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Neuregulin Rescues PC12-ErbB4 Cells from Cell Death Induced by H2O2

REGULATION OF REACTIVE OXYGEN SPECIES LEVELS BY PHOSPHATIDYLINOSITOL 3-KINASE*
  • Yona Goldshmit
    Footnotes
    Affiliations
    Department of Neurobiochemistry. Tel-Aviv University, Ramat-Aviv, 69978, Israel
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  • Shlomit Erlich
    Footnotes
    Affiliations
    Department of Neurobiochemistry. Tel-Aviv University, Ramat-Aviv, 69978, Israel
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  • Ronit Pinkas-Kramarski
    Correspondence
    To whom correspondence should be addressed. Tel.: 972-3-6406801; Fax: 972-3-6407643;
    Affiliations
    Department of Neurobiochemistry. Tel-Aviv University, Ramat-Aviv, 69978, Israel
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  • Author Footnotes
    * This research was supported by Israel Science Foundation Grant 434/01-1 and by the National Institute for Psychobiology in Israel, Charles E. Smith Fund Grant 7-2001.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ The first two authors contributed equally to this work.
Open AccessPublished:December 07, 2001DOI:https://doi.org/10.1074/jbc.M105637200
      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.
      NRG
      neuregulin
      NGF
      nerve growth factor
      DMEM
      Dulbecco's modified Eagle's medium
      Erk
      extracellular signal-regulated kinase
      mAb
      monoclonal antibody
      PBS
      phosphate-buffered saline
      PI3K
      phosphatidylinositol 3-kinase
      ROS
      reactive oxygen species
      PKB
      protein kinase B
      DCF
      2′,7′-dichlorodihydrofluorescein
      DCF-DA
      2′,7′-dichlorodihydrofluorescein diacetate
      FBS
      fetal bovine serum
      HS
      horse serum
      PBS
      phosphate-buffered saline
      MTT
      [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 (
      • Bibel M.
      • Barde Y.A.
      ,
      • Kaplan D.R.
      • Miller F.D.
      ). 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 (
      • Adlkofer K.
      • Lai C.
      ,
      • Burden S.
      • Yarden Y.
      ,
      • Garratt A.N.
      • Britsch S.
      • Birchmeier C.
      ,
      • Gassmann M.L.G.
      ,
      • Pinkas-Kramarski R.
      • Eilam R.
      • Alroy I.
      • Levkowitz G.
      • Lonai P.
      • Yarden Y.
      ,
      • Ullrich A.
      • Schlessinger J.
      ). NRGs along with ErbB-3 and ErbB-4 receptors are highly expressed in the developing and mature nervous system (
      • Pinkas-Kramarski R.
      • Eilam R.
      • Alroy I.
      • Levkowitz G.
      • Lonai P.
      • Yarden Y.
      ,
      • Marchionni 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.
      ,
      • Pinkas-Kramarski R.
      • Eilam R.
      • Spiegler O.
      • Lavi S.
      • Liu N.
      • Chang D.
      • Wen D.
      • Schwartz M.
      • Yarden Y.
      ). After brain insult, the level of NRG (
      • Eilam R.
      • Pinkas-Kramarski R.
      • Menahem S.
      • Yarden Y.
      ) and ErbB-4 receptor (
      • Eilam R.
      • Pinkas-Kramarski R.
      • Menahem S.
      • Yarden Y.
      ,
      • Erlich S.
      • Shohami E.
      • Pinkas-Kramarski R.
      ) 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 (
      • Pinkas-Kramarski R.
      • Eilam R.
      • Spiegler O.
      • Lavi S.
      • Liu N.
      • Chang D.
      • Wen D.
      • Schwartz M.
      • Yarden Y.
      ), oligodendrocytes (
      • Vartanian T.
      • Corfas G.
      • Li Y.
      • Fischbach G.D.
      ), and neurons (
      • Rieff H.I.
      • Raetzman L.T.
      • Sapp D.W.
      • Yeh H.H.
      • Siegel R.E.
      • Corfas G.
      ). Moreover, it was demonstrated that axon-derived neuregulin promotes oligodendrocyte survival in the developing rat optic nerve (
      • Fernandez P.A.
      • Tang D.G.
      • Cheng L.
      • Prochiantz A.
      • Mudge A.W.
      • Raff M.C.
      ) and that neuregulin in the central nervous system diminishes autoimmune demyelination, promotes oligodendrocyte progenitor expansion, and enhances remyelination (
      • Marchionni 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.
      ). In addition, neuregulin affects neuronal survival and neurite outgrowth of developing rat retina (
      • Bermingham-McDonogh O.
      • McCabe K.L.
      • Reh T.A.
      ).
      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 (
      • Dreher D.
      • Junod A.F.
      ,
      • Knight J.A.
      ). In addition, several studies indicate that nontoxic levels of ROS may play an essential role as signaling molecules regulating cell growth and differentiation (
      • Mills E.M.
      • Takeda K., Yu, Z.X.
      • Ferrans V.
      • Katagiri Y.
      • Jiang H.
      • Lavigne M.C.
      • Leto T.L.
      • Guroff G.
      ,
      • Peunova N.
      • Enikolopov G.
      ,
      • Segal R.A.
      • Greenberg M.E.
      ,
      • Suzukawa K.
      • Miura K.
      • Mitsushita J.
      • Resau J.
      • Hirose K.
      • Crystal R.
      • Kamata T.
      ,
      • Guyton K.Z.
      • Liu Y.
      • Gorospe M.
      • Xu Q.
      • Holbrook N.J.
      ). Oxidative stress such as H2O2treatment induces apoptotic cell death in both PC12 cells and cultured neurons (
      • Enokido Y.
      • Hatanaka H.
      ,
      • Ratan R.R.
      • Murphy T.H.
      • Baraban J.M.
      ).
      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 (
      • Chao M.V.
      ,
      • Dichter M.A.
      • Tischler A.S.
      • Greene L.A.
      ,
      • Greene L.A.
      • Tischler A.S.
      ,
      • Huff K.
      • End D.
      • Guroff G.
      ,
      • Marshall C.J.
      ). 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 (
      • Barde Y.A.
      ,
      • Chen M.G.
      • Chen J.S.
      • Calissano P.
      • Levi-Montalcini R.
      ,
      • Greene L.A.
      ). 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 (
      • Greene L.A.
      ,
      • Haviv R.
      • Stein R.
      ,
      • Mesner P.W.
      • Winters T.R.
      • Green S.H.
      ). The survival effect induced by NGFs in PC12 cells requires the activation of the PI3K-signaling pathway (
      • Klesse L.J.
      • Meyers K.A.
      • Marshall C.J.
      • Parada L.F.
      ,
      • Yao R.
      • Cooper G.M.
      ). It was also demonstrated that NGF protects PC12 cells and neurons from oxidative stress-induced death (
      • Kamata H.
      • Tanaka C.
      • Yagisawa H.
      • Hirata H.
      ).
      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 (
      • Vaskovsky A.
      • Lupowitz Z.
      • Erlich S.
      • Pinkas-Kramarski R.
      ). It was also demonstrated that NRG rescues PC12-ErbB-4 cells from apoptosis induced by serum deprivation or tumor necrosis factor treatment (
      • Erlich S.
      • Goldshmidt Y.
      • Lupowitz Z.
      • Pinkas-Kramarski R.
      ). 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.

      DISCUSSION

      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.
      Figure thumbnail gr7
      Figure 7A model of NRG-induced rescue from H2O2-induced cell death. The model depicts the signaling components that mediate NRG-mediated survival from H2O2-induced cell death. The activated signaling component induced by NRG or H2O2 is in black, and the signaling events inhibited are inred. Note that ROS levels are reduced, and cell death is inhibited by activated PI3K (p110-CAAX) and by NRG.
      Oxidative stress including H2O2 treatment results in activation of several signaling pathways (
      • Rhee S.G.
      ). 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 (
      • Pinkas-Kramarski R.
      • Soussan L.
      • Waterman H.
      • Levkowitz G.
      • Alroy I.
      • Klapper L.
      • Lavi S.
      • Seger R.
      • Ratzkin J.B.
      • Sela M.
      • Yarden Y.
      ,
      • Daly J.M.
      • Olayioye M.A.
      • Wong A.M.
      • Neve R.
      • Lane H.A.
      • Maurer F.G.
      • Hynes N.E.
      ). 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 (
      • Marshall C.J.
      ,
      • Vaskovsky A.
      • Lupowitz Z.
      • Erlich S.
      • Pinkas-Kramarski R.
      ,
      • Miyasaka T.
      • Sternberg D.W.
      • Miyasaka J.
      • Sherline P.
      • Saltiel A.R.
      ,
      • Lewin G.R.
      • Barde Y.A.
      ). 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 (
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ). It was recently reported that the PI3K-PKB/Akt pathway delivers an anti-apoptotic signal in human glioblastoma against H2O2-induced apoptosis (
      • Sonoda Y.
      • Watanabe S.
      • Matsumoto Y.
      • Aizu-Yokota E.
      • Kasahara T.
      ). It was also demonstrated that in cardiomyocytes, the PI3K-PKB/Akt-S6K pathway promotes cell survival against oxidative stress-induced apoptosis (
      • Hong F.
      • Kwon S.J.
      • Jhun B.S.
      • Kim S.S.
      • Ha J.
      • Kim S.J.
      • Sohn N.W.
      • Kang C.
      • Kang I.
      ). 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 (
      • Enokido Y.
      • Hatanaka H.
      ,
      • Kamata H.
      • Tanaka C.
      • Yagisawa H.
      • Hirata H.
      ,
      • Yamakawa H.
      • Ito Y.
      • Naganawa T.
      • Banno Y.
      • Nakashima S.
      • Yoshimura S.
      • Sawada M.
      • Nishimura Y.
      • Nozawa Y.
      • Sakai N.
      ,
      • de la Monte S.M.
      • Neely T.R.
      • Cannon J.
      • Wands J.R.
      ). Neurotrophins regulate neuronal survival and are neuroprotective in certain models of injury including oxidative stress (
      • Perez-Polo J.R.
      • Foreman P.J.
      • Jackson G.R.
      • Shan D.
      • Taglialatela G.
      • Thorpe L.W.
      • Werrbach-Perez K.
      ,
      • Zhang Y.
      • Tatsuno T.
      • Carney J.M.
      • Mattson M.P.
      ,
      • Mattson M.P.
      • Lovell M.A.
      • Furukawa K.
      • Markesbery W.R.
      ). It was previously shown that NGF is able to protect PC12 cells from oxidative stress by increasing catalase activity and GSH levels (
      • Jackson G.R.
      • Apffel L.
      • Werrbach-Perez K.
      • Perez-Polo J.R.
      ,
      • Pan Z.
      • Perez-Polo R.
      ). 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.

      ACKNOWLEDGEMENT

      We thank Prof. Y. Kloog for critically reading this manuscript.

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