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Insulin-like Growth Factor-1-mediated Neuroprotection against Oxidative Stress Is Associated with Activation of Nuclear Factor κB*

  • Stefanie Heck
    Footnotes
    Affiliations
    From the Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
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  • Frank Lezoualc'h
    Footnotes
    Affiliations
    From the Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
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  • Stefanie Engert
    Affiliations
    From the Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
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  • Christian Behl
    Correspondence
    To whom correspondence should be addressed. Tel.: 49-89-30622-246; Fax.: 49-89-30622-642;
    Affiliations
    From the Max Planck Institute of Psychiatry, Kraepelinstrasse 2-10, 80804 Munich, Germany
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  • Author Footnotes
    * This work was supported in part by a grant from the Deutsche Hirnliga e.V. (to C. B.).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.
    ‡ These authors contributed equally to this work.
    § Present address: INSERM U 446, Faculty of Pharmacy, 92296 Chatenay-Malabry, France.
Open AccessPublished:April 02, 1999DOI:https://doi.org/10.1074/jbc.274.14.9828
      The role of insulin-like growth factor 1 (IGF-1) for the treatment of neurodegenerative disorders, such as Alzheimer's disease, has recently gained attention. The present study demonstrates that IGF-1 promotes the survival of rat primary cerebellar neurons and of immortalized hypothalamic rat GT1–7 cells after challenge with oxidative stress induced by hydrogen peroxide (H2O2). Neuroprotective concentrations of IGF-1 specifically induce the transcriptional activity and the DNA binding activity of nuclear factor κB (NF-κB), a transcription factor that has been suggested to play a neuroprotective role. This induction is associated with increased nuclear translocation of the p65 subunit of NF-κB and with degradation of the NF-κB inhibitory protein IκBα. IGF-1-mediated protection of GT1–7 cells against oxidative challenges was mimicked by overexpression of the NF-κB subunit c-Rel. Partial inhibition of NF-κB baseline activity by overexpression of a dominant-negative IκBα mutant enhanced the toxicity of H2O2 in GT1–7 cells. The pathway by which IGF-1 promotes neuronal survival and activation of NF-κB involves the phosphoinositol (PI) 3-kinase, because both effects of IGF-1 are blocked by LY294002 and wortmannin, two specific PI 3-kinase inhibitors. Taken together, our results provide evidence for a novel molecular link between IGF-1-mediated neuroprotection and induction of NF-κB that is dependent on the PI 3-kinase pathway.
      IGF-1
      insulin-like growth factor
      amyloid β protein
      PI 3-kinase
      phosphatidylinositol 3-kinase
      NF-κB
      nuclear factor κB
      IκB
      inhibitor of κB
      Luc
      luciferase
      Tk
      thymidine kinase
      MTT
      3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
      PEI
      polyethylenimine
      EMSA
      electrophoretic mobility shift assay
      Insulin-like growth factor 1 (IGF-1)1 is a pleiotropic factor with a wide spectrum of action in the central nervous system and also in the peripheral nervous system (
      • de Pablo F.
      • de la Rosa E.J.
      ,
      • LeRoith D.
      • Werner H.
      • Beitner-Johnson D.
      • Roberts C.T.
      ). It belongs to a superfamily of structurally related proteins that includes insulin and IGF-2. The biological functions of the IGFs and insulin are mediated by specific membrane receptors, designated the IGF-1, IGF-2, and insulin receptors (
      • LeRoith D.
      • Werner H.
      • Beitner-Johnson D.
      • Roberts C.T.
      ,
      • Jones J.I.
      • Clemmons D.R.
      ). The IGF-1 receptor is the primary mediator of the action of IGF-1.
      Recently, IGF-1 has gained increasing attention for the treatment of neurodegenerative disorders, such as the amyotrophic lateral sclerosis, which is characterized by the progressive loss of motor neurons (
      • Murphy M.F.
      • Felice K.
      • Gawel M.
      • Gelinas D.
      • Kratz R.
      • Lai F.
      • Lange D.
      • Natter H.
      • Rudnicki S.
      • the ALS/North America Myotrophin group
      ,
      • Yuen E.C.
      • Mobley W.C.
      ). Moreover, with respect to the central nervous system, IGF-1 has been found to protect hippocampal neurons against the toxicity of the Alzheimer's disease-associated amyloid β protein (Aβ) (
      • Doré S.
      • Kar S.
      • Quirion R.
      ). Aβ is the main component of the senile plaques in the brain of Alzheimer's disease patients and its cytotoxic action on neurons results from oxidative damage to susceptible cells (
      • Behl C.
      • Davis J.B.
      • Lesley R.
      • Schubert D.
      ). Specifically, it has been shown that hydrogen peroxide (H2O2) is an important intermediate in Aβ neurotoxicity (
      • Behl C.
      • Davis J.B.
      • Lesley R.
      • Schubert D.
      ). In accordance with a neuroprotective role of IGF-1 against oxidative injury, this growth factor is also effective in protecting neurons against glutathione depletion, suggesting a more general protective potency of IGF-1 against oxidative stress (
      • Sortino M.A.
      • Canonico P.L.
      ).
      The molecular signaling pathways by which IGF-1 promotes survival, in particular survival of neurons of the central nervous system, are not well characterized. Dudek et al. (
      • 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.
      ) revealed a critical function of the serine-threonine protein kinase Akt to mediate the protective effects of IGF-1 on the survival of cerebellar neurons against serum deprivation. Moreover, the inhibition of apoptosis by IGF-1 has been shown to require the activation of signaling molecules such as the phosphatidylinositol (PI) 3-kinase (
      • 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.
      ,
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ).
      The nuclear factor κB (NF-κB) is composed of homo- and heterodimers of members of the Rel family of related transcription factors that are well characterized for controlling the expression of numerous immune and inflammatory response genes (
      • Baeuerle P.A.
      • Henkel T.
      ,
      • Baeuerle P.A.
      • Baltimore D.
      ). Frequently, NF-κB is present as a heterodimer comprising a 50-kDa (p50) and a 60-kDa (p65) subunit that is sequestered in the cytoplasm by an inhibitory protein of the IκB family, with IκBα being the best characterized member of this family (
      • Baldwin A.S.
      ,
      • Verma I.M.
      • Stevenson J.-K.
      • Schwarz E.M.
      • van Antwerp D.
      • Miyamoto S.
      ). NF-κB-inducing agents, such as cytokines, viruses, phorbol esters, and UV light, result in the phosphorylation and degradation of the IκB inhibitory protein (
      • Israel A.
      ,
      • Thanos D.
      • Maniatis T.
      ) allowing free NF-κB to enter the nucleus, to bind to its cognate DNA sequences, and to induce target gene transcription.
      As for IGF-1, an important role for NF-κB during cell death has been suggested (
      • Wu M.
      • Lee H.Y.
      • Bellas R.E.
      • Schauer S.L.
      • Arsura M.
      • Katz D.
      • Fitzgerald M.J.
      • Rothstein T.L.
      • Sherr D.H.
      • Sonenshein G.E.
      ). Several reports have shown that NF-κB counteracts the induction of apoptosis by the cytokine tumor necrosis factor-α (
      • Beg A.A.
      • Baltimore D.
      ,
      • Liu Z.-G.
      • Hsu H.
      • Goeddel D.V.
      • Karin M.
      ,
      • Van Antwerp D.J.
      • Martin S.J.
      • Kafri T.
      • Green D.R.
      • Verma I.M.
      ), by ionizing radiation, and by the cancer chemotherapeutic agent daunorubicin (
      • Wang C.-Y.
      • Mayo M.W.
      • Baldwin Jr., A.S.
      ). Recently, we have demonstrated that constitutively increased NF-κB activity mediates the protection of neuronal cells against oxidative stress (
      • Lezoualc'h F.
      • Sagara Y.
      • Holsboer F.
      • Behl C.
      ). This prompted us to investigate whether IGF-1 offers neuroprotection against oxidative insult by affecting the activity of NF-κB as downstream target in neurons. For this study, two IGF-1 receptor-expressing neuronal cell systems were employed, the gonadotropin-releasing hormone-secreting neuronal cell line GT1–7 (
      • Tsai P.S.
      • Werner S.
      • Weiner R.I.
      ) and primary cultures of rat cerebellar granule neurons (
      • Torres-Aleman I.
      • Naftolin F.
      • Robbins R.J.
      ,
      • Torres-Aleman I.
      • Pons S.
      • Arevalo M.A.
      ). We demonstrate that IGF-1 exerts a neuroprotective effect against oxidative stress, mediated by a PI 3-kinase-dependent pathway that finally leads to the activation of NF-κB.

      DISCUSSION

      The aim of the present study was to investigate the ability of IGF-1 to protect neurons against H2O2-induced neuronal cell death and to examine a possible involvement of NF-κB in this function. We employed GT1–7 hypothalamic neuronal cells and mature primary cerebellar granule neurons that have been shown to express functional IGF-1 receptors (
      • 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.
      ,
      • Olson B.R.
      • Scott D.C.
      • Wetsel W.C.
      • Elliot S.J.
      • Tomic M.
      • Stojilkovic S.
      • Nieman L.K.
      • Wray S.
      ). As previously reported for other oxidative stressors, such as the Alzheimer's disease-associated Aβ and the glutathione depleting agent buthionine sulfoximine (
      • Doré S.
      • Kar S.
      • Quirion R.
      ,
      • Sortino M.A.
      • Canonico P.L.
      ), we found that IGF-1 is effective in protecting neuronal cells against oxidative injury induced by H2O2. A 24-h pretreatment with 50 ng/ml of IGF-1 caused an enhanced cell survival of about 30% after H2O2 challenge in GT1–7 cells and primary mature postmitotic neurons of 5 days in vitro. IGF-1 did not significantly affect GT1–7 cell growth in serum-free conditions (data not shown), confirming that the increased cell survival is not due to a proliferative effect of IGF-1 in this neuronal system.
      The fact that IGF-1 is a potent neuroprotective agent against oxidative stress induced by the lipid peroxidizing agent H2O2 may offer a new therapeutic avenue for the treatment of neurodegenerative disorders in which free radicals have been implicated. For instance, IGF-1 has been shown to reduce neuronal cell loss observed in vivo following ischemic insults and is beneficial in the treatment of amyotrophic lateral sclerosis (
      • Doré S.
      • Kar S.
      • Quirion R.
      ,
      • Loddick S.A.
      • Liu X.J.
      • Lu Z.X.
      • Liu C.
      • Behan D.P.
      • Chalmers D.C.
      • Foster A.C.
      • Vale W.W.
      • Ling N.
      • De Souza E.B.
      ). With respect to its potency to rescue neurons against Aβ toxicity (
      • Doré S.
      • Kar S.
      • Quirion R.
      ), one can likewise imagine that the development of IGF-1-related compounds could be a promising strategy toward the treatment of Alzheimer's disease.
      The PI 3-kinase pathway has been shown to be implicated in the signaling of IGF-1-mediated cell survival, including survival of neurons (
      • 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.
      ,
      • Cross D.A.
      • Alessi D.R.
      • Cohen P.
      • Andjelkovich M.
      • Hemmings B.A.
      ). PI 3-kinase phosphorylates inositol lipids that act as second messengers for several targets, such as the serine-threonine kinase Akt (
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ), which has been shown accordingly to be involved in anti-apoptotic signaling (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordan J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Crowder R.J.
      • Freeman R.S.
      ). Experiments with pharmacological inhibitors demonstrated that IGF-1-mediated neuroprotection against serum deprivation in neuronal cells is dependent on PI 3-kinase activity (
      • 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.
      ,
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ).
      The data obtained in the present study extend this observation showing that the PI 3-kinase pathway is also involved in the protective effect of IGF-1 against H2O2-induced cell death. Two potent PI 3-kinase inhibitors, LY294002 (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ) and wortmannin (
      • Arcaro A.
      • Wymann M.P.
      ), were able to block the cellular protection afforded by IGF-1 against oxidative challenge in GT1–7 cells. The data from Parizzas et al. (
      • Parrizas M.
      • Saltiel A.R.
      • LeRoith D.
      ) additionally suggest a role of the mitogen-activated protein kinase pathway in IGF-1-mediated neuroprotection because the anti-apoptotic function of IGF-1 could not completely be blocked by inhibition of the PI 3-kinase pathway in the PC12 cells employed in their study. In our cellular system, the neuroprotective effect of IGF-1 was entirely prevented by blocking PI 3-kinase, arguing against the involvement of other signal transduction pathways in IGF-1-mediated neuroprotection.
      Because the transcription factor NF-κB can modulate neuronal vulnerability and has recently gained great interest for its potential role in neuroprotection (
      • Lipton S.A.
      ,
      • Lezoualc'h F.
      • Behl C.
      ,
      • Taglialatela G.
      • Kaufmann J.A.
      • Trevino A.
      • Perez-Polo J.R.
      ), we tested the ability of IGF-1 to induce NF-κB activation. We found that increasing concentrations of IGF-1 in GT1–7 cells stimulate the NF-κB transcriptional activity with a substantial effect at 50 ng/ml. These results were also confirmed in postmitotic cerebellar granule neurons, indicating that the enhancement of NF-κB activity by IGF-1 is not dependent on the cell cycle. Consistent with this increase in NF-κB transcriptional activity, NF-κB DNA binding activity could also be enhanced upon stimulation with 50 ng/ml IGF-1 in both neuronal systems. In agreement with these findings, we observed increased nuclear translocation of the p65-subunit of NF-κB and a parallel decrease in the amount of the NF-κB cytoplasmic inhibitory protein, IκBα. Thus, the induction of NF-κB-driven protective transcriptional programs by IGF-1 could be one way for the neurons to protect themselves against exogenous insults. In support of this hypothesis, we have recently shown that in a clone of the sympathetic precursor-like cell line PC12 selected for its resistance against Aβ and H2O2, constitutively increased NF-κB activity mediates this resistance. The suppression of NF-κB activation in these cells reverses the oxidative stress resistance phenotype (
      • Lezoualc'h F.
      • Sagara Y.
      • Holsboer F.
      • Behl C.
      ). Consistently, the inhibition of NF-κB activation results in apoptosis in PC12 cells, and nerve growth factor did not protect from apoptosis when NF-κB activation is blocked (
      • Taglialatela G.
      • Robinson R.
      • Perez-Polo J.R.
      ).
      To confirm the role of NF-κB in IGF-1-mediated neuroprotection, we (i) induced an activation of NF-κB by transient expression of the c-Rel subunit of this transcription factor, and (ii) inhibited NF-κB activity by expression of a dominant-negative form of IκBα. The expression of c-Rel in GT1–7 cells, which mimicked IGF-1 induction of NF-κB activity, protected these cells completely against oxidative stress, consistent with findings from other investigators showing that the c-Rel expression is able to reduce apoptosis in nonneuronal cells (
      • Wu M.
      • Lee H.Y.
      • Bellas R.E.
      • Schauer S.L.
      • Arsura M.
      • Katz D.
      • Fitzgerald M.J.
      • Rothstein T.L.
      • Sherr D.H.
      • Sonenshein G.E.
      ,
      • Liu Z.-G.
      • Hsu H.
      • Goeddel D.V.
      • Karin M.
      ). On the other hand, the dominant-negative IκBα decreased the intrinsic survival of GT1–7 cells against oxidative insult, most likely by reducing the high basal NF-κB activity in these cells. However, this decrease in survival was not significant but rather indicative, which might be due to the fact that either the dominant-negative IκBα was not able to completely abrogate NF-κB activity (supported by the luciferase assay done in parallel) or additional factors, other than NF-κB, are involved in the protection of neuronal cells against oxidative stress. Together, these results suggest that the protection of neuronal cells by IGF-1 may be mediated, at least in part, through the activation of NF-κB.
      In view of the idea that the activation of NF-κB represents an intermediary step in IGF-1-mediated neuroprotection, one would expect that both processes are mediated by a common pathway. Upon investigating the effect of LY294002 and wortmannin on the IGF-1-induced transcriptional activity of NF-κB, it indeed turned out that these PI 3-kinase inhibitors completely blocked the IGF-1-induced increase in NF-κB mediated luciferase activity, indicating that the IGF-1 activation of NF-κB involves the PI 3-kinase pathway. In contrast to these findings, Bertrand et al. (
      • Bertrand F.
      • Atfi A.
      • Cadoret A.
      • L'Allemain G.
      • Robin H.
      • Lascols O.
      • Capeau J.
      • Cherqui G.
      ) reported that an insulin activation of NF-κB in Chinese hamster ovary cells does not involve the PI 3-kinase, but rather occurs via the Raf-1-mediated signal transduction pathway. However, these authors argued that the anti-apoptotic role of insulin in Chinese hamster ovary cells, besides NF-κB activation, additionally requires independent processes that involve PI 3-kinase activity (
      • Bertrand F.
      • Atfi A.
      • Cadoret A.
      • L'Allemain G.
      • Robin H.
      • Lascols O.
      • Capeau J.
      • Cherqui G.
      ). The reasons of these apparent discrepancies remain to be investigated, but might be due to the different cell type and the specific stimuli applied to the cells. So far, besides Raf-1 (
      • Bertrand F.
      • Philippe C.
      • Antoine P.J.
      • Baud L.
      • Groyer A.
      • Capeau J.
      • Cherqui G.
      ), various kinases have been reported to be involved in the pathway leading to activation of NF-κB, such as mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase kinase 1 (
      • Lee F.S.
      • Hagler J.
      • Chen Z.J.
      • Maniatis T.
      ) and the kinase finally phosphorylating IκBα, conserved helix-loop-helix ubiquitous kinase (
      • Regnier C.H.
      • Song H.Y.
      • Gao X.
      • Goeddel D.V.
      • Cao Z.
      • Rothe M.
      ), or IκBα kinase (
      • DiDonato J.A.
      • Hayakawa M.
      • Rothwarf D.M.
      • Zandhi E.
      • Karin M.
      ). Here we report for the first time that also the PI 3-kinase can participate in the induction of NF-κB activity, suggesting that dependent on the stimulus multiple NF-κB activation pathways may exist.
      The mechanisms by which NF-κB may prevent neuronal cell death upon stimulation with IGF-1 remain unknown. The activity of this transcription factor may drive defense programs that afford the protection against oxidative insult. Therefore, in future, it will be important to identify neuroprotective target genes of NF-κB and to investigate their inducibility by exogenous factors, such as IGF-1, to increase the survival of neurons under oxidative stress.

      Acknowledgments

      We thank Drs. R. Weiner for providing the GT1–7 cell line, P. Baeuerle for the NF-κB-luciferase and TK-luciferase constructs, P. Jalinot for the c-Rel construct, and D. W. Ballard for the mutant IκBα construct.

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