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ErbB-4 activation inhibits apoptosis in PC12 cells
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Neuroscience Vol. 107, No. 2, pp. 353^362, 2001 ß 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522 / 01 $20.00+0.00
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ErbB-4 ACTIVATION INHIBITS APOPTOSIS IN PC12 CELLS S. ERLICH, Y. GOLDSHMIT, Z. LUPOWITZ and R. PINKAS-KRAMARSKI* Department of Neurobiochemistry, Tel-Aviv University, Ramat-Aviv 69978, Israel
AbstractöNeuregulins, a large family of polypeptide growth factors, exert various distinctive e¡ects in the nervous system. neuregulins and their receptors are widely expressed in neurons implying important roles in neuronal cell functions. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate neuregulin-induced di¡erentiation. In the present study we demonstrate that in the PC12-ErbB-4 cells, neuregulin rescues cells from apoptosis induced by serum deprivation or tumor necrosis factor (TNF)K treatment. The neuregulin-induced survival is comparable to the e¡ect mediated by the neurotrophic factor nerve growth factor (NGF). Both neuregulin and NGF protect cells from apoptosis induced by serum deprivation and TNFK treatment. Moreover, neuregulin like NGF induces the survival of neuronal di¡erentiated PC12-ErbB-4 cells. The survival e¡ect of neuregulin is probably mediated by the phosphoinositide 3-kinase (PI3K) and protein kinase B/Akt signaling pathways. Neuregulin induces the activation of PI3K and prolonged activation of protein kinase B/Akt. In addition, inhibition of the PI3K activity prevented the neuregulininduced survival e¡ect. Taken together, these results indicate that survival induced by neuregulin in PC12-ErbB-4 cells requires PI3K signaling networks. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: epidermal growth factor, ErbB/HER family, Neu di¡erentiation factor, neuregulin, signal transduction, tyrosine kinase.
1989; Plowman et al., 1990) and HER-4/ErbB-4 (Plowman et al., 1993). The four members of the ErbB family can form di¡erent homo- and heterodimers upon ligand binding that serve to diversify the intracellular signals elicited by receptor activation (Pinkas-Kramarski et al., 1996; Riese et al., 1995). Binding of ligand to a speci¢c ErbB receptor induces rapid receptor dimerization and phosphorylation followed by the recruitment of cytoplasmic molecules, which bind to phosphorylated tyrosine residues of the receptor and initiate a cascade of signaling events (Ullrich and Schlessinger, 1990). ErbBmediated responses are a¡ected by the multiplicity of the ligands (Pinkas-Kramarski et al., 1997a). EGF, heparin binding-EGF (HB-EGF), amphiregulin, epiregulin, betacellulin and transforming growth factor (TGF)K are typical ErbB-1 ligands. Ligands for ErbB-2 have not yet been identi¢ed. The family of growth factor isoforms called neuregulins (NRGs) also known as Neu di¡erentiation factor (NDF), are typical ligands of ErbB-3 and ErbB-4 (Peles et al., 1992; Pinkas-Kramarski and Yarden, 1995; Wen et al., 1992). Three ligands of ErbB-1, betacellulin, HB-EGF and epiregulin, bind also the ErbB-4 receptor (Beerli and Hynes, 1996; Elenius et al., 1997; Riese et al., 1996; Shelly et al., 1998). NRGs and their receptors (ErbB-3 and ErbB-4) are widely expressed in the nervous system (Marchionni et al., 1993; Orr-Urtreger et al., 1993; Pinkas-Kramarski et al., 1997b, 1994; Gassmann et al., 1995). Several examples exist which demonstrate the variety of biological e¡ects of NRG on neurons and glial cells. For example, NRGs were shown to be mitogenic for Schwann and glial cells (Brockes et al., 1980; Ra¡ et al., 1978). Like-
The ErbB subfamily of receptor tyrosine kinases includes four members (Heldin, 1995; Peles and Yarden, 1993): the epidermal growth factor (EGF) receptor (also called ErbB-1/HER-1) (Ullrich et al., 1984), Neu/HER-2/ErbB2 (Bargmann et al., 1986; Coussens et al., 1985; Yamamoto et al., 1986), HER-3/ErbB-3 (Kraus et al.,
*Corresponding author: Tel.: +972-3-6406801; fax: +972-36407643. E-mail address: [email protected] (R. Pinkas-Kramarski). Abbreviations : DAPI, 4P,6-diamidine-2P-phenylindol dihydrochloride ; DMEM, Dulbecco's modi¢ed Eagle's medium; EDTA, ethylenediaminetetra-acetate ; EGF, epidermal growth factor; EGTA, ethyleneglyco-bis(2-aminoethyl-ether)-N,N,NP,NP-tetraacetic acid; Erk, extracellular signal-regulated kinase ; FBS, fetal bovine serum ; FGF, ¢broblast growth factor; GF109203X, 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-maleimide ; HB, heparin binding ; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulphonic acid); HS, horse serum ; LY294002, [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]; MAPK, mitogen-activated protein kinase ; MTT, [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl]tetrazolium bromide; NGF, nerve growth factor; NRG, neuregulin; NT, neurotrophin; PBS, phosphatebu¡ered saline; PD98059, 2P-amino-3P-methoxy£avone; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; SB203580, [4-(3-iodophenyl)-2-(4-methylsul¢nylphenyl)-5-(4-pyridyl)1H-imidazole]; SDS^PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; TrK, tyrosine kinase. 353
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wise, the factor can promote maturation of astrocytes in primary cultures of embryonic brain cells (PinkasKramarski et al., 1994), and the di¡erentiation of oligodendrocytes from O2A progenitor cells (Vartanian et al., 1994). Another study, that made use of clonal cultures of neural crest stem cells, demonstrated that NRG can inhibit neuronal cell di¡erentiation by favoring glial cells (Shah et al., 1994), and that NRG can promote neurotrophin-3 (NT3) production by glial cells, thus promoting neuronal survival (Verdi et al., 1996). Additional biological activities of NRG have been demonstrated in the neuromuscular junction (Corfas et al., 1993; Corfas and Fischbach, 1993). Furthermore, in cerebellar granule cells it was demonstrated that NRG induces cell migration along the radial glial ¢bers (Rio et al., 1997), induces GABA(A) receptor expression and induces neurite outgrowth through ErbB-4 receptor (Rie¡ et al., 1999) as well as the regulation of N-methyl-D-aspartate (NMDA)-2C receptor expression (Ozaki et al., 2000). The PC12 cell line, derived from a rat adrenal medullary pheochromocytoma tumor, has become a widely used paradigm for studies on the regulation of neural di¡erentiation and survival (Greene and Tischler, 1976). PC12 cells can be induced to di¡erentiate by neurotrophic factors such as nerve growth factor (NGF) and basic ¢broblast growth factor (FGF), that bind and activate their respective tyrosine kinases trkA and FGF receptors through the Ras/mitogen-activated protein kinase (MAPK) pathway (Cowley et al., 1994; Marshall, 1995). Other studies showed that EGF is a mild mitogen for these cells and that like NGF it induces extracellular signal-regulated kinase (Erk) activation via the Ras pathway but unlike NGF, EGF-induced Erk activation is transient (Marshall, 1995). It was also demonstrated that many growth factors and neurotrophins can promote neuronal survival of several classes of neurons. Among these factors are insulin, insulin-like growth factor-1, brain-derived neurotrophic factor (BDNF), NGF, NT3 and NT4/5 (Barde, 1989; Chen et al., 1977; Greene, 1978). The PC12 system has also been used to study the e¡ects of neurotrophic factors on cell survival. Following stimulation by tumor necrosis factor (TNF)K or when deprived of growth factors, these cells die apoptotically, and NGF can maintain their long-term survival (Greene, 1978; Haviv and Stein, 1999; Mesner et al., 1992). The survival e¡ect induced by NGF in PC12 cells, requires the activation of phosphoinositide 3-kinase (PI3K) signaling pathway (Klesse et al., 1999; Yao and Cooper, 1995). Recently, we have shown that NRG like NGF can induce the di¡erentiation of PC12-ErbB-4 cells (Vaskovsky et al., 2000). The accumulated data on NRG function in neuronal and glial cells indicate an important function of NRGs and their receptors in neural development and maintenance. In spite of that, it is not yet clear whether ErbB-4 receptor alone or in combination with other ErbB family members can mediate neuroprotective signals. The objective of this study was to evaluate the survival e¡ect of NRG and to explore the mechanism by which NRG induces survival of PC12-ErbB-4 cells.
EXPERIMENTAL PROCEDURES
Materials and bu¡ers EGF (human recombinant) was purchased from Sigma (Rehovot, Israel). NGF (mouse submaxillary gland) was from Chemicon (Temecula, CA, USA). Recombinant murine TNFK and human recombinant NRGL were from RpD Systems (Oxon, UK). A monoclonal anti-phosphotyrosine antibody (PY-20) was purchased from Santa-Cruz Biotechnology (Santa Cruz, CA, USA). A polyclonal anti-PI3K-p85 antibody was from Upstate Biotechnology (Lake Placid, NY, USA). LY294002, SB203580 and wortmannin were purchased from Calbiochem (La Jolla, CA, USA). GF109203X was purchased from Sigma. PD98059 was purchased from Promega (Madison, MI, USA). All other reagents were from Sigma (Rehovot, Israel). HNTG bu¡er contained 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol. Solubilization bu¡er contained 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 1.5 mM MgCl2 , 10% glycerol, 0.2 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl £uoride (PMSF), 10 Wg/ml aprotinin, and 10 Wg/ml leupeptin. Cell lines The rat pheochromocytoma cells (PC12) which express ErbB1, ErbB-2 and ErbB-3 receptors were used for expressing the ErbB-4 receptor. Expression vector LXSHD-ErbB-4 containing the coding region of ErbB-4 (Shelly et al., 1998) was introduced by infection into PC12 cells as previously described and was termed PC12-ErbB-4 cell line (Vaskovsky et al., 2000). The two cell lines were grown in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with antibiotics, 10% heatinactivated fetal bovine serum (FBS) and 10% horse serum (HS). Cells were incubated at 37³C in 5% CO2 in air, and the medium was changed every 3^4 days. Cells were passaged when 90% con£uent using trypsin/disodium EDTA (Biological Industries, Kibbutz Beit Haemek, Israel). Cells were induced to di¡erentiate by growing on collagen-coated plates at 2U104 cells/ml in the presence of 50 ng/ml NGF for 7 days. Before experiments were performed, cells were washed twice with phosphate-bu¡ered saline (PBS). Cell survival assays Cells were resuspended and seeded on collagen-coated 96-well plates at 1.2U104 cells/well. Cultures were grown either in serum-free DMEM or in DMEM supplemented with 2.5% FBS and 2.5% HS (for the analysis of TNFK e¡ect), and treated without or with NRG, NGF or EGF at 100 ng/ml for comparison of long-term factors activity. Cell survival was determined by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide (MTT) assay, which determines mitochondrial activity in living cells (Mosmann, 1983). MTT 0.1 mg/ml was incubated with the analyzed cells for 2 h at 37³C. Living cells can transform the tetrazolium ring into dark blue formazan crystals, which can be quanti¢ed by reading the optical density at 550^650 nm after lysis of the cells with acidic isopropanol. Staining of nuclei with the £uorescent DNA dye 4P,6-diamidine2P-phenylindol dihydrochloride (DAPI) was used to estimate the number of dying cells. Cells were scored for apoptosis by nuclear morphology. Treated cells were ¢xed in 4% formaldehyde, washed three times with PBS and then stained with DAPI solution (10 Wg/ml) for 10 min. Following washing with PBS and mounting, the cells were photographed. The instrument used was Olympus optical inverted phase-contrast microscope Model IX70 (U20 magni¢cation). Cell cycle analysis For cell cycle analysis, cells were seeded at 105 cells/ml, with or without growth factors (100 ng/ml), in serum-free DMEM or
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Fig. 1. Ligand-induced cell growth and survival of PC12-ErbB-4 cells. (A) Cells were deprived of serum and plated in 96-well plates at a density of 1.2U104 cells/well in media containing 100 ng/ml of EGF (closed circles), NRG (closed squares) or NGF (open triangles). For control (CON), we used mock-treated cells (closed triangles). The extent of cell proliferation was determined daily by using the colorimetric MTT assay. Results are presented as fold induction over the control untreated cells at day 0, before cells were treated (t = 0), and is the mean þ S.D. of six determinations. Each experiment was repeated at least three times with similar results. (B) 2U103 cells/well were induced to di¡erentiate by 50 ng/ml NGF treatment for 7 days in DMEM containing 2.5% FBS and 2.5% HS. Wells were then washed twice with PBS and serum-free medium containing 100 ng/ml of EGF (closed circles), NRG (closed squares) or NGF (open triangles) or medium alone (closed triangles) was added. The extent of cell survival was determined daily as described in (A) using the di¡erentiated untreated cells at the start point as control t = 0. (C) Cells were plated at a density of 1.2U104 cells/well in DMEM containing 2.5% FBS and 2.5% HS. Cells were treated with 50 ng/ml TNFK either alone (CON) or together with 100 ng/ml NRG, NGF or EGF. The e¡ect of the treatments on cell survival was then determined as described in (A). (D) 2U103 cells/well were induced to di¡erentiate by 50 ng/ml NGF treatment for 7 days in DMEM containing 2.5% FBS and 2.5% HS. Wells were then washed twice with PBS and DMEM containing 2.5% FBS, 2.5% HS and 50 ng/ml TNFK without (CON) or with 100 ng/ml of EGF (closed circles), NRG (closed squares) or NGF (open triangles). The extent of cell survival was determined daily as described in (A).
in DMEM supplemented with 2.5% FBS and 2.5% HS (for the analysis of TNFK e¡ect). TNFK concentration was 50 ng/ml. After 24 h, 106 treated cells were detached from the plates (by 0.5 mM EDTA), and centrifuged (200Ug). The pellet was washed once with PBS and resuspended in 1 ml of hypotonic £uorochrome solution (50 Wg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) and stored overnight in the dark at 4³C prior to £ow cytometric analysis. The stained cells were analyzed in a £uorescence cell sorter (FACSort; Becton and Dickinson) excited at 488-nm wavelength and collected through a 570-nm BP ¢lter. Data were analyzed using WinMDI 2.1.4 computer software. The percentage of cells in sub-G1 phase of the cell cycle was designated as apoptotic cell population. Lysate preparation and immunoprecipitation Cells were exposed to the indicated stimuli. After treatment, cells were solubilized in lysis bu¡er. Lysates were cleared by
centrifugation. For direct electrophoretic analysis, boiling gel sample bu¡er was added to cell lysates. For other experiments, lysates were ¢rst subjected to immunoprecipitation with antiphosphotyrosine antibodies (PY-20). Antibodies were ¢rst coupled to rabbit anti-mouse immunoglobulin G and then to protein A-Sepharose by the same procedure. The proteins in the lysate supernatant were immunoprecipitated with aliquots of the protein A-Sepharose^antibody complexes for 2 h at 4³C. The immunoprecipitates were washed three times with HNTG, resolved by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) through 7.5% gels and electrophoretically transferred to nitrocellulose membrane. Membranes were blocked for 2 h in TBST bu¡er (0.02 M Tris^HCl pH 7.5, 0.15 M NaCl and 0.05% Tween 20) containing 6% milk, blotted with 1 Wg/ml primary antibody for 2 h, followed by 0.5 Wg/ml secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected with the enhanced chemiluminescence reagent (Amersham Corp, Bukinghamshire, UK).
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Fig. 2. NRG-induced cell survival of PC12-ErbB-4 cells. PC12-ErbB-4 cells were tested for cell survival by using £ow cytometry analysis. Cultures of 106 cells were treated as indicated and as described in Fig. 1A and C. Cells were harvested after 24 h and analyzed for DNA content by £ow cytometry. The percentage of cells at the sub-G1 phase of the cell cycle is indicated (Ap). This cell fraction represents cells undergoing apoptosis. The experiments were repeated three times with similar results.
RESULTS
Induction of cell survival by NRG, EGF and NGF PC12 cells express ErbB-1, ErbB-2 and ErbB-3, but not ErbB-4 receptors (Vaskovsky et al., 2000). ErbB-4 was introduced into PC12 cells by retroviral infection and stable ErbB-4 expressing clones were then selected and a representative clone was chosen for further analysis (PC12-ErbB-4) (Vaskovsky et al., 2000). To determine the e¡ect of the NRG, EGF and NGF ligands on long-term survival of PC12-ErbB-4 cells we have used the MTT assay (Fig. 1). Figure 1 represents survival e¡ect induced by the various ligands in naive or di¡erentiated cells. The cell death inducers are serum starvation (Fig. 1A and B) or TNFK (Fig. 1C and D). The parameter used to determine cell viability was the percentage of the apparent cell number at the indicated day compare to day 0 (% of t = 0). As shown, under conditions of serum deprivation both NRG and NGF induced cell survival that was greater than the e¡ect induced by EGF (Fig. 1A). Under the same conditions the control serum deprived untreated cells die (Fig. 1A). Moreover, when cells were induced to die by TNFK treatment, both NGF and NRG rescued cells from death (Fig. 1C). We next examined whether NRG could also protect di¡erentiated cells from death. For this aim, similar experiments were performed on PC12ErbB-4 cells with sympathetic neuronal cell phenotype induced by NGF treatment for 1 week (prior to the experiment performance). As shown, incubation of differentiated PC12-ErbB-4 cells with NRG inhibited cell death induced by both serum deprivation and TNFK treatment (Fig. 1B and D). The e¡ect of NRG on di¡erentiated PC12-ErbB-4 cells was similar to the e¡ect of NGF but not of EGF (Fig. 1). However, NRG could not
rescue di¡erentiated and naive PC12 cells from apoptosis induced by both serum deprivation and TNFK treatment (data not shown). Clearly then, NRG, that activates ErbB-4 receptors in PC12-ErbB-4 cells, mediates survival responses. These results encouraged us to further characterize the e¡ect of NRG on cell survival. NRG rescue from apoptosis induced by serum deprivation and TNFK The responses of PC12 cells to EGF and NGF have been well characterized. While EGF is a potent mitogen (Mark et al., 1995), NGF is a di¡erentiation and survival factor for PC12 cells (Greene and Tischler, 1976). To further analyze the e¡ect of NRG on survival of PC12ErbB-4 cells, we determined the fraction of cells that underwent apoptosis by performing either £ow cytometry or by staining the cells with the DNA-speci¢c dye DAPI. Evidently, the fraction of sub-G1 cells that represents cells undergoing apoptosis was increased by serum deprivation or by incubation with TNFK to 55% and 20% respectively (Fig. 2). Addition of NRG reduced the percentage of apoptotic cells to 4% and 1.5% in serum-deprived cells or in TNFK-treated cells respectively (Fig. 2). The e¡ect of NRG on cell survival was also evident by DAPI staining of cell nuclei (Fig. 3). Condensed or fragmented nuclei represent cells undergoing apoptosis. In the control cells, serum deprived and TNFK treated for 24 h, condensed and fragmented nuclei were observed indicating that cells underwent apoptosis (Fig. 3). As shown, less apoptotic cells were present in NRG-treated cells compared to control (serum-deprived) untreated or TNFK-treated cells indicating that NRG protected these cells from apoptosis. Moreover, it was previously shown that following di¡erentiation, the removal of NGF and serum from PC12
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Fig. 3. NRG rescue from apoptosis induced by serum deprivation or TNFK treatment. (A) PC12-ErbB-4 cultures were grown in 96-well plates at 1.2U104 cells/well. Separate wells were then treated for 24 h in serum-free medium containing either EGF, NRG, NGF at 100 ng/ml, or left untreated (control, CON). For experiments with di¡erentiated cells, cells were grown 1 week in the presence of 50 ng/ml NGF and then washed twice with PBS prior to growth factors treatment for additional 24 h as described above. (B) Sister cultures were grown in DMEM containing 2.5% FBS and 2.5% HS. Separate wells were then treated with either EGF, NRG or NGF at 100 ng/ml in the presence or absence of TNFK (50 ng/ml) for 24 h. For experiments with di¡erentiated cells, cells were pretreated as described in (A). The incubation was terminated by cell ¢xation and staining with the DNA intercalating dye DAPI. The resulting £uorescent photomicrographs are shown. Scale bar = 20 Wm.
cells leads to apoptosis (Teng and Green, 1994). As shown in Fig. 3, the protective e¡ect of NRG was also evident in di¡erentiated PC12-ErbB-4 cells. In addition, in EGF-treated cells apoptotic nuclei were observed although the number of live cells was higher and comparable to the control untreated cell, indicating that EGF only partially rescues cells from apoptosis and induces cell division. Taken together, the results shown in Fig. 3 indicate that NRG may protect di¡erentiated and non-di¡erentiated PC12-ErbB-4 cells from apoptosis induced by both serum deprivation and TNFK treatment. Analysis of the signaling pathway that mediates NRG-induced survival Since NRG proved in this cell system to protect cells from apoptosis we next employed various pharmacological inhibitors to test through which signaling pathway NRG exerts its e¡ect. In order to examine whether Erk activation is required for the survival e¡ect induced by
NRG, we examined the e¡ect of Erk pathway inhibitor, PD98059, which inhibits MAPKK, on the NRG-mediated survival e¡ect. PC12-ErbB-4 cells were incubated with 100 ng/ml NRG in the absence or in the presence of PD98059 (50 WM). As shown (Fig. 4) the MAPKK inhibitor did not a¡ect the survival induced by NRG. It was previously shown that PD98059 inhibits the di¡erentiation induced by NRG and by NGF in these cells (Vaskovsky et al., 2000), suggesting that the sustained MAPK activation is required for di¡erentiation in PC12-ErbB-4. Our results implicate that activation of the MAPK pathway is not required for NRG-mediated survival e¡ect. Several studies showed that atypical protein kinase C (aPKC) are involved in NGF-induced survival of serumdeprived PC12 cells (Wooten et al., 2000) and overexpression of aPKCs enhances NGF responsiveness as well as survival of di¡erentiated cells (Wooten et al., 1999). In addition, it was previously shown that the PKC inhibitor GF109203X inhibits the morphological changes induced by NRG or by NGF in PC12-ErbB-4
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Fig. 4. The e¡ect of various drugs on NRG-induced cell survival. Cells were plated in 96-well plates and induced to di¡erentiate as described in Fig. 1. (A) Separate wells, grown in serum-free medium, were treated with either EGF, NRG or NGF at 100 ng/ml in the absence or presence of 1 WM GF109203X (GF), 50 WM PD98059 (PD), 4 WM SB203580 (SB), 20 nM rapamycin (RAP) and 20 WM LY294002 (LY) as indicated. (B) Separate cultures were grown in medium containing 2.5% HS and 2.5% FBS in the presence or absence of TNFK (50 ng/ml), and treated with either EGF, NRG or NGF at 100 ng/ml in the absence or presence of 1 WM GF109203X (GF), 50 WM PD98059 (PD), 4 WM SB203580 (SB), 20 nM rapamycin (RAP) and 20 WM LY294002 (LY) as indicated. The MTT assay was performed 48 h later. Results are presented as fold induction over the control untreated cells in (A), and over the TNFK-treated cells in (B), and are the mean þ S.D. of six determinations. Each experiment was repeated at least three times with similar results. CON, control.
cells (Vaskovsky et al., 2000). In the present study we were interested to study whether PKCs are involved in NRG-mediated survival e¡ect. As shown in Fig. 4, the PKC inhibitor GF109203X (1 WM) which blocked the morphological changes induced by NRG or by NGF in PC12-ErbB-4 cells (Vaskovsky et al., 2000), did not inhibit the survival e¡ect induced by NRG. It was previously shown that selective p38 inhibitors promoted the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent manner (Horstmann et al., 1998). In the present study we examined the involvement of p38 in NRG-mediated survival. For this aim, we examined the ability of a p38-speci¢c inhibitor to a¡ect the NRG-mediated survival in PC12ErbB-4 cells. We found that SB203580 (4 WM) did not a¡ect NRG-mediated survival (Fig. 4). Taken together, these results suggest that Erk, PKC and p38 activation are not required for this process. Since PI3K has been previously implicated in survival signaling (Yao and Cooper, 1995), we examined the e¡ect of a speci¢c PI3K inhibitor LY294002 (20 WM) on NRG-induced survival. PI3K inhibition induced apoptosis of PC12-ErbB-4 cells, namely cell death was observed also in the presence of NGF or NRG (Fig. 4). Dimethylsulfoxide (DMSO) treatment at a concentration used to solubilize LY294002 had no e¡ect on cell viability and served as a control. The addition of LY294002 as well as other inhibitors (Fig. 4A) by themselves could reduce cell viability. These results suggest that inhibition of PI3K pathway (and also other signaling pathways)
has a toxic e¡ect on the cells and that the toxic e¡ect of PI3K inhibitor is not prevented by activation of ErbB4. However, the toxic e¡ect of the other inhibitors used was prevented by NRG. The inability of NRG to rescue cells following treatment with LY294002 encouraged us to further investigate the PI3K activation induced by NRG. The p85 regulatory subunit of PI3K contains two SH2 domains which bind speci¢c phosphorylated tyrosine residues in receptor tyrosine kinases. As shown in Fig. 5A, immunoblotting of speci¢c phoshotyrosine immunoprecipitates with anti-p85 antibodies suggested that p85 is mainly associated with ErbB-4 upon NRG stimulation. These ¢ndings suggest that activation of ErbB-4 receptors by NRG results in activation of PI3K. Several studies demonstrate that PKB/Akt, a serine threonine kinase that functions as an e¡ector of PI3K, is involved in cell survival (Klesse et al., 1999). PI3K inhibitor LY294002 blocks the activity of Akt as well as cell survival (Ashcroft et al., 1999). For these reasons we have analyzed the kinetics of Akt activation following NGF, NRG and EGF (each at 100 ng/ml) treatments in PC12 and PC12-ErbB-4 cells. As shown (Fig. 5B), NRG and NGF induced prolonged Akt activation that lasted for 2 h in PC12-ErbB-4 cells. EGF, however, induced transient Akt activation (Fig. 5B). The prolonged Akt activation induced by NRG and NGF correlated with the ability of these ligands to rescue cells from apoptosis. These results may indicate that PI3K, as well as its downstream e¡ector Akt, may be the signaling pathway responsible for NRG-induced survival.
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Fig. 5. Ligand-induced activation of PI3K and PKB/Akt. (A) Whole cell lysates from cells treated for 10 min at 37³C with either EGF, NRG or NGF at 100 ng/ml were immunoprecipitated (IP) with mouse monoclonal PY20 antibody (P-TYR) as indicated. After SDS^PAGE, blots were probed with a rabbit polyclonal anti-p85 antibody. (B) Whole cell lysates were prepared from cells treated for the indicated time points with EGF, NRG or NGF at concentration of 100 ng/ml. Lysates were analyzed by immunoblotting with a monoclonal antibody to phosphorylated PKB/Akt. Signal detection was performed by using a chemiluminescence kit. (C) Whole cell lysates were prepared from PC12 or PC12-ErbB-4 cells treated for the indicated time points with EGF, NRG or NGF at concentration of 100 ng/ml. Lysates were analyzed by immunoblotting with a monoclonal antibody to phosphorylated PKB/Akt. Quanti¢cation of the phosphorylated PKB/AKT intensity was performed using TINA 2.0 computer program. Data are presented as fold induction over the control untreated cells and is the mean þ S.D. of three experiments. CON, control. DISCUSSION
NRG receptors ErbB-3 and ErbB-4, unlike other members of the EGF receptor family, are characterized by a relatively limited expression pattern. ErbB-4 expression is relatively restricted to the developing CNS and to the developing heart (Kraus et al., 1989; Pinkas-Kramarski et al., 1997b; Plowman et al., 1993). NRGs play an important role in the nervous system, primarily in communicating nerve cells to non-neuronal cells. NRGs act as the most potent mitogens for precursor Schwann cells in vitro (Dong et al., 1995), and they reduce apoptosis of mature Schwann cells upon axotomy (Grinspan et al., 1996). Myelin-forming cells of the CNS, the oligodendrocytes, are also targets of NRGs (Canoll et al., 1996; Vartanian et al., 1994), as are embryonic astroglia, whose maturation and survival are a¡ected (Pinkas-
Kramarski et al., 1994). Another signal of NRG for glial di¡erentiation, on the expense of a neuronal fate, was demonstrated by using neural crest stem cells (Shah et al., 1994). Recently it was demonstrated that ErbB-4 receptor when expressed in PC12 cells can induce neuronal di¡erentiation (Vaskovsky et al., 2000). In the present study we demonstrated that ErbB-4 receptors in PC12-ErbB-4 cells mediate also NRG-induced survival. The NRG-mediated survival e¡ect is indistinguishable from the e¡ect mediated by the NGF-activated tyrosine kinase (Trk) receptors (Fig. 1). Accordingly, activation of the ErbB-4 receptors by NRG resulted in signi¢cant ErbB-4 receptor phosphorylation (Vaskovsky et al., 2000), induced a sustained activation of Akt (Fig. 5B) and survival signals (Figs. 1 and 2). These are among the typical responses of PC12 cells when they are exposed to NGF (Connolly et al., 1984; Greene
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and Tischler, 1976; Hu¡ et al., 1981). Moreover, we ¢nd that the NRG-induced survival in PC12-ErbB-4 cells is probably mediated through the PI3K pathway since NRG failed to rescue cells in the presence of PI3K inhibitor (Fig. 4). PI3K-dependent survival is also a known characteristic of NGF-induced survival in PC12 cells (Klesse et al., 1999). We therefore suggest that, in PC12-ErbB-4 cells, the NRG-activated ErbB-4 receptors share with the NGF-activated Trk receptors a similar set of signaling events leading to survival. By employing the PC12 cells as a model system for sympathetic neurons, our results suggest that one of the e¡ects of NRG on neurons may be to protect them from apoptosis. The e¡ect of NRG on cell survival is evident by three independent observations. First, NRG increased cell viability as measured by MTT assay (Fig. 1). Second, FACS analyses demonstrated that NRG treatment reduced the percentage of apoptotic cells (Fig. 2). Third, evaluation of apoptosis following DAPI staining showed the characteristics of apoptotic cells namely chromatin condensation and nuclear segmentation. NRG treatment signi¢cantly reduced the number of apoptotic cells (Fig. 3). NRG did not prevent apoptosis of untransfected PC12 cells (data not shown) indicating that NRG-induced cell survival in PC12ErbB-4 cells is mediated by the ErbB-4 receptors. It was previously shown that NRG indirectly a¡ects neuroblast survival and di¡erentiation (Verdi et al., 1996). Also paracrine mechanisms may underlie the previously reported survival and di¡erentiation e¡ect of NRG on rat retinal neurons, whose number of neurite extensions, but not their size, was increased by NRG (BerminghamMcDonogh et al., 1996). Furthermore, it was demonstrated that in cerebellar granule cells NRG induces cell migration along the radial glial ¢bers (Rio et al., 1997) and induces GABA(A) receptor expression and neurite outgrowth through ErbB-4 receptor (Rie¡ et al., 1999). Our results demonstrate that NRG can support neuronal survival of ErbB-4 expressing PC12 cells. The e¡ect of NRG on PC12-ErbB-4 cells, as demonstrated, resembles the e¡ect of NGF in supporting cell survival. Taken together, these results demonstrate that NRG can act as neurotrophic factor a¡ecting neuronal di¡erentiation and survival. Previously we reported that NRG activates the phosphorylation of ErbB-4 receptors in these cells (Vaskovsky et al., 2000). Activation of ErbB-4 leads to
neuronal di¡erentiation through the activation of the Ras/MAPK pathway (Vaskovsky et al., 2000). In this respect NRG-induced di¡erentiation resembles the e¡ect of NGF. A growing body of data accumulates concerning the survival e¡ect mediated by NGF in neurons. In PC12 cells NGF protects cells from apoptosis induced by various stimuli (Greene, 1978; Haviv and Stein, 1999; Mesner et al., 1992). It was previously shown that the protective e¡ect of NGF, on serum-deprived PC12 cells, is mediated by the PI3K signaling pathway (Yao and Cooper, 1995). Moreover, it was demonstrated that PI3K activation and subsequent activation of the downstream signaling e¡ector, Akt, function to promote cell survival in serum-deprived PC12 cells (Ashcroft et al., 1999). Our results demonstrate that the NRG-induced cell survival in PC12 cells expressing the ErbB-4 receptors, is probably mediated by PI3K pathway since PI3K inhibition blocked the survival e¡ect induced by NRG (Fig. 4). In addition, the kinetics of Akt activation induced by NRG is similar to that induced by NGF but not of EGF, indicating that Akt activation may be important for NRG-induced survival e¡ect.
CONCLUSION
The present study strengthens the hypothesis that ErbB-4 receptors when activated by NRG can play an important role in neuronal survival. It is important to note that the e¡ect of NRG on cell survival may not be restricted to ErbB-4 receptor. In the present study we analyzed the e¡ect of ErbB-4, which is known to be highly expressed in neurons. Our results, however, do not exclude the possibilities that only overexpression of ErbB-4 can provide survival signals and that such e¡ects may be also mediated by ErbB-2 or ErbB-3 receptors. Nevertheless, this model system, PC12-ErbB-4 cells, can serve future studies on ErbB-4-mediated e¡ects and signal transduction pathways involved in neuronal survival and function. AcknowledgementsöThis work was supported in part by The National Institute for Psychobiology in Israel, The Charles E. Smith Foundation, grant 7-2001 No. 34-01 and by the Adams super-center for brain studies, No. 1190, Israel. We thank Prof. A. Dvir and Prof. R. Stein for critically reading this manuscript.
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ErbB-4 ACTIVATION INHIBITS APOPTOSIS IN PC12 CELLS S. ERLICH, Y. GOLDSHMIT, Z. LUPOWITZ and R. PINKAS-KRAMARSKI* Department of Neurobiochemistry, Tel-Aviv University, Ramat-Aviv 69978, Israel
AbstractöNeuregulins, a large family of polypeptide growth factors, exert various distinctive e¡ects in the nervous system. neuregulins and their receptors are widely expressed in neurons implying important roles in neuronal cell functions. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate neuregulin-induced di¡erentiation. In the present study we demonstrate that in the PC12-ErbB-4 cells, neuregulin rescues cells from apoptosis induced by serum deprivation or tumor necrosis factor (TNF)K treatment. The neuregulin-induced survival is comparable to the e¡ect mediated by the neurotrophic factor nerve growth factor (NGF). Both neuregulin and NGF protect cells from apoptosis induced by serum deprivation and TNFK treatment. Moreover, neuregulin like NGF induces the survival of neuronal di¡erentiated PC12-ErbB-4 cells. The survival e¡ect of neuregulin is probably mediated by the phosphoinositide 3-kinase (PI3K) and protein kinase B/Akt signaling pathways. Neuregulin induces the activation of PI3K and prolonged activation of protein kinase B/Akt. In addition, inhibition of the PI3K activity prevented the neuregulininduced survival e¡ect. Taken together, these results indicate that survival induced by neuregulin in PC12-ErbB-4 cells requires PI3K signaling networks. ß 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: epidermal growth factor, ErbB/HER family, Neu di¡erentiation factor, neuregulin, signal transduction, tyrosine kinase.
1989; Plowman et al., 1990) and HER-4/ErbB-4 (Plowman et al., 1993). The four members of the ErbB family can form di¡erent homo- and heterodimers upon ligand binding that serve to diversify the intracellular signals elicited by receptor activation (Pinkas-Kramarski et al., 1996; Riese et al., 1995). Binding of ligand to a speci¢c ErbB receptor induces rapid receptor dimerization and phosphorylation followed by the recruitment of cytoplasmic molecules, which bind to phosphorylated tyrosine residues of the receptor and initiate a cascade of signaling events (Ullrich and Schlessinger, 1990). ErbBmediated responses are a¡ected by the multiplicity of the ligands (Pinkas-Kramarski et al., 1997a). EGF, heparin binding-EGF (HB-EGF), amphiregulin, epiregulin, betacellulin and transforming growth factor (TGF)K are typical ErbB-1 ligands. Ligands for ErbB-2 have not yet been identi¢ed. The family of growth factor isoforms called neuregulins (NRGs) also known as Neu di¡erentiation factor (NDF), are typical ligands of ErbB-3 and ErbB-4 (Peles et al., 1992; Pinkas-Kramarski and Yarden, 1995; Wen et al., 1992). Three ligands of ErbB-1, betacellulin, HB-EGF and epiregulin, bind also the ErbB-4 receptor (Beerli and Hynes, 1996; Elenius et al., 1997; Riese et al., 1996; Shelly et al., 1998). NRGs and their receptors (ErbB-3 and ErbB-4) are widely expressed in the nervous system (Marchionni et al., 1993; Orr-Urtreger et al., 1993; Pinkas-Kramarski et al., 1997b, 1994; Gassmann et al., 1995). Several examples exist which demonstrate the variety of biological e¡ects of NRG on neurons and glial cells. For example, NRGs were shown to be mitogenic for Schwann and glial cells (Brockes et al., 1980; Ra¡ et al., 1978). Like-
The ErbB subfamily of receptor tyrosine kinases includes four members (Heldin, 1995; Peles and Yarden, 1993): the epidermal growth factor (EGF) receptor (also called ErbB-1/HER-1) (Ullrich et al., 1984), Neu/HER-2/ErbB2 (Bargmann et al., 1986; Coussens et al., 1985; Yamamoto et al., 1986), HER-3/ErbB-3 (Kraus et al.,
*Corresponding author: Tel.: +972-3-6406801; fax: +972-36407643. E-mail address: [email protected] (R. Pinkas-Kramarski). Abbreviations : DAPI, 4P,6-diamidine-2P-phenylindol dihydrochloride ; DMEM, Dulbecco's modi¢ed Eagle's medium; EDTA, ethylenediaminetetra-acetate ; EGF, epidermal growth factor; EGTA, ethyleneglyco-bis(2-aminoethyl-ether)-N,N,NP,NP-tetraacetic acid; Erk, extracellular signal-regulated kinase ; FBS, fetal bovine serum ; FGF, ¢broblast growth factor; GF109203X, 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-maleimide ; HB, heparin binding ; HEPES, N-(2-hydroxyethyl)piperazine-NP-(2-ethanesulphonic acid); HS, horse serum ; LY294002, [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one]; MAPK, mitogen-activated protein kinase ; MTT, [3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl]tetrazolium bromide; NGF, nerve growth factor; NRG, neuregulin; NT, neurotrophin; PBS, phosphatebu¡ered saline; PD98059, 2P-amino-3P-methoxy£avone; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; SB203580, [4-(3-iodophenyl)-2-(4-methylsul¢nylphenyl)-5-(4-pyridyl)1H-imidazole]; SDS^PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; TrK, tyrosine kinase. 353
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wise, the factor can promote maturation of astrocytes in primary cultures of embryonic brain cells (PinkasKramarski et al., 1994), and the di¡erentiation of oligodendrocytes from O2A progenitor cells (Vartanian et al., 1994). Another study, that made use of clonal cultures of neural crest stem cells, demonstrated that NRG can inhibit neuronal cell di¡erentiation by favoring glial cells (Shah et al., 1994), and that NRG can promote neurotrophin-3 (NT3) production by glial cells, thus promoting neuronal survival (Verdi et al., 1996). Additional biological activities of NRG have been demonstrated in the neuromuscular junction (Corfas et al., 1993; Corfas and Fischbach, 1993). Furthermore, in cerebellar granule cells it was demonstrated that NRG induces cell migration along the radial glial ¢bers (Rio et al., 1997), induces GABA(A) receptor expression and induces neurite outgrowth through ErbB-4 receptor (Rie¡ et al., 1999) as well as the regulation of N-methyl-D-aspartate (NMDA)-2C receptor expression (Ozaki et al., 2000). The PC12 cell line, derived from a rat adrenal medullary pheochromocytoma tumor, has become a widely used paradigm for studies on the regulation of neural di¡erentiation and survival (Greene and Tischler, 1976). PC12 cells can be induced to di¡erentiate by neurotrophic factors such as nerve growth factor (NGF) and basic ¢broblast growth factor (FGF), that bind and activate their respective tyrosine kinases trkA and FGF receptors through the Ras/mitogen-activated protein kinase (MAPK) pathway (Cowley et al., 1994; Marshall, 1995). Other studies showed that EGF is a mild mitogen for these cells and that like NGF it induces extracellular signal-regulated kinase (Erk) activation via the Ras pathway but unlike NGF, EGF-induced Erk activation is transient (Marshall, 1995). It was also demonstrated that many growth factors and neurotrophins can promote neuronal survival of several classes of neurons. Among these factors are insulin, insulin-like growth factor-1, brain-derived neurotrophic factor (BDNF), NGF, NT3 and NT4/5 (Barde, 1989; Chen et al., 1977; Greene, 1978). The PC12 system has also been used to study the e¡ects of neurotrophic factors on cell survival. Following stimulation by tumor necrosis factor (TNF)K or when deprived of growth factors, these cells die apoptotically, and NGF can maintain their long-term survival (Greene, 1978; Haviv and Stein, 1999; Mesner et al., 1992). The survival e¡ect induced by NGF in PC12 cells, requires the activation of phosphoinositide 3-kinase (PI3K) signaling pathway (Klesse et al., 1999; Yao and Cooper, 1995). Recently, we have shown that NRG like NGF can induce the di¡erentiation of PC12-ErbB-4 cells (Vaskovsky et al., 2000). The accumulated data on NRG function in neuronal and glial cells indicate an important function of NRGs and their receptors in neural development and maintenance. In spite of that, it is not yet clear whether ErbB-4 receptor alone or in combination with other ErbB family members can mediate neuroprotective signals. The objective of this study was to evaluate the survival e¡ect of NRG and to explore the mechanism by which NRG induces survival of PC12-ErbB-4 cells.
EXPERIMENTAL PROCEDURES
Materials and bu¡ers EGF (human recombinant) was purchased from Sigma (Rehovot, Israel). NGF (mouse submaxillary gland) was from Chemicon (Temecula, CA, USA). Recombinant murine TNFK and human recombinant NRGL were from RpD Systems (Oxon, UK). A monoclonal anti-phosphotyrosine antibody (PY-20) was purchased from Santa-Cruz Biotechnology (Santa Cruz, CA, USA). A polyclonal anti-PI3K-p85 antibody was from Upstate Biotechnology (Lake Placid, NY, USA). LY294002, SB203580 and wortmannin were purchased from Calbiochem (La Jolla, CA, USA). GF109203X was purchased from Sigma. PD98059 was purchased from Promega (Madison, MI, USA). All other reagents were from Sigma (Rehovot, Israel). HNTG bu¡er contained 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol. Solubilization bu¡er contained 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 1.5 mM MgCl2 , 10% glycerol, 0.2 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl £uoride (PMSF), 10 Wg/ml aprotinin, and 10 Wg/ml leupeptin. Cell lines The rat pheochromocytoma cells (PC12) which express ErbB1, ErbB-2 and ErbB-3 receptors were used for expressing the ErbB-4 receptor. Expression vector LXSHD-ErbB-4 containing the coding region of ErbB-4 (Shelly et al., 1998) was introduced by infection into PC12 cells as previously described and was termed PC12-ErbB-4 cell line (Vaskovsky et al., 2000). The two cell lines were grown in Dulbecco's modi¢ed Eagle's medium (DMEM) supplemented with antibiotics, 10% heatinactivated fetal bovine serum (FBS) and 10% horse serum (HS). Cells were incubated at 37³C in 5% CO2 in air, and the medium was changed every 3^4 days. Cells were passaged when 90% con£uent using trypsin/disodium EDTA (Biological Industries, Kibbutz Beit Haemek, Israel). Cells were induced to di¡erentiate by growing on collagen-coated plates at 2U104 cells/ml in the presence of 50 ng/ml NGF for 7 days. Before experiments were performed, cells were washed twice with phosphate-bu¡ered saline (PBS). Cell survival assays Cells were resuspended and seeded on collagen-coated 96-well plates at 1.2U104 cells/well. Cultures were grown either in serum-free DMEM or in DMEM supplemented with 2.5% FBS and 2.5% HS (for the analysis of TNFK e¡ect), and treated without or with NRG, NGF or EGF at 100 ng/ml for comparison of long-term factors activity. Cell survival was determined by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide (MTT) assay, which determines mitochondrial activity in living cells (Mosmann, 1983). MTT 0.1 mg/ml was incubated with the analyzed cells for 2 h at 37³C. Living cells can transform the tetrazolium ring into dark blue formazan crystals, which can be quanti¢ed by reading the optical density at 550^650 nm after lysis of the cells with acidic isopropanol. Staining of nuclei with the £uorescent DNA dye 4P,6-diamidine2P-phenylindol dihydrochloride (DAPI) was used to estimate the number of dying cells. Cells were scored for apoptosis by nuclear morphology. Treated cells were ¢xed in 4% formaldehyde, washed three times with PBS and then stained with DAPI solution (10 Wg/ml) for 10 min. Following washing with PBS and mounting, the cells were photographed. The instrument used was Olympus optical inverted phase-contrast microscope Model IX70 (U20 magni¢cation). Cell cycle analysis For cell cycle analysis, cells were seeded at 105 cells/ml, with or without growth factors (100 ng/ml), in serum-free DMEM or
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Fig. 1. Ligand-induced cell growth and survival of PC12-ErbB-4 cells. (A) Cells were deprived of serum and plated in 96-well plates at a density of 1.2U104 cells/well in media containing 100 ng/ml of EGF (closed circles), NRG (closed squares) or NGF (open triangles). For control (CON), we used mock-treated cells (closed triangles). The extent of cell proliferation was determined daily by using the colorimetric MTT assay. Results are presented as fold induction over the control untreated cells at day 0, before cells were treated (t = 0), and is the mean þ S.D. of six determinations. Each experiment was repeated at least three times with similar results. (B) 2U103 cells/well were induced to di¡erentiate by 50 ng/ml NGF treatment for 7 days in DMEM containing 2.5% FBS and 2.5% HS. Wells were then washed twice with PBS and serum-free medium containing 100 ng/ml of EGF (closed circles), NRG (closed squares) or NGF (open triangles) or medium alone (closed triangles) was added. The extent of cell survival was determined daily as described in (A) using the di¡erentiated untreated cells at the start point as control t = 0. (C) Cells were plated at a density of 1.2U104 cells/well in DMEM containing 2.5% FBS and 2.5% HS. Cells were treated with 50 ng/ml TNFK either alone (CON) or together with 100 ng/ml NRG, NGF or EGF. The e¡ect of the treatments on cell survival was then determined as described in (A). (D) 2U103 cells/well were induced to di¡erentiate by 50 ng/ml NGF treatment for 7 days in DMEM containing 2.5% FBS and 2.5% HS. Wells were then washed twice with PBS and DMEM containing 2.5% FBS, 2.5% HS and 50 ng/ml TNFK without (CON) or with 100 ng/ml of EGF (closed circles), NRG (closed squares) or NGF (open triangles). The extent of cell survival was determined daily as described in (A).
in DMEM supplemented with 2.5% FBS and 2.5% HS (for the analysis of TNFK e¡ect). TNFK concentration was 50 ng/ml. After 24 h, 106 treated cells were detached from the plates (by 0.5 mM EDTA), and centrifuged (200Ug). The pellet was washed once with PBS and resuspended in 1 ml of hypotonic £uorochrome solution (50 Wg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100) and stored overnight in the dark at 4³C prior to £ow cytometric analysis. The stained cells were analyzed in a £uorescence cell sorter (FACSort; Becton and Dickinson) excited at 488-nm wavelength and collected through a 570-nm BP ¢lter. Data were analyzed using WinMDI 2.1.4 computer software. The percentage of cells in sub-G1 phase of the cell cycle was designated as apoptotic cell population. Lysate preparation and immunoprecipitation Cells were exposed to the indicated stimuli. After treatment, cells were solubilized in lysis bu¡er. Lysates were cleared by
centrifugation. For direct electrophoretic analysis, boiling gel sample bu¡er was added to cell lysates. For other experiments, lysates were ¢rst subjected to immunoprecipitation with antiphosphotyrosine antibodies (PY-20). Antibodies were ¢rst coupled to rabbit anti-mouse immunoglobulin G and then to protein A-Sepharose by the same procedure. The proteins in the lysate supernatant were immunoprecipitated with aliquots of the protein A-Sepharose^antibody complexes for 2 h at 4³C. The immunoprecipitates were washed three times with HNTG, resolved by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) through 7.5% gels and electrophoretically transferred to nitrocellulose membrane. Membranes were blocked for 2 h in TBST bu¡er (0.02 M Tris^HCl pH 7.5, 0.15 M NaCl and 0.05% Tween 20) containing 6% milk, blotted with 1 Wg/ml primary antibody for 2 h, followed by 0.5 Wg/ml secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected with the enhanced chemiluminescence reagent (Amersham Corp, Bukinghamshire, UK).
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Fig. 2. NRG-induced cell survival of PC12-ErbB-4 cells. PC12-ErbB-4 cells were tested for cell survival by using £ow cytometry analysis. Cultures of 106 cells were treated as indicated and as described in Fig. 1A and C. Cells were harvested after 24 h and analyzed for DNA content by £ow cytometry. The percentage of cells at the sub-G1 phase of the cell cycle is indicated (Ap). This cell fraction represents cells undergoing apoptosis. The experiments were repeated three times with similar results.
RESULTS
Induction of cell survival by NRG, EGF and NGF PC12 cells express ErbB-1, ErbB-2 and ErbB-3, but not ErbB-4 receptors (Vaskovsky et al., 2000). ErbB-4 was introduced into PC12 cells by retroviral infection and stable ErbB-4 expressing clones were then selected and a representative clone was chosen for further analysis (PC12-ErbB-4) (Vaskovsky et al., 2000). To determine the e¡ect of the NRG, EGF and NGF ligands on long-term survival of PC12-ErbB-4 cells we have used the MTT assay (Fig. 1). Figure 1 represents survival e¡ect induced by the various ligands in naive or di¡erentiated cells. The cell death inducers are serum starvation (Fig. 1A and B) or TNFK (Fig. 1C and D). The parameter used to determine cell viability was the percentage of the apparent cell number at the indicated day compare to day 0 (% of t = 0). As shown, under conditions of serum deprivation both NRG and NGF induced cell survival that was greater than the e¡ect induced by EGF (Fig. 1A). Under the same conditions the control serum deprived untreated cells die (Fig. 1A). Moreover, when cells were induced to die by TNFK treatment, both NGF and NRG rescued cells from death (Fig. 1C). We next examined whether NRG could also protect di¡erentiated cells from death. For this aim, similar experiments were performed on PC12ErbB-4 cells with sympathetic neuronal cell phenotype induced by NGF treatment for 1 week (prior to the experiment performance). As shown, incubation of differentiated PC12-ErbB-4 cells with NRG inhibited cell death induced by both serum deprivation and TNFK treatment (Fig. 1B and D). The e¡ect of NRG on di¡erentiated PC12-ErbB-4 cells was similar to the e¡ect of NGF but not of EGF (Fig. 1). However, NRG could not
rescue di¡erentiated and naive PC12 cells from apoptosis induced by both serum deprivation and TNFK treatment (data not shown). Clearly then, NRG, that activates ErbB-4 receptors in PC12-ErbB-4 cells, mediates survival responses. These results encouraged us to further characterize the e¡ect of NRG on cell survival. NRG rescue from apoptosis induced by serum deprivation and TNFK The responses of PC12 cells to EGF and NGF have been well characterized. While EGF is a potent mitogen (Mark et al., 1995), NGF is a di¡erentiation and survival factor for PC12 cells (Greene and Tischler, 1976). To further analyze the e¡ect of NRG on survival of PC12ErbB-4 cells, we determined the fraction of cells that underwent apoptosis by performing either £ow cytometry or by staining the cells with the DNA-speci¢c dye DAPI. Evidently, the fraction of sub-G1 cells that represents cells undergoing apoptosis was increased by serum deprivation or by incubation with TNFK to 55% and 20% respectively (Fig. 2). Addition of NRG reduced the percentage of apoptotic cells to 4% and 1.5% in serum-deprived cells or in TNFK-treated cells respectively (Fig. 2). The e¡ect of NRG on cell survival was also evident by DAPI staining of cell nuclei (Fig. 3). Condensed or fragmented nuclei represent cells undergoing apoptosis. In the control cells, serum deprived and TNFK treated for 24 h, condensed and fragmented nuclei were observed indicating that cells underwent apoptosis (Fig. 3). As shown, less apoptotic cells were present in NRG-treated cells compared to control (serum-deprived) untreated or TNFK-treated cells indicating that NRG protected these cells from apoptosis. Moreover, it was previously shown that following di¡erentiation, the removal of NGF and serum from PC12
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Fig. 3. NRG rescue from apoptosis induced by serum deprivation or TNFK treatment. (A) PC12-ErbB-4 cultures were grown in 96-well plates at 1.2U104 cells/well. Separate wells were then treated for 24 h in serum-free medium containing either EGF, NRG, NGF at 100 ng/ml, or left untreated (control, CON). For experiments with di¡erentiated cells, cells were grown 1 week in the presence of 50 ng/ml NGF and then washed twice with PBS prior to growth factors treatment for additional 24 h as described above. (B) Sister cultures were grown in DMEM containing 2.5% FBS and 2.5% HS. Separate wells were then treated with either EGF, NRG or NGF at 100 ng/ml in the presence or absence of TNFK (50 ng/ml) for 24 h. For experiments with di¡erentiated cells, cells were pretreated as described in (A). The incubation was terminated by cell ¢xation and staining with the DNA intercalating dye DAPI. The resulting £uorescent photomicrographs are shown. Scale bar = 20 Wm.
cells leads to apoptosis (Teng and Green, 1994). As shown in Fig. 3, the protective e¡ect of NRG was also evident in di¡erentiated PC12-ErbB-4 cells. In addition, in EGF-treated cells apoptotic nuclei were observed although the number of live cells was higher and comparable to the control untreated cell, indicating that EGF only partially rescues cells from apoptosis and induces cell division. Taken together, the results shown in Fig. 3 indicate that NRG may protect di¡erentiated and non-di¡erentiated PC12-ErbB-4 cells from apoptosis induced by both serum deprivation and TNFK treatment. Analysis of the signaling pathway that mediates NRG-induced survival Since NRG proved in this cell system to protect cells from apoptosis we next employed various pharmacological inhibitors to test through which signaling pathway NRG exerts its e¡ect. In order to examine whether Erk activation is required for the survival e¡ect induced by
NRG, we examined the e¡ect of Erk pathway inhibitor, PD98059, which inhibits MAPKK, on the NRG-mediated survival e¡ect. PC12-ErbB-4 cells were incubated with 100 ng/ml NRG in the absence or in the presence of PD98059 (50 WM). As shown (Fig. 4) the MAPKK inhibitor did not a¡ect the survival induced by NRG. It was previously shown that PD98059 inhibits the di¡erentiation induced by NRG and by NGF in these cells (Vaskovsky et al., 2000), suggesting that the sustained MAPK activation is required for di¡erentiation in PC12-ErbB-4. Our results implicate that activation of the MAPK pathway is not required for NRG-mediated survival e¡ect. Several studies showed that atypical protein kinase C (aPKC) are involved in NGF-induced survival of serumdeprived PC12 cells (Wooten et al., 2000) and overexpression of aPKCs enhances NGF responsiveness as well as survival of di¡erentiated cells (Wooten et al., 1999). In addition, it was previously shown that the PKC inhibitor GF109203X inhibits the morphological changes induced by NRG or by NGF in PC12-ErbB-4
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Fig. 4. The e¡ect of various drugs on NRG-induced cell survival. Cells were plated in 96-well plates and induced to di¡erentiate as described in Fig. 1. (A) Separate wells, grown in serum-free medium, were treated with either EGF, NRG or NGF at 100 ng/ml in the absence or presence of 1 WM GF109203X (GF), 50 WM PD98059 (PD), 4 WM SB203580 (SB), 20 nM rapamycin (RAP) and 20 WM LY294002 (LY) as indicated. (B) Separate cultures were grown in medium containing 2.5% HS and 2.5% FBS in the presence or absence of TNFK (50 ng/ml), and treated with either EGF, NRG or NGF at 100 ng/ml in the absence or presence of 1 WM GF109203X (GF), 50 WM PD98059 (PD), 4 WM SB203580 (SB), 20 nM rapamycin (RAP) and 20 WM LY294002 (LY) as indicated. The MTT assay was performed 48 h later. Results are presented as fold induction over the control untreated cells in (A), and over the TNFK-treated cells in (B), and are the mean þ S.D. of six determinations. Each experiment was repeated at least three times with similar results. CON, control.
cells (Vaskovsky et al., 2000). In the present study we were interested to study whether PKCs are involved in NRG-mediated survival e¡ect. As shown in Fig. 4, the PKC inhibitor GF109203X (1 WM) which blocked the morphological changes induced by NRG or by NGF in PC12-ErbB-4 cells (Vaskovsky et al., 2000), did not inhibit the survival e¡ect induced by NRG. It was previously shown that selective p38 inhibitors promoted the in vitro survival of sensory, sympathetic, ciliary and motor neurons in a dose-dependent manner (Horstmann et al., 1998). In the present study we examined the involvement of p38 in NRG-mediated survival. For this aim, we examined the ability of a p38-speci¢c inhibitor to a¡ect the NRG-mediated survival in PC12ErbB-4 cells. We found that SB203580 (4 WM) did not a¡ect NRG-mediated survival (Fig. 4). Taken together, these results suggest that Erk, PKC and p38 activation are not required for this process. Since PI3K has been previously implicated in survival signaling (Yao and Cooper, 1995), we examined the e¡ect of a speci¢c PI3K inhibitor LY294002 (20 WM) on NRG-induced survival. PI3K inhibition induced apoptosis of PC12-ErbB-4 cells, namely cell death was observed also in the presence of NGF or NRG (Fig. 4). Dimethylsulfoxide (DMSO) treatment at a concentration used to solubilize LY294002 had no e¡ect on cell viability and served as a control. The addition of LY294002 as well as other inhibitors (Fig. 4A) by themselves could reduce cell viability. These results suggest that inhibition of PI3K pathway (and also other signaling pathways)
has a toxic e¡ect on the cells and that the toxic e¡ect of PI3K inhibitor is not prevented by activation of ErbB4. However, the toxic e¡ect of the other inhibitors used was prevented by NRG. The inability of NRG to rescue cells following treatment with LY294002 encouraged us to further investigate the PI3K activation induced by NRG. The p85 regulatory subunit of PI3K contains two SH2 domains which bind speci¢c phosphorylated tyrosine residues in receptor tyrosine kinases. As shown in Fig. 5A, immunoblotting of speci¢c phoshotyrosine immunoprecipitates with anti-p85 antibodies suggested that p85 is mainly associated with ErbB-4 upon NRG stimulation. These ¢ndings suggest that activation of ErbB-4 receptors by NRG results in activation of PI3K. Several studies demonstrate that PKB/Akt, a serine threonine kinase that functions as an e¡ector of PI3K, is involved in cell survival (Klesse et al., 1999). PI3K inhibitor LY294002 blocks the activity of Akt as well as cell survival (Ashcroft et al., 1999). For these reasons we have analyzed the kinetics of Akt activation following NGF, NRG and EGF (each at 100 ng/ml) treatments in PC12 and PC12-ErbB-4 cells. As shown (Fig. 5B), NRG and NGF induced prolonged Akt activation that lasted for 2 h in PC12-ErbB-4 cells. EGF, however, induced transient Akt activation (Fig. 5B). The prolonged Akt activation induced by NRG and NGF correlated with the ability of these ligands to rescue cells from apoptosis. These results may indicate that PI3K, as well as its downstream e¡ector Akt, may be the signaling pathway responsible for NRG-induced survival.
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Fig. 5. Ligand-induced activation of PI3K and PKB/Akt. (A) Whole cell lysates from cells treated for 10 min at 37³C with either EGF, NRG or NGF at 100 ng/ml were immunoprecipitated (IP) with mouse monoclonal PY20 antibody (P-TYR) as indicated. After SDS^PAGE, blots were probed with a rabbit polyclonal anti-p85 antibody. (B) Whole cell lysates were prepared from cells treated for the indicated time points with EGF, NRG or NGF at concentration of 100 ng/ml. Lysates were analyzed by immunoblotting with a monoclonal antibody to phosphorylated PKB/Akt. Signal detection was performed by using a chemiluminescence kit. (C) Whole cell lysates were prepared from PC12 or PC12-ErbB-4 cells treated for the indicated time points with EGF, NRG or NGF at concentration of 100 ng/ml. Lysates were analyzed by immunoblotting with a monoclonal antibody to phosphorylated PKB/Akt. Quanti¢cation of the phosphorylated PKB/AKT intensity was performed using TINA 2.0 computer program. Data are presented as fold induction over the control untreated cells and is the mean þ S.D. of three experiments. CON, control. DISCUSSION
NRG receptors ErbB-3 and ErbB-4, unlike other members of the EGF receptor family, are characterized by a relatively limited expression pattern. ErbB-4 expression is relatively restricted to the developing CNS and to the developing heart (Kraus et al., 1989; Pinkas-Kramarski et al., 1997b; Plowman et al., 1993). NRGs play an important role in the nervous system, primarily in communicating nerve cells to non-neuronal cells. NRGs act as the most potent mitogens for precursor Schwann cells in vitro (Dong et al., 1995), and they reduce apoptosis of mature Schwann cells upon axotomy (Grinspan et al., 1996). Myelin-forming cells of the CNS, the oligodendrocytes, are also targets of NRGs (Canoll et al., 1996; Vartanian et al., 1994), as are embryonic astroglia, whose maturation and survival are a¡ected (Pinkas-
Kramarski et al., 1994). Another signal of NRG for glial di¡erentiation, on the expense of a neuronal fate, was demonstrated by using neural crest stem cells (Shah et al., 1994). Recently it was demonstrated that ErbB-4 receptor when expressed in PC12 cells can induce neuronal di¡erentiation (Vaskovsky et al., 2000). In the present study we demonstrated that ErbB-4 receptors in PC12-ErbB-4 cells mediate also NRG-induced survival. The NRG-mediated survival e¡ect is indistinguishable from the e¡ect mediated by the NGF-activated tyrosine kinase (Trk) receptors (Fig. 1). Accordingly, activation of the ErbB-4 receptors by NRG resulted in signi¢cant ErbB-4 receptor phosphorylation (Vaskovsky et al., 2000), induced a sustained activation of Akt (Fig. 5B) and survival signals (Figs. 1 and 2). These are among the typical responses of PC12 cells when they are exposed to NGF (Connolly et al., 1984; Greene
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and Tischler, 1976; Hu¡ et al., 1981). Moreover, we ¢nd that the NRG-induced survival in PC12-ErbB-4 cells is probably mediated through the PI3K pathway since NRG failed to rescue cells in the presence of PI3K inhibitor (Fig. 4). PI3K-dependent survival is also a known characteristic of NGF-induced survival in PC12 cells (Klesse et al., 1999). We therefore suggest that, in PC12-ErbB-4 cells, the NRG-activated ErbB-4 receptors share with the NGF-activated Trk receptors a similar set of signaling events leading to survival. By employing the PC12 cells as a model system for sympathetic neurons, our results suggest that one of the e¡ects of NRG on neurons may be to protect them from apoptosis. The e¡ect of NRG on cell survival is evident by three independent observations. First, NRG increased cell viability as measured by MTT assay (Fig. 1). Second, FACS analyses demonstrated that NRG treatment reduced the percentage of apoptotic cells (Fig. 2). Third, evaluation of apoptosis following DAPI staining showed the characteristics of apoptotic cells namely chromatin condensation and nuclear segmentation. NRG treatment signi¢cantly reduced the number of apoptotic cells (Fig. 3). NRG did not prevent apoptosis of untransfected PC12 cells (data not shown) indicating that NRG-induced cell survival in PC12ErbB-4 cells is mediated by the ErbB-4 receptors. It was previously shown that NRG indirectly a¡ects neuroblast survival and di¡erentiation (Verdi et al., 1996). Also paracrine mechanisms may underlie the previously reported survival and di¡erentiation e¡ect of NRG on rat retinal neurons, whose number of neurite extensions, but not their size, was increased by NRG (BerminghamMcDonogh et al., 1996). Furthermore, it was demonstrated that in cerebellar granule cells NRG induces cell migration along the radial glial ¢bers (Rio et al., 1997) and induces GABA(A) receptor expression and neurite outgrowth through ErbB-4 receptor (Rie¡ et al., 1999). Our results demonstrate that NRG can support neuronal survival of ErbB-4 expressing PC12 cells. The e¡ect of NRG on PC12-ErbB-4 cells, as demonstrated, resembles the e¡ect of NGF in supporting cell survival. Taken together, these results demonstrate that NRG can act as neurotrophic factor a¡ecting neuronal di¡erentiation and survival. Previously we reported that NRG activates the phosphorylation of ErbB-4 receptors in these cells (Vaskovsky et al., 2000). Activation of ErbB-4 leads to
neuronal di¡erentiation through the activation of the Ras/MAPK pathway (Vaskovsky et al., 2000). In this respect NRG-induced di¡erentiation resembles the e¡ect of NGF. A growing body of data accumulates concerning the survival e¡ect mediated by NGF in neurons. In PC12 cells NGF protects cells from apoptosis induced by various stimuli (Greene, 1978; Haviv and Stein, 1999; Mesner et al., 1992). It was previously shown that the protective e¡ect of NGF, on serum-deprived PC12 cells, is mediated by the PI3K signaling pathway (Yao and Cooper, 1995). Moreover, it was demonstrated that PI3K activation and subsequent activation of the downstream signaling e¡ector, Akt, function to promote cell survival in serum-deprived PC12 cells (Ashcroft et al., 1999). Our results demonstrate that the NRG-induced cell survival in PC12 cells expressing the ErbB-4 receptors, is probably mediated by PI3K pathway since PI3K inhibition blocked the survival e¡ect induced by NRG (Fig. 4). In addition, the kinetics of Akt activation induced by NRG is similar to that induced by NGF but not of EGF, indicating that Akt activation may be important for NRG-induced survival e¡ect.
CONCLUSION
The present study strengthens the hypothesis that ErbB-4 receptors when activated by NRG can play an important role in neuronal survival. It is important to note that the e¡ect of NRG on cell survival may not be restricted to ErbB-4 receptor. In the present study we analyzed the e¡ect of ErbB-4, which is known to be highly expressed in neurons. Our results, however, do not exclude the possibilities that only overexpression of ErbB-4 can provide survival signals and that such e¡ects may be also mediated by ErbB-2 or ErbB-3 receptors. Nevertheless, this model system, PC12-ErbB-4 cells, can serve future studies on ErbB-4-mediated e¡ects and signal transduction pathways involved in neuronal survival and function. AcknowledgementsöThis work was supported in part by The National Institute for Psychobiology in Israel, The Charles E. Smith Foundation, grant 7-2001 No. 34-01 and by the Adams super-center for brain studies, No. 1190, Israel. We thank Prof. A. Dvir and Prof. R. Stein for critically reading this manuscript.
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