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• W1980283113 abstract "Activation of the JNK pathway and induction of the AP-1 transcription factor c-Jun are critical for neuronal apoptosis caused by a variety of insults. Ara-C-induced DNA damage caused rapid sympathetic neuronal death that was associated with an increase of c-jun expression. In addition, c-Jun was phosphorylated in its N-terminal transactivation domain, which is important for c-Jun-mediated gene transcription. Blocking c-Jun activation by JNK pathway inhibition prevented neuronal death after stress. In contrast, neither the JNK inhibitor SP600125 nor the mixed lineage kinase inhibitor CEP-1347 prevented cytosine arabinoside-induced neuronal death, demonstrating that the JNK pathway was not necessary for DNA damage-induced neuronal apoptosis. Surprisingly, SP600125 or CEP-1347 could not block c-Jun induction or phosphorylation after DNA damage. Pharmacological inhibitors of cyclin-dependent kinase (CDK) activity completely prevented c-Jun phosphorylation after DNA damage. These results demonstrate that c-Jun activation during DNA damage-induced neuronal apoptosis was independent of the classical JNK pathway and was mediated by a novel c-Jun kinase. Based on pharmacological criteria, DNA damage-induced neuronal c-Jun kinase may be a member of the CDK family or be activated by a CDK-like kinase. Activation of this novel kinase and subsequent phosphorylation of c-Jun may be important in neuronal death after DNA damage. Activation of the JNK pathway and induction of the AP-1 transcription factor c-Jun are critical for neuronal apoptosis caused by a variety of insults. Ara-C-induced DNA damage caused rapid sympathetic neuronal death that was associated with an increase of c-jun expression. In addition, c-Jun was phosphorylated in its N-terminal transactivation domain, which is important for c-Jun-mediated gene transcription. Blocking c-Jun activation by JNK pathway inhibition prevented neuronal death after stress. In contrast, neither the JNK inhibitor SP600125 nor the mixed lineage kinase inhibitor CEP-1347 prevented cytosine arabinoside-induced neuronal death, demonstrating that the JNK pathway was not necessary for DNA damage-induced neuronal apoptosis. Surprisingly, SP600125 or CEP-1347 could not block c-Jun induction or phosphorylation after DNA damage. Pharmacological inhibitors of cyclin-dependent kinase (CDK) activity completely prevented c-Jun phosphorylation after DNA damage. These results demonstrate that c-Jun activation during DNA damage-induced neuronal apoptosis was independent of the classical JNK pathway and was mediated by a novel c-Jun kinase. Based on pharmacological criteria, DNA damage-induced neuronal c-Jun kinase may be a member of the CDK family or be activated by a CDK-like kinase. Activation of this novel kinase and subsequent phosphorylation of c-Jun may be important in neuronal death after DNA damage. Cells experience DNA damage caused by several environmental stresses such as UV irradiation and oxidative stress. When DNA damage is no longer reparable, various cell death pathways are activated before mitosis to prevent the delivery of damaged DNA to offspring. Thus, many chemotherapy regimens are based on DNA-damaging agents. However, DNA damage can also kill postmitotic cells such as neurons. Central nervous system neurotoxicity is a major dose-limiting factor in high dose ara-C treatment for refractory leukemias (1Winkelman M.D. Hines J.D. Ann. Neurol. 1983; 14: 520-527Crossref PubMed Scopus (113) Google Scholar, 2Sylvester R.K. Fisher A.J. Lobell M. Drug Intell. Clin. Pharm. 1987; 21: 177-180PubMed Google Scholar, 3Lazarus H.M. Herzig R.H. Herzig G.P. Phillips G.L. Roessmann U. Fishman D.J. Cancer (Phila.). 1981; 48: 2577-2582Crossref PubMed Scopus (159) Google Scholar, 4Vogel H. Horoupian D.S. Cancer (Phila.). 1993; 71: 1303-1308Crossref PubMed Scopus (33) Google Scholar, 5Resar L.M. Phillips P.C. Kastan M.B. Leventhal B.G. Bowman P.W. Civin C.I. Cancer (Phila.). 1993; 71: 117-123Crossref PubMed Scopus (57) Google Scholar). In vitro, ara-C is toxic to postmitotic sympathetic, parasympathetic, and sensory neurons of the peripheral nervous system as well as cerebellar and cortical neurons of the central nervous system (6Dessi F. Pollard H. Moreau J. Ben-Ari Y. Charriaut-Marlangue C. J. Neurochem. 1995; 64: 1980-1987Crossref PubMed Scopus (66) Google Scholar, 7Geller H.M. Cheng K.Y. Goldsmith N.K. Romero A.A. Zhang A.L. Morris E.J. Grandison L. J. Neurochem. 2001; 78: 265-275Crossref PubMed Scopus (104) Google Scholar, 8Martin D.P. Wallace T.L. Johnson Jr., E.M. J. Neurosci. 1990; 10: 184-193Crossref PubMed Google Scholar, 9Tomkins C.E. Edwards S.N. Tolkovsky A.M. J. Cell Sci. 1994; 107: 1499-1507Crossref PubMed Google Scholar, 10Wallace T.L. Johnson Jr., E.M. J. Neurosci. 1989; 9: 115-124Crossref PubMed Google Scholar). In sympathetic neurons, ara-C exposure activates two separate cell death pathways (67Besirli C.G. Deckwerth T.L. Crowder R.J. Freeman R.S. Johnson Jr., E.M. Cell Death Differ. 2003; (in press)PubMed Google Scholar). The default pathway is similar to the programmed cell death of neurons upon withdrawal of their trophic factor, nerve growth factor (NGF). 1The abbreviations used are: NGF, nerve growth factor; CDK, cyclin-dependent kinase; DIV, days in vitro; JNK, Jun-N-terminal kinase; MKK, MAP kinase kinase; MAPK, MAP kinase; MLK, mixed lineage kinase; MEK kinase, MAPK/extracellular signal-regulated kinase kinase; JNK, c-Jun N-terminal kinase; ara-C, cytosine arabinoside. 1The abbreviations used are: NGF, nerve growth factor; CDK, cyclin-dependent kinase; DIV, days in vitro; JNK, Jun-N-terminal kinase; MKK, MAP kinase kinase; MAPK, MAP kinase; MLK, mixed lineage kinase; MEK kinase, MAPK/extracellular signal-regulated kinase kinase; JNK, c-Jun N-terminal kinase; ara-C, cytosine arabinoside. This pathway kills neurons rapidly and requires the proapoptotic Bcl-2 family member, Bax. Ara-C exposure leads to Bax-mediated mitochondrial cytochrome c release and caspase activation. The second pathway is only observed in the absence of Bax. Activation of cell death in Bax-null neurons is significantly delayed and proceeds with a protracted time course compared with wild type neurons. This slower death is similar to the delayed death observed in camptothecin-treated embryonic cortical neurons in the presence of caspase inhibitors or after Bax deletion (11Stefanis L. Park D.S. Friedman W.J. Greene L.A. J. Neurosci. 1999; 19: 6235-6247Crossref PubMed Google Scholar, 12Morris E.J. Keramaris E. Rideout H.J. Slack R.S. Dyson N.J. Stefanis L. Park D.S. J. Neurosci. 2001; 21: 5017-5026Crossref PubMed Google Scholar). AP-1 transcription factor c-Jun is induced in many neuronal cell death paradigms (reviewed in Refs. 13Ham J. Eilers A. Whitfield J. Neame S.J. Shah B. Biochem. Pharmacol. 2000; 60: 1015-1021Crossref PubMed Scopus (214) Google Scholar and 14Herdegen T. Skene P. Bahr M. Trends Neurosci. 1997; 20: 227-231Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar) and appears to be important for the transcription of proapoptotic genes, such as the BH3-only Bcl-2 family members Bim and Dp5 (15Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar, 16Whitfield J. Neame S.J. Paquet L. Bernard O. Ham J. Neuron. 2001; 29: 629-643Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar). Inhibition of c-Jun activity by dominant-negative overexpression, neutralizing antibody injection, and genetic deletion prevents sympathetic neuronal apoptosis after NGF deprivation (17Ham J. Babij C. Whitfield J. Pfarr C.M. Lallemand D. Yaniv M. Rubin L.L. Neuron. 1995; 14: 927-939Abstract Full Text PDF PubMed Scopus (757) Google Scholar, 18Estus S. Zaks W.J. Freeman R.S. Gruda M. Bravo R. Johnson Jr., E.M. J. Cell Biol. 1994; 127: 1717-1727Crossref PubMed Scopus (786) Google Scholar, 19Palmada M. Kanwal S. Rutkoski N.J. Gufstafson-Brown C. Johnson R.S. Wisdom R. Carter B.D. J. Cell Biol. 2002; 158: 453-461Crossref PubMed Scopus (108) Google Scholar). Moreover, hippocampal neurons carrying a mutant c-Jun gene (JunAA) that lacks the two critical N-terminal phosphorylation sites show increased resistance to kainate exposure (20Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (594) Google Scholar). The N-terminal transactivation domain of c-Jun is phosphorylated by c-Jun N-terminal kinase (JNK) (21Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1708) Google Scholar). JNK is part of a three-sequential kinase signaling cascade (22Mielke K. Herdegen T. Prog. Neurobiol. 2000; 61: 45-60Crossref PubMed Scopus (434) Google Scholar, 23Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar). MAP kinase kinase (MKK) activates JNK by dual Tyr and Thr phosphorylation. MKK activation is mediated by MAP kinase kinase kinases, including the mixed lineage kinase (MLK) family in neurons. Selective inhibition of the MLK family in sympathetic neurons by the K252a analog, CEP-1347, demonstrates that MLKs mediate JNK activation after trophic factor deprivation, UV irradiation, and oxidative stress (24Maroney A.C. Finn J.P. Bozyczko-Coyne D. O'Kane T.M. Neff N.T. Tolkovsky A.M. Park D.S. Yan C.Y. Troy C.M. Greene L.A. J. Neurochem. 1999; 73: 1901-1912PubMed Google Scholar, 25Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: 25302-25308Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 26Harris C.A. Deshmukh M. Tsui-Pierchala B. Maroney A.C. Johnson Jr., E.M. J. Neurosci. 2002; 22: 103-113Crossref PubMed Google Scholar). In this study, we examined the changes in c-Jun and JNK pathway activity in sympathetic neurons during DNA damage-induced apoptosis. DNA damage caused c-Jun activation in neurons by two separate mechanisms: induction of c-Jun expression and N-terminal c-Jun phosphorylation. Surprisingly, DNA damage-induced c-Jun expression and phosphorylation were maintained in the absence of neuronal JNK activity. In addition, the JNK pathway was not necessary for DNA damage-induced apoptosis because pharmacological inhibition of the JNK pathway by CEP-1347 or SP600125 did not increase neuronal survival. These results demonstrate that DNA damage-induced neuronal c-Jun activation was independent of the classical JNK pathway. c-Jun activation was blocked by several structurally distinct pharmacological CDK inhibitors, suggesting that a CDK-like activity may be responsible for mediating c-Jun activation and neuronal death after DNA damage. Animals and Materials—All reagents were purchased from Sigma unless otherwise stated. Timed-pregnant Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN). Animal use and treatment complied fully with the Animal Studies Committee of Washington University and the United States Animal Welfare Act (1985). Collagenase and trypsin were purchased from Worthington. Mouse 2.5S NGF was from Harlan Bioproducts (Indianapolis, IN). The goat anti-mouse 2.5 S NGF neutralizing antiserum has been characterized previously (27Ruit K.G. Elliott J.L. Osborne P.A. Yan Q. Snider W.D. Neuron. 1992; 8: 573-587Abstract Full Text PDF PubMed Scopus (209) Google Scholar). CEP-1347 was a kind gift of Cephalon (Westchester, PA). SP600125 was provided by Celgene Inc., San Diego, CA. Etoposide and olomoucine were purchased from Biomol (Plymouth Meeting, PA). Alsterpaullone was obtained from Calbiochem. Neuronal Culture—Primary sympathetic neuronal cultures were prepared from P0 to P1 rat superior cervical ganglia by using methods described previously (28Johnson M.I. Argiro V. Methods Enzymol. 1983; 103: 334-347Crossref PubMed Scopus (68) Google Scholar, 29Deckwerth T.L. Johnson Jr., E.M. J. Cell Biol. 1993; 123: 1207-1222Crossref PubMed Scopus (515) Google Scholar). Briefly, superior cervical ganglia were dissected from newborn animals and incubated for 30 min each with 1 mg/ml collagenase and 2.5 mg/ml trypsin at 37 °C. The ganglia were dissociated by triturating with a P200 micropipet tip; cells were plated on collagen-coated plastic tissue culture plates at appropriate densities. Cultures were maintained in AM50 medium (90% minimum essential medium (Invitrogen), 2 mm glutamine, 10% fetal bovine serum (Hyclone, Logan, UT), 50 ng/ml 2.5 S NGF, 20 μm fluorodeoxyuridine, 20 μm uridine, 100 units/ml penicillin, and 100 units/ml streptomycin), supplemented with 3.3 μg/ml aphidicolin (AG Scientific, Inc., San Diego, CA) for the first 5 days in vitro (DIV) to reduce the number of non-neuronal cells. To deprive neurons of NGF, 5-day-old neuronal cultures were washed three times with AM0 (AM50 medium lacking NGF) and fed with fresh AM0 containing 0.01% anti-NGF antiserum. For the treatment of neurons with various DNA-damaging agents, the culture medium was replaced with fresh AM50 containing the indicated concentrations of the particular DNA toxin. In neuronal cultures used to harvest protein, 25–50 μm of broad spectrum caspase inhibitor, bocaspartyl-(OMe)-fluoromethyl ketone (Enzyme Systems Products, Livermore, CA), was included in all treatment conditions to inhibit neuronal death. Neuronal Survival—The number of viable cells was assessed after fixing the cultures with 4% paraformaldehyde (Fisher) in phosphate-buffered saline and staining with crystal violet. Neurons were scored as viable by a naïve observer if the crystal violet-positive cells had large, well defined cellular outlines. Dead neurons and debris stain faintly or show no staining with crystal violet. Percentage viability was calculated by dividing the number of crystal violet-positive neurons at each time point by the total number of neurons in NGF-maintained, untreated sister cultures. Immunocytochemistry—Cultures were fixed with fresh 4% paraformaldehyde in phosphate-buffered saline, washed with Tris-buffered saline (TBS: 0.1 m Tris-HCl (pH 7.6), 0.9% NaCl), and incubated in blocking solution (5% normal goat serum in TBS, containing 0.3% Triton X-100) for 1 h at room temperature. The cultures were then incubated with primary antibodies in antibody solution (1% normal goat serum in TBS, containing 0.3% Triton X-100) overnight at 4 °C. The following primary antibodies were used: phospho-c-Jun (Ser63 II) (1:1000, Cell Signaling, Beverly, MA) and active caspase-3 (1:250, Promega, Madison, WI). The cultures were next washed three times with TBS and incubated in antibody solution containing Cy-3-labeled secondary antibodies (1:400, Jackson ImmunoResearch, West Grove, PA) for 4 h at 4 °C and counterstained with 1 μg/ml bisbenzimide (Hoechst 33258, Molecular Probes, Eugene, OR). After four washes with TBS, the cultures were mounted for fluorescence microscopy. Western Blot Analysis—Neuronal cultures were rinsed twice with cold phosphate-buffered saline, lysed in reducing sample buffer (125 mm Tris-HCl (pH 6.8), 10% 2-mercaptoethanol, 4% SDS, 0.1% bromphenol blue, and 20% glycerol), boiled for 5 min, and stored at –20 °C until use. Proteins were separated by SDS-PAGE on Tris glycine mini-gels (Invitrogen) and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA). Blots were blocked for1hat room temperature with TBST (10 mm Tris-HCl (pH 7.5), 100 mm NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk and incubated overnight at 4 °C with primary antibody diluted in blocking solution recommended by manufacturer. The following primary antibodies were used: c-Jun (0.25 μg/ml, Transduction Laboratories, San Diego, CA), c-Jun (1:1000, Cell Signaling), phospho-c-Jun (Ser63 II) (1:1000, Cell Signaling), phospho-c-Jun (Ser73) (1:1000, Cell Signaling), phospho-SAPK/JNK (Thr183/Tyr185) (1:1000, Cell Signaling), phospho—SEK1/MKK4 (Thr261) (1:1000, Cell Signaling), and tubulin (Clone DM 1A, 1:50,000, Sigma). After washing, blots were incubated for 1 h at room temperature with HRP-linked secondary antibodies (Cell Signaling) diluted 1:2500–1:10,000 in blocking solution. The blots were washed 3 times with TBST and developed with a chemiluminescent substrate (Supersignal, Pierce). To strip and reprobe blots, the membranes were incubated in 100 mm glycine (pH 2.5) twice for 25 min and then washed with TBST. The Western analysis was then repeated. To semiquantitate immunoblots, the films were digitally scanned into Adobe Photoshop, and the selected bands were subjected to pixel analysis by using the UN-SCAN-IT software (Silk Scientific, Orem, UT). All values were normalized using the values obtained for tubulin loading control. c-Jun Expression Is Induced in Neuronal Apoptosis after DNA Damage—We have reported previously that ara-C kills sympathetic neurons in vitro (8Martin D.P. Wallace T.L. Johnson Jr., E.M. J. Neurosci. 1990; 10: 184-193Crossref PubMed Google Scholar, 10Wallace T.L. Johnson Jr., E.M. J. Neurosci. 1989; 9: 115-124Crossref PubMed Google Scholar). A recent study (7Geller H.M. Cheng K.Y. Goldsmith N.K. Romero A.A. Zhang A.L. Morris E.J. Grandison L. J. Neurochem. 2001; 78: 265-275Crossref PubMed Scopus (104) Google Scholar) has shown that ara-C causes oxidative stress in embryonic cortical neurons and leads to DNA damage and cell death. We determined whether this DNA damage can activate proapoptotic signals in sympathetic neurons. Several lines of evidence suggest that c-Jun has an essential role in neuronal apoptosis. Therefore, we examined the expression of c-Jun during DNA damage-induced neuronal apoptosis. In sympathetic neurons, ara-C exposure caused an increase in the levels of c-Jun protein (Fig. 1). This increase was apparent at 24 h, thus preceding the loss of viability (8Martin D.P. Wallace T.L. Johnson Jr., E.M. J. Neurosci. 1990; 10: 184-193Crossref PubMed Google Scholar). The increased levels of c-Jun protein remained heightened throughout the period of ara-C exposure. After 48 h of ara-C treatment, the migration of neuronal c-Jun became slower in SDS-PAGE, suggesting that c-Jun may have been phosphorylated during cell death. c-Jun Is Phosphorylated in Its N-terminal Transactivation Domain after DNA Damage—One of the most critical steps in c-Jun regulation involves phosphorylation of two specific serine residues, Ser63 and Ser73, located in the N-terminal transactivation domain. Phosphorylation of Ser63 and Ser73 enhances c-Jun-mediated gene transcription (30Kallunki T. Deng T. Hibi M. Karin M. Cell. 1996; 87: 929-939Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). To determine whether neuronal c-Jun is subject to posttranslational modification after DNA damage, we examined the potential c-Jun N-terminal phosphorylation using an antibody that specifically detects c-Jun only when Ser63 is phosphorylated. When sympathetic neurons were exposed to increasing concentrations of ara-C, neuronal c-Jun was phosphorylated beginning at 11.1 μm of ara-C, and the levels of phospho-c-Jun were augmented with increasing ara-C concentrations (Fig. 2A). This concentration dependence correlated well with the dose-response curve of ara-C killing in sympathetic neurons (8Martin D.P. Wallace T.L. Johnson Jr., E.M. J. Neurosci. 1990; 10: 184-193Crossref PubMed Google Scholar). Moreover, neurons exposed to ara-C showed increased levels of phospho-c-Jun over time (Fig. 2B). Because ara-C exposure induced c-Jun expression and N-terminal phosphorylation simultaneously, increased phosphorylation after DNA damage might have been an indirect effect of elevated neuronal c-Jun levels. Therefore, we measured the relative ratio of phosphorylated c-Jun to total c-Jun during ara-C-induced neuronal apoptosis (Fig. 2C). The phospho-c-Jun/c-Jun ratio increased by 3-fold after 48 h of ara-C exposure, thus demonstrating that N-terminal c-Jun phosphorylation was not an indirect effect of increased protein levels. Localization of phospho-c-Jun by immunocytochemistry demonstrated that activated c-Jun was exclusively nuclear during DNA damage-induced neuronal apoptosis (Fig. 2D). About 50% of sympathetic neurons lose their viability after 48 h of ara-C exposure, and most neurons are dead by 96 h (67Besirli C.G. Deckwerth T.L. Crowder R.J. Freeman R.S. Johnson Jr., E.M. Cell Death Differ. 2003; (in press)PubMed Google Scholar). Therefore, the time course of N-terminal c-Jun phosphorylation preceded the time course of ara-C-induced sympathetic neuronal death because the number of phospho-c-Jun-positive neurons started to increase at 24 h, at which time all neurons are still alive, and continued until 48 h with 70% of neurons containing nuclear phospho-c-Jun (Fig. 2E). Phosphorylation of Ser73, located in the N-terminal transactivation domain, is also critical for increasing c-Jun-mediated transcription. Similar to Ser63, DNA damage induced by ara-C treatment also led to increased levels of Ser73-phosphorylated c-Jun in sympathetic neurons (Fig. 2F). These results demonstrate that c-Jun was activated by N-terminal phosphorylation after DNA damage. In addition, the time course of c-Jun activation is consistent with increased c-Jun activity being a critical step in neuronal cell death after DNA damage (Fig. 2E). Neuronal c-Jun Activation Is a Common Stress Response Seen after DNA Damage—Besides causing DNA damage, ara-C can also affect several other metabolic events in neurons, including the synthesis of membrane lipids and glycoproteins, potentially influencing the intracellular levels of lipid second messengers (10Wallace T.L. Johnson Jr., E.M. J. Neurosci. 1989; 9: 115-124Crossref PubMed Google Scholar). To determine whether c-Jun induction and N-terminal phosphorylation occurred as a specific response to DNA damage after ara-C exposure, we examined the effects of other DNA-damaging agents in sympathetic neurons. Etoposide is a DNA topoisomerase II inhibitor that damages DNA by generating double-stranded DNA breaks. Sympathetic neurons exposed to 10 μm etoposide exhibited a significant increase in c-Jun levels starting at 6 h (Fig. 3A). This increase was associated with rapid neuronal death (data not shown). Etoposide-induced c-Jun protein also migrated more slowly in SDS-PAGE, indicating post-translational modification by increased phosphorylation. Immunoblot analysis of neuronal phospho-c-Jun levels showed that etoposide-induced DNA damage also caused enhanced N-terminal phosphorylation of c-Jun on Ser63 and Ser73 (Fig. 3, B and C). We also exposed sympathetic neurons to another nucleoside analog, ara-A. Similar to ara-C, ara-A interferes with DNA metabolism, but it does not share other DNA-independent metabolic actions of ara-C. Ara-A-induced sympathetic neuronal death was also associated with c-Jun activation, as shown by increased phospho-c-Jun Ser63 levels in neuronal nuclei after 96 h of treatment (Fig. 3D). Compared with ara-C and etoposide, ara-A-induced N-terminal c-Jun phosphorylation was delayed. However, this delay was consistent with the time course of ara-A-induced neuronal death, which was also considerably delayed compared with ara-C-induced death. 2C. G. Besirli and E. M. Johnson, Jr., unpublished observations. Induction and phosphorylation of c-Jun after both etoposide and ara-A-induced DNA damage indicate that our initial observation of ara-C-induced c-Jun activation was caused by neuronal DNA damage. Topoisomerase I inhibitor camptothecin also kills sympathetic neurons. Compared with ara-C and etoposide, death of sympathetic neurons after camptothecin exposure shows a slower time course (50% death at 72 h (31Park D.S. Morris E.J. Greene L.A. Geller H.M. J. Neurosci. 1997; 17: 1256-1270Crossref PubMed Google Scholar), data not shown). Surprisingly, camptothecin exposure did not lead to the phosphorylation of c-Jun in sympathetic neurons (Fig. 3E). Therefore, c-Jun activation was a common event during neuronal death induced by multiple, but not all, DNA-damaging agents. DNA Damage-induced c-Jun Induction and Phosphorylation Are Primarily Mediated by a JNK-independent Signaling Pathway—Phosphorylation of c-Jun N-terminal transactivation domain is believed to be performed exclusively by JNKs after cellular stress (13Ham J. Eilers A. Whitfield J. Neame S.J. Shah B. Biochem. Pharmacol. 2000; 60: 1015-1021Crossref PubMed Scopus (214) Google Scholar, 22Mielke K. Herdegen T. Prog. Neurobiol. 2000; 61: 45-60Crossref PubMed Scopus (434) Google Scholar, 23Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar). This JNK-mediated N-terminal phosphorylation is critical for the transcriptional activity of c-Jun (30Kallunki T. Deng T. Hibi M. Karin M. Cell. 1996; 87: 929-939Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Neuronal c-Jun phosphorylation induced by ara-C and other DNA-damaging agents suggests that the JNK pathway was activated during DNA-damage-induced neuronal apoptosis. During neuronal cell death caused by trophic factor deprivation, UV irradiation, or oxidative stress, MLKs mediate phosphorylation-coupled JNK activation by stimulating JNK-kinase MKK4/7 (24Maroney A.C. Finn J.P. Bozyczko-Coyne D. O'Kane T.M. Neff N.T. Tolkovsky A.M. Park D.S. Yan C.Y. Troy C.M. Greene L.A. J. Neurochem. 1999; 73: 1901-1912PubMed Google Scholar, 32Xu Z. Maroney A.C. Dobrzanski P. Kukekov N.V. Greene L.A. Mol. Cell Biol. 2001; 21: 4713-4724Crossref PubMed Scopus (221) Google Scholar). Phosphorylation of MKKs and JNKs is required for catalytic activity of these kinases. Therefore, activation of the JNK pathway can be indirectly examined by analyzing the levels of phosphorylated kinases, as increased phosphorylation is coupled to increased catalytic activity. We determined the levels of phospho-MKK4 in sympathetic neurons by using an antibody that specifically recognizes Thr261-phosphorylated MKK4, which is important for kinase activity. Although we did not detect any significant increase above basal levels after DNA damage at 12 h (Fig. 4A), our data indicated that there might be a slight increase in phospho-MKK4 levels in ara-C-treated neurons at 24 and 36 h. However, quantitative data showed that there was no significant difference in phospho-MKK4 levels between NGF-maintained and ara-C-treated neurons at these time points (Fig. 4B). In contrast, NGF-deprived neurons, in which the JNK pathway is activated, showed significantly higher phospho-MKK4 levels compared with NGF-maintained, untreated neurons (Fig. 4A). Similar to phospho-MKK4, we did not detect any increase in the amount of phospho-JNKs after ara-C treatment (Fig. 4C). Indeed, the amount of phospho-JNK declined during DNA damage-induced neuronal death, because sympathetic neurons showed high basal levels of JNK phosphorylation when maintained in NGF. These results suggest that activity of the JNK pathway was not increased by DNA damage and, in fact, was decreased and that JNKs did not mediate c-Jun phosphorylation. However, MKK7 might have been the JNK kinase activated after DNA damage, and phospho-MKK4 antibody might not have detected phosphorylated MKK7 in neurons. In addition, even though the total phospho-JNK levels were decreased in ara-C-exposed sympathetic neurons, a subset of JNKs might have been activated by DNA damage, as the JNK family consists of 3 isoforms and 10 splice variants. The detection of this potential isoform-specific JNK activation with a pan-phospho-JNK antibody may not be possible. Therefore, we determined whether JNK pathway activation was responsible for c-Jun phosphorylation in DNA-damaged neurons by assessing the effect of pharmacological JNK pathway inhibition on c-Jun activation. CEP-1347 is a selective MLK inhibitor that prevents JNK activity and neuronal apoptosis (15Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar, 24Maroney A.C. Finn J.P. Bozyczko-Coyne D. O'Kane T.M. Neff N.T. Tolkovsky A.M. Park D.S. Yan C.Y. Troy C.M. Greene L.A. J. Neurochem. 1999; 73: 1901-1912PubMed Google Scholar, 25Maroney A.C. Finn J.P. Connors T.J. Durkin J.T. Angeles T. Gessner G. Xu Z. Meyer S.L. Savage M.J. Greene L.A. Scott R.W. Vaught J.L. J. Biol. Chem. 2001; 276: 25302-25308Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 26Harris C.A. Deshmukh M. Tsui-Pierchala B. Maroney A.C. Johnson Jr., E.M. J. Neurosci. 2002; 22: 103-113Crossref PubMed Google Scholar). When we treated sympathetic neurons with ara-C in the presence of 1.3 μm CEP-1347, the majority of N-terminal c-Jun phosphorylation was maintained (Fig. 5, A and B). In contrast, N-terminal c-Jun phosphorylation induced by NGF deprivation was completely prevented in sibling neuronal cultures (Fig. 5, A and B), demonstrating that CEP-1347 fully prevented MLK-dependent activation of JNKs in neurons as reported previously (26Harris C.A. Deshmukh M. Tsui-Pierchala B. Maroney A.C. Johnson Jr., E.M. J. Neurosci. 2002; 22: 103-113Crossref PubMed Google Scholar).Fig. 5c-Jun induction and phosphorylation during DNA damage-induced neuronal death is independent of the classical JNK pathway. 5-DIV sympathetic neurons were exposed to 100 μm ara-C, deprived of NGF, or left untreated and were fixed after 48 h. In some neuronal cultures, ara-C treatment or NGF deprivation was performed in the presence of 1.3 μm CEP-1347 (CEP) or 14 μm SP600125 (SP). N-terminal c-Jun phosphorylation was examined by the detection of neuronal phospho-c-Jun Ser63 immunocytochemically. Concurrent staining with bisbenzimide was performed to detect the nuclei of neurons. Neither MLK-inhibitor CEP-1347 (A and B) nor JNK-inhibitor SP600125 (C and D) blocked the majority of c-Jun phosphorylation after ara-C-induced DNA damage. Mean ± S.D., n = 3–4. Similar results were obtained in at least two independent experiments.View Large" @default.
• W1980283113 created "2016-06-24" @default.
• W1980283113 creator A5000274726 @default.
• W1980283113 creator A5086652241 @default.
• W1980283113 date "2003-06-01" @default.
• W1980283113 modified "2023-09-27" @default.
• W1980283113 title "JNK-independent Activation of c-Jun during Neuronal Apoptosis Induced by Multiple DNA-damaging Agents" @default.
• W1980283113 cites W1491979926 @default.
• W1980283113 cites W1506543054 @default.
• W1980283113 cites W1515821225 @default.
• W1980283113 cites W1528310990 @default.
• W1980283113 cites W1530284699 @default.
• W1980283113 cites W1539880650 @default.
• W1980283113 cites W1549081254 @default.
• W1980283113 cites W1567753257 @default.
• W1980283113 cites W1611726375 @default.
• W1980283113 cites W1646788158 @default.
• W1980283113 cites W1668168210 @default.
• W1980283113 cites W1672183542 @default.
• W1980283113 cites W1888982058 @default.
• W1980283113 cites W1893523030 @default.
• W1980283113 cites W1908458283 @default.
• W1980283113 cites W1964893456 @default.
• W1980283113 cites W1966406567 @default.
• W1980283113 cites W1968015127 @default.
• W1980283113 cites W1968867081 @default.
• W1980283113 cites W1970458627 @default.
• W1980283113 cites W1970937164 @default.
• W1980283113 cites W1973227020 @default.
• W1980283113 cites W1990144221 @default.
• W1980283113 cites W1999319877 @default.
• W1980283113 cites W2004126223 @default.
• W1980283113 cites W2005423627 @default.
• W1980283113 cites W2007410245 @default.
• W1980283113 cites W2017686829 @default.
• W1980283113 cites W2036804890 @default.
• W1980283113 cites W2044200046 @default.
• W1980283113 cites W2060982272 @default.
• W1980283113 cites W2065477683 @default.
• W1980283113 cites W2070044720 @default.
• W1980283113 cites W2076485183 @default.
• W1980283113 cites W2077880395 @default.
• W1980283113 cites W2081567150 @default.
• W1980283113 cites W2082559835 @default.
• W1980283113 cites W2084810833 @default.
• W1980283113 cites W2086525709 @default.
• W1980283113 cites W2089038077 @default.
• W1980283113 cites W2089528428 @default.
• W1980283113 cites W2091604918 @default.
• W1980283113 cites W2094155854 @default.
• W1980283113 cites W2096885631 @default.
• W1980283113 cites W2096931514 @default.
• W1980283113 cites W2099405729 @default.
• W1980283113 cites W2099571311 @default.
• W1980283113 cites W2104373322 @default.
• W1980283113 cites W2107528191 @default.
• W1980283113 cites W2108678943 @default.
• W1980283113 cites W2121955382 @default.
• W1980283113 cites W2122716676 @default.
• W1980283113 cites W2131871435 @default.
• W1980283113 cites W2132970977 @default.
• W1980283113 cites W2134882450 @default.
• W1980283113 cites W2139454515 @default.
• W1980283113 cites W2145828270 @default.
• W1980283113 cites W2147347134 @default.
• W1980283113 cites W2153385508 @default.
• W1980283113 cites W2156024250 @default.
• W1980283113 cites W2167083565 @default.
• W1980283113 cites W2182684865 @default.
• W1980283113 cites W2191713065 @default.
• W1980283113 cites W2326067170 @default.
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