Opposing Roles for ERK1/2 in Neuronal Oxidative Toxicity

Abstract

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress. Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress. Oxidative stress can contribute to neuronal toxicity and has been implicated in both acute injury and chronic neuropathological conditions (1Pettmann B. Henderson C.E. Neuron. 1998; 20: 633-647Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 2Simonian N.A. Coyle J.T. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 83-106Crossref PubMed Scopus (1002) Google Scholar). Many in vitro models have been used to examine the mechanistic basis for neuronal cell death induced by oxidative stress. For example, oxidative toxicity can be induced by glutamate treatment in the HT22 mouse hippocampal cell line (3Davis J.B. Maher P. Brain Res. 1994; 652: 169-173Crossref PubMed Scopus (306) Google Scholar, 4Li Y. Maher P. Schubert D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7748-7753Crossref PubMed Scopus (101) Google Scholar, 5Stanciu M. Wang Y. Kentor R. Burke N. Watkins S. Kress G. Reynolds I. Klann E. Angiolieri M.R. Johnson J.W. DeFranco D.B. J. Biol. Chem. 2000; 275: 12200-12206Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar) and immature primary embryonic rat cortical neuron cultures (4Li Y. Maher P. Schubert D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7748-7753Crossref PubMed Scopus (101) Google Scholar, 6Ratan R.R. Murphy T.H. Baraban J.M. J. Neurochem. 1994; 62: 376-379Crossref PubMed Scopus (512) Google Scholar, 7Levinthal D.J. DeFranco D.B. J. Biol. Chem. 2004; 279: 11206-11213Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). In these models, glutamate treatment leads to glutathione (GSH) 2The abbreviations used are: GSH, glutathione; MAPK, mitogen-activated protein kinase; DN, dominant negative; ERK, extracellular signal-regulated kinase; eYFP, enhanced yellow fluorescent protein; mGluRI, type I metabotropic glutamate receptor; mt-eYFP, mitochondrial-targeted eYFP; PI, propidium iodide; ROS, reactive oxygen species; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MES, 4-morpholineethanesulfonic acid; UAS, upstream activator sequence; AIDA, (R,S)-1-aminoindan-1,5-dicarboxylic acid; DHPG, (R,S)-3,5-dihydroxyphenylglycine; GCS, γ-glutamylcysteine synthetase. depletion and subsequent accumulation of reactive oxygen species (ROS) (8Tan S. Sagara Y. Liu Y. Maher P. Schubert D. J. Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (663) Google Scholar). Many intracellular second messenger pathways are required for oxidative toxicity in HT22 cells including arachidonic acid metabolites, cyclic GMP (cGMP), and calcium (8Tan S. Sagara Y. Liu Y. Maher P. Schubert D. J. Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (663) Google Scholar). In addition, signaling kinases, such as mitogen-activated protein kinases (MAPK), are activated upon glutamate-induced oxidative stress in HT22 cells and primary neurons and are likely to affect targets that either limit or promote oxidative toxicity. Extracellular signal-regulated kinases-1/2 (ERK1/2) has been implicated in glutamate-induced neuronal oxidative toxicity based upon the neuroprotective effects of U0126, a specific inhibitor of the ERK1/2-activating kinase, MEK1/2 (5Stanciu M. Wang Y. Kentor R. Burke N. Watkins S. Kress G. Reynolds I. Klann E. Angiolieri M.R. Johnson J.W. DeFranco D.B. J. Biol. Chem. 2000; 275: 12200-12206Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). U0126 is also effective at reducing brain injury following focal ischemia in rodents suggesting that ERK1/2 may also promote neuronal cell death resulting from acute injury in vivo (9Namura S. Iihara K. Takami S. Nagata I. Kikuchi H. Matsushita K. Moskowitz M.A. Bonventre J.V. Alessandrini A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11569-11574Crossref PubMed Scopus (342) Google Scholar). However, the role of ERK1/2 in neuronal cell death remains controversial (10Chu C.T. Levinthal D.J. Kulich S.M. Chalovich E.M. DeFranco D.B. Eur. J. Biochem. 2004; 271: 2060-2066Crossref PubMed Scopus (246) Google Scholar, 11Hetman M. Gozdz A. Eur. J. Biochem. 2004; 271: 2050-2055Crossref PubMed Scopus (242) Google Scholar, 12Cavanaugh J.E. Eur. J. Biochem. 2004; 271: 2056-2059Crossref PubMed Scopus (74) Google Scholar). In a number of studies, ERK1/2 has been found to promote neuronal survival and reduce cell death induced by various insults (13Grewal S.S. York R.D. Stork P.J. Curr. Opin. Neurobiol. 1999; 9: 544-553Crossref PubMed Scopus (509) Google Scholar, 14Spencer J.P. Rice-Evans C. Williams R.J. J. Biol. Chem. 2003; 278: 34783-34793Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 15Colucci-D'Amato L. Perrone-Capano C. di Porzio U. BioEssays. 2003; 25: 1085-1095Crossref PubMed Scopus (174) Google Scholar). Furthermore, even in HT22 cells, ERK1/2 may limit toxicity in response to specific insults such as serum withdrawal (16Rossler O.G. Giehl K.M. Thiel G. J. Neurochem. 2004; 88: 1240-1252Crossref PubMed Scopus (73) Google Scholar). Distinct kinetic profiles of ERK1/2 activation are observed in response to different extracellular signals and also can be associated with differential compartmentalization of active ERK1/2 within the cell. For example, in PC12 cells, prolonged, nerve growth factor-induced activation of ERK1/2 leads to its persistent accumulation within nuclei; however, rapid and transient epidermal growth factor-induced activation of ERK1/2 is unable to trigger its efficient nuclear translocation (15Colucci-D'Amato L. Perrone-Capano C. di Porzio U. BioEssays. 2003; 25: 1085-1095Crossref PubMed Scopus (174) Google Scholar, 17Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4267) Google Scholar). Interestingly, increasing evidence suggests that the kinetics and duration of ERK1/2 activation, as well as its subcellular localization, may direct ERK1/2 toward downstream targets that will either promote or limit neuronal survival (10Chu C.T. Levinthal D.J. Kulich S.M. Chalovich E.M. DeFranco D.B. Eur. J. Biochem. 2004; 271: 2060-2066Crossref PubMed Scopus (246) Google Scholar, 15Colucci-D'Amato L. Perrone-Capano C. di Porzio U. BioEssays. 2003; 25: 1085-1095Crossref PubMed Scopus (174) Google Scholar, 18Kulich S.M. Chu C.T. J. Neurochem. 2001; 77: 1058-1066Crossref PubMed Scopus (174) Google Scholar). In HT22 cells and primary neurons, glutamate induces a biphasic activation of ERK1/2 (5Stanciu M. Wang Y. Kentor R. Burke N. Watkins S. Kress G. Reynolds I. Klann E. Angiolieri M.R. Johnson J.W. DeFranco D.B. J. Biol. Chem. 2000; 275: 12200-12206Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). Persistent activation of ERK1/2 in these cells is mediated primarily through the oxidative inhibition of select ERK phosphatases D.J. D.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the for the initial rapid activation of ERK1/2 by glutamate in HT22 cells and primary neurons are not Furthermore, the of the of ERK1/2 activation the response of HT22 cells and primary neurons to oxidative stress has not been In this report, we that rapid activation of ERK1/2 by glutamate in HT22 cells is by type I metabotropic glutamate Furthermore, mGluRI activation of ERK1/2 a cellular response that to limit glutathione depletion resulting from glutamate-induced cysteine protective response mediated by ERK1/2 is unable to an and chronic oxidative stress that ERK1/2 in its stages to promote neuronal cell death. Thus, ERK1/2 activation may roles in neuronal oxidative toxicity acting through effects glutathione to limit oxidative stress as a to trigger cell death cellular oxidative stress is expression for and from E. M.A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). an dominant negative of MEK1 by J.E. Eur. J. Biochem. 2004; 271: 2056-2059Crossref PubMed Scopus (74) Google Scholar). The mitochondrial-targeted enhanced yellow fluorescent protein expression a from and the expression for the the and the from Cell cells, a mouse hippocampal cell in with serum of and at and Cell cells in for min with 1 of a of propidium to or cells D.J. DeFranco D.B. J. Biol. Chem. 2004; 279: 11206-11213Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). observed an with for in at For that not the of cells to by phase In transfection the of in transfected and transfected with using conditions by the cells in and h of to cells with the following cells at for and in with of a inhibitor The at for min at of 30 by and to with in with primary ERK, from Cell at with min with and to the for 1 h at min with and by enhanced using different times used for to of using a to the of the In used to in HT22 cells D.J. D.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transfected with an expression for an a the to a a expression and various MEK1 expression (i.e. In this is a of the of and with the using a to the from at to the within glutathione from used to glutathione from and in of (i.e. 1 at for min at the for with the of a of cell that used to protein to in the because of and protein acid in and from used to of to and for at in a for min at of of of or the in the to with the following and of the to of the and The in the an at at or using a min used to and the glutathione to the by the glutathione to the protein within of by of with for of using a to be and using for ERK1/2 Activation for Glutamate-induced Oxidative in HT22 has been implicated in both neuronal cell survival and cell death the basis for its effects is not In even in HT22 cells, roles for ERK1/2 have been (5Stanciu M. Wang Y. Kentor R. Burke N. Watkins S. Kress G. Reynolds I. Klann E. Angiolieri M.R. Johnson J.W. DeFranco D.B. J. Biol. Chem. 2000; 275: 12200-12206Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, O.G. Giehl K.M. Thiel G. J. Neurochem. 2004; 88: 1240-1252Crossref PubMed Scopus (73) Google Scholar, P. J. 2001; PubMed Google Scholar). part of the for the role of ERK1/2 in neuronal cell survival may be because of effects of pharmacological agents used to block ERK1/2 activation, we used a to definitively the role of ERK1/2 in HT22 cell response to oxidative stress. HT22 cells with dominant negative MEK1 and mitochondrial-targeted enhanced yellow fluorescent protein expression the cells with cells likely to a at (i.e. to that its kinase J.M. S. K. 1994; PubMed Scopus Google Scholar). a the with activation of type Cell death in transfected cells h after glutamate treatment using a D.J. DeFranco D.B. J. Biol. Chem. 2004; 279: 11206-11213Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). in glutamate-induced oxidative toxicity in HT22 results with a pharmacological inhibitor of MEK1 (i.e. and demonstrate that ERK1/2 activation is for glutamate toxicity. the effect of ERK1/2 activation, we used a to ERK1/2 in HT22 cells transfected with the expression with an expression for an protein and a the of a upstream activator that is an nuclear of enhanced resulting from its by ERK1/2 is through the of the D.J. D.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in U0126 treatment from in both and glutamate treatment HT22 from the induced following a treatment with glutamate with results of ERK1/2 activation using this D.J. D.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The expression of reduced from the in glutamate cells The reduced of to block to U0126 treatment likely results from expression of the within transfected these results the inhibition of ERK1/2 in the of HT22 cells upon of ERK Activation to in HT22 chronic activation of ERK sufficient to toxicity in HT22 cells, we used constitutively active ERK2 is a protein with ERK2 that in the E. M.A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). In the constitutively active ERK2 in MEK1 that are for its nuclear have been to M. I. Y. E. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). a the protein is in the E. M.A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). Furthermore, by of its within a ERK2 E. M.A. Curr. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). the activation of ERK signaling by the in vivo used to expression with an expression for an protein and the transfected HT22 Cell and h after in can ERK1/2 activation with that of The enhanced activation of the in cells likely results from its efficient within We the effects of HT22 cell using the in both and cells enhanced toxicity as with HT22 ERK activation upon of also not toxicity in HT22 cells to a of glutamate will be glutamate can trigger cell death conditions of Thus, ERK activation is not sufficient to toxicity in HT22 cells cells to toxicity conditions of glutamate in Glutamate-induced Oxidative cells both type 1 and type metabotropic glutamate and Y. Schubert D. J. 1998; PubMed Google Scholar). are likely to be activated during glutamate that oxidative stress. have been to play a protective role in glutamate toxicity Y. Schubert D. J. 1998; PubMed Google we to examine role in or toxicity in HT22 HT22 cells with a mGluRI (R,S)-1-aminoindan-1,5-dicarboxylic acid or mGluRI at or 30 min later at or at a that h after glutamate and h after glutamate cell by glutamate treatment of HT22 cells for h results in a in cell as by However, a with toxicity in the of glutamate resulting in cell death after h In contrast, a of HT22 cells with reduced the toxicity induced by glutamate treatment to The toxicity observed in HT22 cells with glutamate and or reduced by U0126 that the toxicity by the remains In HT22 cells with and have effects as observed in cell results that play a role in oxidative toxicity induced by glutamate treatment Y. Schubert D. J. 1998; PubMed Google Scholar) and establish that mGluRI activation toxicity at levels of of Activation the ERK1/2 Activation in Glutamate-induced Oxidative the protective effects of mGluRI activation oxidative toxicity in HT22 cells, we to mGluRI ERK1/2 with from HT22 cells with glutamate and or and to the kinetics of ERK1/2 activation the various protein at different (i.e. 30 and following glutamate In the of ERK1/2 activation to ERK1/2 from in treatment of HT22 cells with glutamate induced a activation of ERK1/2 that within 30 ERK1/2 activation not and reduced to levels following h of treatment with glutamate to glutamate (i.e. h a second peak of ERK1/2 activation and role for in rapid transient activation of ERK1/2 by glutamate by the observed of this activation by a with the early phase of ERK1/2 activation (i.e. within 30 min of glutamate reduced by The of with glutamate not to a effect the initial phase of ERK1/2 activation levels of activated ERK1/2 during the between early and phases of activation that observed in we the of ERK1/2 activation at h to h of glutamate treatment to the phase of activation this we observed that enhanced the second phase of activation However, of HT22 cells to glutamate and not a second peak of ERK1/2 activation results a dual role for in ERK1/2 Activation of by glutamate activation of ERK1/2 that may to limit toxicity. However, in the of mGluRI activation, a activation of ERK1/2 is enhanced that to promote cell death. The of ERK1/2 in oxidative toxicity is by glutathione depletion (8Tan S. Sagara Y. Liu Y. Maher P. Schubert D. J. Cell Biol. 1998; 141: 1423-1432Crossref PubMed Scopus (663) Google Scholar). role for ERK1/2 in this of glutamate toxicity has not been We intracellular glutathione at various times after glutamate treatment in the or of with studies, a glutamate treatment induced glutathione depletion in HT22 cells levels to in h and in h to U0126 HT22 cells from glutamate not trigger the restoration of levels in HT22 cells In this U0126 with glutamate and both early and phases of ERK1/2 However, U0126 h following glutamate treatment to for the early not phase of ERK1/2 activation, levels to of line levels within h in U0126 treatment HT22 cells from glutamate toxicity (5Stanciu M. Wang Y. Kentor R. Burke N. Watkins S. Kress G. Reynolds I. Klann E. Angiolieri M.R. Johnson J.W. DeFranco D.B. J. Biol. Chem. 2000; 275: 12200-12206Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). Thus, ERK1/2 activation signaling within the of glutamate treatment is required for HT22 cells to levels that reduced to cysteine a (i.e. of glutamate toxicity in HT22 cells that is following we the kinetics of depletion in response to a of these ERK1/2 activation is observed at early times after glutamate glutamate induced an early depletion of in of line is to the of depletion in However, a in levels between to line levels Interestingly, inhibition of mGluRI activation by a with the in levels that in response to a glutamate levels (i.e. of line from to h these the phase of ERK1/2 activation is Thus, in the of mGluRI activation, a that to levels is not and cell death can in HT22 cells from even a to ERK1/2 and Cell is well that ERK1/2 roles in neurons, acting in to promote cell survival J.P. Rice-Evans C. Williams R.J. J. Biol. Chem. 2003; 278: 34783-34793Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, P. D.B. G. C. K. R. Biol. Cell. 1999; PubMed Scopus Google Scholar) also in neuronal cell death and the of (10Chu C.T. Levinthal D.J. Kulich S.M. Chalovich E.M. DeFranco D.B. Eur. J. Biochem. 2004; 271: 2060-2066Crossref PubMed Scopus (246) Google Scholar). a mechanistic basis for effects of ERK1/2 in neurons is to is to of results in different cell or neuronal cell In this we have both the and of ERK1/2 in neuronal cell line to a We (5Stanciu M. Wang Y. Kentor R. Burke N. Watkins S. Kress G. Reynolds I. Klann E. Angiolieri M.R. Johnson J.W. DeFranco D.B. J. Biol. Chem. 2000; 275: 12200-12206Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar) and M.A. D. Biochem. 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Thus, at the ERK1/2 the effects of this signaling within cells and oxidative stress is a contributing in cell death. We and Schubert for of cells or