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• W1963697539 abstract "Neuregulins (NRGs), a large family of transmembrane polypeptide growth factors, mediate various cellular responses depending on the cell type and receptor expression. We previously showed that NRG mediates survival of PC12-ErbB4 cells from apoptosis induced by serum deprivation or tumor necrosis factor-α treatment. In the present study we show that NRG induces a significant protective effect from H2O2-induced death. This effect of NRG is mediated by the phosphatidylinositol 3-kinase (PI3K)-signaling pathway since NRG failed to rescue cells from H2O2 insult in the presence of the PI3K inhibitor, LY294002. Furthermore, the downstream effector of PI3K, protein kinase B/AKT, is activated by NRG in the presence of H2O2, and protein kinase B/AKT activation is inhibited by LY294002. In addition, our results demonstrate that reactive oxygen species (ROS) elevation induced by H2O2 is inhibited by NRG. LY294002, which blocks NRG-mediated rescue, increases ROS levels. Moreover, both H2O2-induced ROS elevation and cell death are reduced by expression of activated PI3K. These results suggest that PI3K-dependent pathways may regulate toxic levels of ROS generated by oxidative stress. Neuregulins (NRGs), a large family of transmembrane polypeptide growth factors, mediate various cellular responses depending on the cell type and receptor expression. We previously showed that NRG mediates survival of PC12-ErbB4 cells from apoptosis induced by serum deprivation or tumor necrosis factor-α treatment. In the present study we show that NRG induces a significant protective effect from H2O2-induced death. This effect of NRG is mediated by the phosphatidylinositol 3-kinase (PI3K)-signaling pathway since NRG failed to rescue cells from H2O2 insult in the presence of the PI3K inhibitor, LY294002. Furthermore, the downstream effector of PI3K, protein kinase B/AKT, is activated by NRG in the presence of H2O2, and protein kinase B/AKT activation is inhibited by LY294002. In addition, our results demonstrate that reactive oxygen species (ROS) elevation induced by H2O2 is inhibited by NRG. LY294002, which blocks NRG-mediated rescue, increases ROS levels. Moreover, both H2O2-induced ROS elevation and cell death are reduced by expression of activated PI3K. These results suggest that PI3K-dependent pathways may regulate toxic levels of ROS generated by oxidative stress. neuregulin nerve growth factor Dulbecco's modified Eagle's medium extracellular signal-regulated kinase monoclonal antibody phosphate-buffered saline phosphatidylinositol 3-kinase reactive oxygen species protein kinase B 2′,7′-dichlorodihydrofluorescein 2′,7′-dichlorodihydrofluorescein diacetate fetal bovine serum horse serum phosphate-buffered saline [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl]tetrazolium bromide Growth factors activate various signaling pathways that are critical for neuronal cell growth and survival (1Bibel M. Barde Y.A. Genes Dev. 2000; 14: 2919-2937Crossref PubMed Scopus (881) Google Scholar, 2Kaplan D.R. Miller F.D. Curr. Opin. Neurobiol. 2000; 10: 381-391Crossref PubMed Scopus (1653) Google Scholar). The neuregulins (NRGs)1 are a family of growth and differentiation factors that bind to members of the epidermal growth factor family of tyrosine kinase receptors and result in many important effects on neurons and glial cell development (3Adlkofer K. Lai C. Glia. 2000; 15: 104-111Crossref Scopus (158) Google Scholar, 4Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 5Garratt A.N. Britsch S. Birchmeier C. Bioessays. 2000; 22: 987-996Crossref PubMed Scopus (252) Google Scholar, 6Gassmann M.L.G. Curr. Opin. Neurobiol. 1997; 7: 87-92Crossref PubMed Scopus (108) Google Scholar, 7Pinkas-Kramarski R. Eilam R. Alroy I. Levkowitz G. Lonai P. Yarden Y. Oncogene. 1997; 15: 2803-2815Crossref PubMed Scopus (114) Google Scholar, 8Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4604) Google Scholar). NRGs along with ErbB-3 and ErbB-4 receptors are highly expressed in the developing and mature nervous system (7Pinkas-Kramarski R. Eilam R. Alroy I. Levkowitz G. Lonai P. Yarden Y. Oncogene. 1997; 15: 2803-2815Crossref PubMed Scopus (114) Google Scholar, 9Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Denehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBury R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (682) Google Scholar, 10Pinkas-Kramarski R. Eilam R. Spiegler O. Lavi S. Liu N. Chang D. Wen D. Schwartz M. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9387-9391Crossref PubMed Scopus (139) Google Scholar). After brain insult, the level of NRG (11Eilam R. Pinkas-Kramarski R. Menahem S. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1888-1893Crossref PubMed Scopus (92) Google Scholar) and ErbB-4 receptor (11Eilam R. Pinkas-Kramarski R. Menahem S. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1888-1893Crossref PubMed Scopus (92) Google Scholar, 12Erlich S. Shohami E. Pinkas-Kramarski R. Mol. Cell. Neurosci. 2000; 16: 597-608Crossref PubMed Scopus (38) Google Scholar) elevates, indicating that ErbB-4 and NRG may function in either synaptic plasticity or neuroprotection. NRG and its receptors mediate various biological effects depending on the cell type examined. Several studies exist to demonstrate that NRG can serve as a differentiation factor for astrocytes (10Pinkas-Kramarski R. Eilam R. Spiegler O. Lavi S. Liu N. Chang D. Wen D. Schwartz M. Yarden Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9387-9391Crossref PubMed Scopus (139) Google Scholar), oligodendrocytes (13Vartanian T. Corfas G. Li Y. Fischbach G.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 91: 11626-11630Crossref Scopus (96) Google Scholar), and neurons (14Rieff H.I. Raetzman L.T. Sapp D.W. Yeh H.H. Siegel R.E. Corfas G. J. Neurosci. 1999; 19: 10757-10766Crossref PubMed Google Scholar). Moreover, it was demonstrated that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve (15Fernandez P.A. Tang D.G. Cheng L. Prochiantz A. Mudge A.W. Raff M.C. Neuron. 2000; 28: 81-90Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and that neuregulin in the central nervous system diminishes autoimmune demyelination, promotes oligodendrocyte progenitor expansion, and enhances remyelination (16Marchionni M.A. Cannella B. Hoban C. Gao Y.L. Garcia-Arenas R. Lawson D. Happel E. Noel F. Tofilon P. Gwynne D. Raine C.S. Adv. Exp. Med. Biol. 1999; 468: 283-295Crossref PubMed Google Scholar). In addition, neuregulin affects neuronal survival and neurite outgrowth of developing rat retina (17Bermingham-McDonogh O. McCabe K.L. Reh T.A. Development. 1996; 122: 1427-1438PubMed Google Scholar). Reactive oxygen species (ROS) are produced by several cellular metabolic reactions. Cells also possess antioxidant systems to control the redox state, which is important for their survival. ROS that cause oxidative stress have been implicated in several diseases including cancer and neurodegenerative disorders (18Dreher D. Junod A.F. Eur. J. Cancer. 1996; 32: 30-38Abstract Full Text PDF Scopus (770) Google Scholar, 19Knight J.A. Ann. Clin. Lab. Sci. 1997; 27: 93-104PubMed Google Scholar). In addition, several studies indicate that nontoxic levels of ROS may play an essential role as signaling molecules regulating cell growth and differentiation (20Mills E.M. Takeda K., Yu, Z.X. Ferrans V. Katagiri Y. Jiang H. Lavigne M.C. Leto T.L. Guroff G. J. Biol. Chem. 1998; 273: 22165-22168Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 21Peunova N. Enikolopov G. Nature. 1995; 375: 68-73Crossref PubMed Scopus (473) Google Scholar, 22Segal R.A. Greenberg M.E. Annu. Rev. Neurosci. 1996; 19: 463-489Crossref PubMed Scopus (904) Google Scholar, 23Suzukawa K. Miura K. Mitsushita J. Resau J. Hirose K. Crystal R. Kamata T. J. Biol. Chem. 2000; 275: 13175-13178Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 24Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1139) Google Scholar). Oxidative stress such as H2O2treatment induces apoptotic cell death in both PC12 cells and cultured neurons (25Enokido Y. Hatanaka H. Brain Res. 1990; 536: 23-29Crossref PubMed Scopus (33) Google Scholar, 26Ratan R.R. Murphy T.H. Baraban J.M. J. Neurochem. 1994; 62: 376-379Crossref PubMed Scopus (508) Google Scholar). The PC12 cell model has been extensively used to study the signaling pathways leading to neuronal differentiation induced by neurotrophins such as NGF compare with the signaling pathways leading to mitogenesis induced by growth factors such as epidermal growth factor (27Chao M.V. Cell. 1992; 68: 995-997Abstract Full Text PDF PubMed Scopus (268) Google Scholar, 28Dichter M.A. Tischler A.S. Greene L.A. Nature. 1977; 268: 501-504Crossref PubMed Scopus (288) Google Scholar, 29Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4852) Google Scholar, 30Huff K. End D. Guroff G. J. Cell Biol. 1981; 88: 189-198Crossref PubMed Scopus (206) Google Scholar, 31Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4230) Google Scholar). 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, NGF, NT3, and NT4/5 (32Barde Y.A. Neuron. 1989; 2: 1525-1534Abstract Full Text PDF PubMed Scopus (1444) Google Scholar, 33Chen M.G. Chen J.S. Calissano P. Levi-Montalcini R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5559-5563Crossref PubMed Scopus (19) Google Scholar, 34Greene L.A. J. Cell Biol. 1978; 78: 747-755Crossref PubMed Scopus (373) Google Scholar). The PC12 system has also been used to study the effects of neurotrophic factors on cell survival. After stimulation by tumor necrosis factor-α or when deprived of growth factors, these cells die apoptotically, and NGFs can maintain their long term survival (34Greene L.A. J. Cell Biol. 1978; 78: 747-755Crossref PubMed Scopus (373) Google Scholar, 35Haviv R. Stein R. J. Neurosci. Res. 1999; 55: 269-277Crossref PubMed Scopus (39) Google Scholar, 36Mesner P.W. Winters T.R. Green S.H. J. Cell Biol. 1992; 119: 1669-1680Crossref PubMed Scopus (210) Google Scholar). The survival effect induced by NGFs in PC12 cells requires the activation of the PI3K-signaling pathway (37Klesse L.J. Meyers K.A. Marshall C.J. Parada L.F. Oncogene. 1999; 18: 2055-2068Crossref PubMed Scopus (184) Google Scholar, 38Yao R. Cooper G.M. Science. 1995; 267: 2003-2006Crossref PubMed Scopus (1292) Google Scholar). It was also demonstrated that NGF protects PC12 cells and neurons from oxidative stress-induced death (39Kamata H. Tanaka C. Yagisawa H. Hirata H. Neurosci. Lett. 1996; 212: 179-182Crossref PubMed Scopus (62) Google Scholar). Recently, it has been demonstrated that ErbB-4 receptor stably expressed in PC12 cells mediate NRG-induced signals and neurite outgrowth that is indistinguishable from those mediated by NGF-activated Trk receptor (40Vaskovsky A. Lupowitz Z. Erlich S. Pinkas-Kramarski R. J. Neurochem. 2000; 74: 979-987Crossref PubMed Scopus (81) Google Scholar). It was also demonstrated that NRG rescues PC12-ErbB-4 cells from apoptosis induced by serum deprivation or tumor necrosis factor treatment (41Erlich S. Goldshmidt Y. Lupowitz Z. Pinkas-Kramarski R. Neuroscience. 2001; 107: 353-362Crossref PubMed Scopus (45) Google Scholar). Because in various pathological conditions oxidative stress may contribute to neuronal dysfunction and NRGs mediate similar effects as NGFs, we examined the effect of NRGs on H2O2-induced toxicity and analyzed the intracellular mechanism of the NRG-mediated protection from oxidative stress. Epidermal growth factor (human recombinant) was purchased from Sigma. NGF (mouse submaxillary gland) was from Chemicon (Temecula, CA). Human recombinant NRGβ was from R&D Systems Inc. A monoclonal anti-phosphorylated PKB/Akt and a monoclonal anti Pan-PKB/Akt antibody were from New England BioLabs Inc. 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was purchased from Sigma. PD98059 was purchased from Promega (Madison, MI). LY294002 and SB203580 were purchased from Calbiochem. All other reagents were from Sigma. Solubilization buffer 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 mmphenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. PC12 cells and PC12 cells that express ErbB-4 (40Vaskovsky A. Lupowitz Z. Erlich S. Pinkas-Kramarski R. J. Neurochem. 2000; 74: 979-987Crossref PubMed Scopus (81) Google Scholar) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with antibiotics, 7.5% heat-inactivated fetal bovine serum (FBS), and 7.5% horse serum (HS). Cells were incubated at 37 °C in 5% CO2 in air, and the medium was changed every 3–4 days. Cells were passaged when 90% confluent using 0.5 mm EDTA in PBS. Cells were induced to differentiate by growing on collagen-coated plates at 2 × 104 cells/ml in the presence of 50 ng/ml NGF for 7 days. Before experiments were performed, cells were washed twice with PBS. Cells were resuspended and seeded on collagen-coated 96-well plates at 7.5 × 103cells/well in DMEM supplemented with 2.5% FBS and 2.5% HS and treated without or with H2O2 for 30 min in the presence or absence of either NRG or NGF at 50 ng/ml for comparison of long term factor 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 (42Mosman T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (46225) Google Scholar). 0.1 mg/ml MTT was incubated with the analyzed cells for 2 h at 37 °C. Living cells can transform the tetrazolium ring into dark blue formazan crystals, which can be quantified by reading the optical density at 550–650 nm after lysis of the cells with acidic isopropanol. Staining of nuclei with the fluorescent DNA dye 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) was used to estimate the number of dying cells. Cells were scored for apoptosis by nuclear morphology. Treated cells were fixed in 4% formaldehyde, washed three times with PBS, and then stained with DAPI solution (1 μg/ml) for 10 min. After washing with PBS and mounting, the cells were photographed. The instrument used was an Olympus optical inverted phase-contrast microscope model IX70 (×20 magnification). Cells were exposed to the indicated stimuli. After treatment, cells were solubilized in lysis buffer. Lysates were cleared by centrifugation. For direct electrophoretic analysis, boiling gel sample buffer was added to cell lysates. Lysates were resolved by SDS-polyacrylamide gel electrophoresis through 10% gels and electrophoretically transferred to nitrocellulose membrane. Membranes were blocked for 1 h in 0.02m Tris HCl, pH 7.5, 0.15 m NaCl, and 0.05% Tween 20) containing 6% milk and blotted with 1 μg/ml primary antibodies for 2 h followed by 0.5 μg/ml secondary antibody linked to horseradish peroxidase. Immunoreactive bands were detected with the enhanced chemiluminescence reagent (Amersham Pharmacia Biotech). Measurement of intracellular ROS generation was determined using the DCFH-DA assay (43Wang H. Joseph J.A. Free Radic. Biol. Med. 1999; 27: 612-616Crossref PubMed Scopus (1974) Google Scholar). DCHF-DA was dissolved in Me2SO and diluted with DMEM lacking phenol red to a final concentration of 100 μm. Cells were plated on collagen-coated 48-well culture plates at a density of 1 × 105 cells/well in DMEM supplemented with 7.5% FBS and 7.5% HS. After 24 h, medium was replaced by DMEM containing 2.5% FBS and 2.5% HS for 1 h. Cells were treated for 30 min with 0.5 mm H2O2 in the presence or absence of the indicated growth factors or drugs. The medium was then replaced with fresh medium containing growth factors or drugs for the indicated time periods. Cells were then loaded with 100 μm DCFH-DA for 30 min at 37 °C. The loading was terminated by washing the cells with DMEM lacking phenol red containing 2.5% FBS and 2.5% HS. The cells were either photographed by an Olympus optical inverted phase-contrast microscope model IX70 (×100 magnification), or the DCF fluorescence was monitored using a microplate fluorescence reader (Bio-TEK Instruments, FL500) with an excitation wavelength of 485 nm and an emission wavelength 530 nm. The increase in fluorescence for each treatment was calculated by the relative fluorescence of each treatment compared with the control untreated cells normalized by the number of cells as determined by the MTT assay. One day before transfection, PC12-ErbB-4 cells were seeded at a density of 2 × 105 cells/well in 24-well plates. To each well 300 μl of DNA-LipofectAMINE mixture (2 μg of DNA and 30 μg of LipofectAMINE in 1 ml of Opti-MEM (Life Technologies, Inc.)) were added according to the manufacture's instructions. Each transfection was performed four times. After incubation of cells for 5 h with the DNA LipofectAMINE mixture, equal volume of fresh medium was added, and incubation was continued. Medium was replaced 24 h later, and cells were treated with H2O2 for 30 min. The viability of the transfected cells in all experiments was monitored 72 h after transfection by measuring the activity of SEAP in the medium of the transfected cells. The ratio of the different DNA species in each transfection was 10:1 for pSG5/5′MycTp110αCAAX or pcDNA3 and reporter plasmid (secreted alkaline phosphatase (SEAP)). The vector pSG5/5′MycTp110αCAAX was obtained from Prof. J. Downward (Imperial Cancer Research Fund, London) (44Wennström S. Downward J. Mol. Cell. Biol. 1999; 19: 4279-4288Crossref PubMed Scopus (254) Google Scholar). pCMV-SEAP was obtained from Prof. R. Stein (Tel-Aviv University, Israel). SEAP activity was assayed as described previously (45Berger J. Hauber J. Hauber R. Geiger R. Cullen B.R. Gene. 1988; 66: 1-10Crossref PubMed Scopus (580) Google Scholar). Briefly, culture medium (200 μl) from transfected cells was collected and spun for 2 min at 10,000 ×g. The supernatant was incubated at 65 °C for 10 min, and then aliquots (25 μl) from each treatment were incubated with 200 μl of SEAP buffer (1 m diethanolamine, 0.5 mm MgCl2, and 10 mm l-homoarginine) containing 5 mg/mlp-nitrophenylphosphate at 37 °C until a yellow color developed. The plates were read on a micro-enzyme-linked immunosorbent assay reader at wavelength 405 nm. We used PC12 cells stably expressing the ErbB-4 receptor (40Vaskovsky A. Lupowitz Z. Erlich S. Pinkas-Kramarski R. J. Neurochem. 2000; 74: 979-987Crossref PubMed Scopus (81) Google Scholar) to study the effect of NRG on oxidative stress. Oxidative stress was induced by H2O2. Exposure of the cells to 0.5 mm H2O2 for 30 min induced a marked reduction in cell viability resulting in 80% cell death 3 days post-treatment (Fig. 1). The death induced by H2O2 treatment appeared to have the characteristics of apoptosis; the cells shrunk and their nuclei were condensed or fragmented (Fig. 2). Treatment of the PC12-ErbB-4 cells with NRG (100 ng/ml) completely abolished the H2O2-induced toxicity (Fig. 1). NGF (50 ng/ml) furnished a comparable protection from the H2O2-induced cell death. We next examined whether NRG can also protect differentiated PC12-ErbB-4 cells that exhibit a sympathetic neuronal cell phenotype from H2O2-induced cell death. For this aim, we performed experiments using PC12-ErbB-4 cells that were induced to differentiate by NGF treatment for 1 week. Early experiments showed that the differentiated cells were more resistant to cell death induced by H2O2 (not shown). However H2O2 at a concentration of 1 mminduced cell death comparable with that observed with 0.5 mm in the non-differentiated cells. As shown in Figs. 1 and2, incubation of differentiated PC12-ErbB-4 cells with NRG or with NGF inhibited apoptotic cell death induced by 1 mmH2O2.Figure 2NRG-rescue from apoptosis induced by H2O2 treatment. PC12-ErbB-4 cells were tested for nuclei morphology analysis. PC12-ErbB-4 cultures were grown and treated as described in Fig. 1. The incubation was terminated by cell fixation and staining with the DNA-intercalating dye 4′,6-diamidine-2′-phenylindol dihydrochloride (DAPI). A, the resulting fluorescent photomicrographs are shown. B, quantification of the results presented in A was performed by counting 20 random fields (about 100 cells/field). Condensed or fragmented nuclei were considered as apoptotic cells. The percentage of apoptotic cells were calculated from the total cell number in the field. Note that in H2O2 treatment, more apoptotic nuclei are observed although the total number of cells is reduced because of cell death. The results are presented as the mean ± S.D. of 20 determinations. CON, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To examine which signaling pathway is involved in the protective effect of NRG in H2O2-treated PC12-ErbB-4 cells, we used pharmacological inhibitors of PI3K (LY294002), Erk (PD98059), p38 (SB203580), and protein kinase C (GF109203X). These are prominent signaling pathways known to be activated by NRG (46Pinkas-Kramarski R. Alroy I. Yarden Y. J. Mammary Gland Biol. Neoplasia. 1997; 2: 97-107Crossref PubMed Scopus (100) Google Scholar). Cells were treated for 30 min with H2O2 in the absence or presence of NRG (100 ng/ml) and in the presence of the indicated inhibitors. As shown in Fig. 3, none of the inhibitors except LY294002 could block the NRG-mediated protection from H2O2-induced cell death. This was apparent in both the differentiated and the non-differentiated cells (Fig. 3). We have previously shown that PI3K inhibition induces apoptosis of PC12-ErbB-4 cells, namely, cell death occurs also in the presence of NGF or NRG (41Erlich S. Goldshmidt Y. Lupowitz Z. Pinkas-Kramarski R. Neuroscience. 2001; 107: 353-362Crossref PubMed Scopus (45) Google Scholar). The addition of LY294002 as well as other inhibitors (not shown) by themselves could reduce cell viability (78% cell death induced by LY294002). These results suggest that inhibition of PI3K pathway (and also other signaling pathways) has a toxic effect on the cells and that the toxic effect of PI3K inhibitor is not prevented by activation of ErbB-4. However, the toxic effect of the other inhibitors used was prevented by NRG (41Erlich S. Goldshmidt Y. Lupowitz Z. Pinkas-Kramarski R. Neuroscience. 2001; 107: 353-362Crossref PubMed Scopus (45) Google Scholar). The inability of NRG to rescue cells after treatment with LY294002 indicates that PI3K activity may be required for the NRG protection from H2O2-induced cell death and encouraged us to further investigate the involvement of PI3K activation induced by NRG. Because PI3K and its downstream effector PKB/Akt are frequently associated with cell survival, we examined whether PKB/Akt is involved in the PI3K-dependent protection of NRG from H2O2-induced cell death. As shown in Fig.4 A, NRG, but not H2O2, induced sustained PKB/Akt phosphorylation. Although in the presence of H2O2, NRG-induced activation of PKB/Akt was reduced, a significant sustained activation was observed. Furthermore, the PI3K inhibitor LY294002 inhibited the NRG-induced activation of PKB/Akt both in the presence or absence of H2O2(Fig. 4 B). Taken together the results presented in Figs. 3and 4 indicate that PI3K and PKB/Akt activation may be involved in the NRG-mediated rescue of PC12-ErbB-4 cells from H2O2-induced apoptosis. Previous studies demonstrated that H2O2 could induce ROS elevation in PC12 cells and that a marked elevation in ROS leads to cell death (47Calderon F.H. Bonnefont A. Munoz F.J. Fernandez V. Videla L.A. Inestrosa N.C. J. Neurosci. Res. 1999; 56: 620-631Crossref PubMed Scopus (61) Google Scholar). Because the PI3K-PKB/Akt pathway is involved in NRG-mediated rescue of PC12-ErbB-4 cells from H2O2-induced death, we examined the effect of NRG on ROS generation. Cells were plated in 48-well collagen-coated plates at a density of 105cells/well in media containing 2.5% HS and 2.5% FBS. Wells were pretreated with 20 μm LY294002 for 1 h and then treated with 0.5 mm H2O2 with or without NRG (100 ng/ml) for 30 min. The medium was then replaced, and NRG and LY294002 treatments were continued for 48 h. After 48 h, medium was replaced to DMEM without phenol red, and cells were preloaded with 100 μm DCFH-DA for 30 min. After two washes, DCF fluorescence intensity was measured, and the number of cells was determined by the MTT assay. The relative DCF fluorescence was determined relative to cell number. As shown in Fig.5 A, H2O2 treatment induced a dramatic increase in ROS levels. NRG blocked the H2O2-induced elevation in ROS (Fig. 5). Consistent with the involvement of PI3K in the protection induced by NRG, LY294002 blocked the NRG-mediated reduction in ROS levels as measured by DCF-fluorescence (Fig. 5,B and C). These results indicate that NRG can abolish the H2O2-induced production of ROS and that PI3K mediates this effect. To further examine the role of PI3K in rescue from oxidative stress induced by H2O2we transfected PC12-ErbB-4 cells with the constitutively active PI3K expression vectors (p110-CAAX). The constitutive activity of PI3K in the transfected cells was confirmed by measuring phosphorylation of Akt, as can be seen in Fig.6 A. In subsequent experiments cells were co-transfected with p110-CAAX or empty vector (pcDNA3) and SEAP reporter gene. The effect of constitutively active PI3K on viability of H2O2-treated cells was examined by SEAP activity in the co-transfected cultures. As shown in Fig.6 B, transfection with p110-CAAX increased the activity of SEAP in both H2O2-treated and in untreated cells. SEAP activity in the pcDNA3-transfected cells was reduced after H2O2 treatment (Fig. 6 B). These results show that constitutively active PI3K can rescue PC12-ErbB4 cells from oxidative stress. In addition, the PI3K inhibitor LY294002 blocked the survival effect of activated PI3K (Fig.6 B). In a different set of experiments, cells were transiently co-transfected with p110-CAAX expression vector or empty vector (pcDNA3) and red fluorescent plasmid (pDsRed1). This enabled visualization of the co-transfected cells. One-day post-transfections cells were treated with H2O2 as described above, and 3 h or 2 days post-treatment they were loaded with the fluorophore DCFH-DA. The results of a representative experiment are shown in Fig. 6, C and D. As shown, H2O2 increased ROS levels in the cells at 3 h and 2 days post-H2O2 treatment, and this increase was blocked in cells that express the activated PI3K. These results suggest that PI3K activity can regulate ROS levels even in the absence of NRG. Taken together these results and the ability of PI3K inhibitor to block NRG-mediated rescue and to block NRG inhibition of ROS elevation induced by H2O2 indicate that the PI3K pathway may function as a junction to regulate toxic levels of ROS in the cells. Oxidative stress is thought to contribute to neuronal dysfunction under a variety of pathological conditions. Previous studies showed that NGF and brain-derived neurotrophic factor can rescue neuronal and PC12 cells from death induced by oxidative stress. In the present study, we demonstrate that NRG protects PC12-ErbB-4 cells from oxidative stress via the PI3K-PKB/Akt-dependent pathway. We show that the protective effects of NRG are mediated by modulation of ROS levels in the cells and that this modulation depends on the PI3K pathway. Specifically, we show that H2O2induces ROS elevation and death of PC12-ErbB-4 cells, where activation of PI3K by NRG inhibited ROS production and cell death. Indeed, overexpression of activated PI3K reduced the toxic levels of ROS in the cells and protected them from H2O2-induced death. These results are schematically summarized in Fig.7. Oxidative stress including H2O2 treatment results in activation of several signaling pathways (48Rhee S.G. Exp. Mol. Med. 1999; 31: 53-59Crossref PubMed Scopus (567) Google Scholar). These pathways include the p38, c-Jun NH2-terminal kinase, and Erk pathways, which can mediate a variety of cellular responses. It was previously shown that NRG can activate p38-, Erk-, and PI3K-signaling pathways (49Pinkas-Kramarski R. Soussan L. Waterman H. Levkowitz G. Alroy I. Klapper L. Lavi S. Seger R. Ratzkin J.B. Sela M. Yarden Y. EMBO J. 1996; 15: 2452-2467Crossref PubMed Scopus (697) Google Scholar, 50Daly J.M. Olayioye M.A. Wong A.M. Neve R. Lane H.A. Maurer F.G. Hynes N.E. Oncogene. 1999; 18: 3440-3451Crossref PubMed Scopus (85) Google Scholar). Using the p38 inhibitor SB203580, we demonstrated that inhibition of the p38-signaling pathway does not prevent NRG-mediated rescue from H2O2-induced cell death. Several studies demonstrate that NGF, epidermal growth factor, and NRG can increase Erk activation. It was demonstrated that sustained Erk activation correlates with neuronal differentiation, and transient Erk activation correlates with cell proliferation (31Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4230) Google Scholar, 40Vaskovsky A. Lupowitz Z. Erlich S. Pinkas-Kramarski R. J. Neurochem. 2000; 74: 979-987Crossref PubMed Scopus (81) Google Scholar, 51Miyasaka T. Sternberg D.W. Miyasaka J. Sherline P. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2653-2657Crossref PubMed Scopus (52) Google Scholar, 52Lewin G.R. Barde Y.A. Annu. Rev. Neurosci. 1996; 19: 289-317Crossref PubMed Scopus (1777) Google Scholar). In the present study we found that PD98059, the MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor, did not inhibit NRG-mediated survival. Together these results indicate that in PC12-ErbB4 cells, p38 and Ras/mitogen-activated protein kinase pathways do not mediate NRG-induced rescue from H2O2. Although PI3K is a well known signaling pathway involved in cell protection under various stresses, little is known about its role in protection against oxidative stress. One of the downstream effectors of PI3K is the serine/threonine kinase PKB/Akt. Akt is involved in promotion of cell survival through inhibition of apoptosis and possibly plays a role in PI3K-mediated neuronal cell survival (53Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 661-665Crossref PubMed Scopus (2217) Google Scholar). It was recently reported that the PI3K-PKB/Akt pathway delivers an anti-apoptotic signal in human glioblastoma against H2O2-induced apoptosis (54Sonoda Y. Watanabe S. Matsumoto Y. Aizu-Yokota E. Kasahara T. J. Biol. Chem. 1999; 274: 10566-10570Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). It was also demonstrated that in cardiomyocytes, the PI3K-PKB/Akt-S6K pathway promotes cell survival against oxidative stress-induced apoptosis (55Hong F. Kwon S.J. Jhun B.S. Kim S.S. Ha J. Kim S.J. Sohn N.W. Kang C. Kang I. Life Sci. 2001; 68: 1095-1105Crossref PubMed Scopus (90) Google Scholar). Our experiments demonstrate that NRG protects PC12-ErbB-4 cells from H2O2-induced death (Figs. 1 and 2). The protection conveyed by NRG is mediated via the PI3K pathway because LY294002 inhibited this effect (Fig. 3). In addition, we show that NRG activates PKB/Akt in the presence of hydrogen peroxide. Also, LY294002 inhibits NRG-induced PKB/Akt activation in the presence and in the absence of H2O2 (Fig. 4). These results indicate that PI3K-PKB/Akt pathways regulate NRG-mediated rescue from H2O2-induced cell death. ROS such as superoxide radicals, hydroxyl radicals, and H2O2 are continuously produced by the cells, and their levels are regulated by a number of enzymes and physiological antioxidants. Excessive generation of ROS has been associated with cytotoxicity in a variety of pathological conditions and in both PC12 and cultured neurons (25Enokido Y. Hatanaka H. Brain Res. 1990; 536: 23-29Crossref PubMed Scopus (33) Google Scholar, 39Kamata H. Tanaka C. Yagisawa H. Hirata H. Neurosci. Lett. 1996; 212: 179-182Crossref PubMed Scopus (62) Google Scholar, 56Yamakawa H. Ito Y. Naganawa T. Banno Y. Nakashima S. Yoshimura S. Sawada M. Nishimura Y. Nozawa Y. Sakai N. Neurol. Res. 2000; 22: 556-564Crossref PubMed Scopus (84) Google Scholar, 57de la Monte S.M. Neely T.R. Cannon J. Wands J.R. Cell. Mol. Life Sci. 2000; 57: 1471-1481Crossref PubMed Scopus (76) Google Scholar). Neurotrophins regulate neuronal survival and are neuroprotective in certain models of injury including oxidative stress (58Perez-Polo J.R. Foreman P.J. Jackson G.R. Shan D. Taglialatela G. Thorpe L.W. Werrbach-Perez K. Mol. Neurobiol. 1990; 4: 57-91Crossref PubMed Scopus (34) Google Scholar, 59Zhang Y. Tatsuno T. Carney J.M. Mattson M.P. J. Cereb. Blood Flow Metab. 1993; 13: 378-388Crossref PubMed Scopus (188) Google Scholar, 60Mattson M.P. Lovell M.A. Furukawa K. Markesbery W.R. J. Neurochem. 1995; 65: 1740-1751Crossref PubMed Scopus (595) Google Scholar). It was previously shown that NGF is able to protect PC12 cells from oxidative stress by increasing catalase activity and GSH levels (61Jackson G.R. Apffel L. Werrbach-Perez K. Perez-Polo J.R. J. Neurosci. Res. 1990; 25: 360-368Crossref PubMed Scopus (160) Google Scholar, 62Pan Z. Perez-Polo R. J. Neurochem. 1993; 61: 1713-1721Crossref PubMed Scopus (158) Google Scholar). Our experiments demonstrate that NRG reduces ROS levels in H2O2-treated PC12-ErbB-4 cells (Fig. 5). These results suggest that the effect of NRG on cell viability may be because of the reduction in ROS levels. Moreover, our results demonstrate that PI3K inhibitor, which in itself blocks NRG-mediated rescue also blocks the NRG-mediated reduction in H2O2-induced ROS elevation. Thus, ROS levels appear to be regulated by PI3K. Consistent with this notion, the H2O2-induced ROS elevation and cell death were blocked by the expression of activated PI3K (Fig. 6). Hence, we suggest that PI3K-dependent pathways may regulate toxic levels of ROS generated by oxidative stress. We thank Prof. Y. Kloog for critically reading this manuscript." @default.
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• W1963697539 title "Neuregulin Rescues PC12-ErbB4 Cells from Cell Death Induced by H2O2" @default.
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