11-670-4692; E-mail: giovanna.gambarotta{at}unito.it.
1 The abbreviations used are: EGF, epidermal growth factor; PI3K, phosphatidylinositol 3-kinase; NRG, neuregulin; HB-EGF, heparin-binding epidermal growth factor-like growth factor; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; RT, reverse transcription; t, time; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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