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Akt pathway
Neuroscience Letters 450 (2009) 45–50
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p62 protects SH-SY5Y neuroblastoma cells against H2 O2 -induced injury through the PDK1/Akt pathway Seong Ryong Heo a , Ah Mi Han b , Yunhee Kim Kwon b,∗ , Insil Joung a,∗∗ a b
Department of Biological Sciences, Hanseo University, Seosan, Chungnam 356-706, Republic of Korea Department of Biology and Department of Life and Nanopharmaceutical Science, Kyunghee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
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Article history: Received 18 September 2008 Received in revised form 29 October 2008 Accepted 6 November 2008 Keywords: Akt H2 O2 p62 PDK1 Survival 14-3-3
a b s t r a c t The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H2 O2 treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H2 O2 -induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H2 O2 -induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinasedefective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3 protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival. © 2008 Elsevier Ireland Ltd. All rights reserved.
p62 is a ubiquitously expressed phosphoprotein composed of several functional modules that are involved in a variety of cellular signaling pathways, including cell proliferation and survival [13,18]. Several reports have shown that the accumulation of p62 is associated with neurodegenerative disease. More specifically, p62 has been detected in neurofibrillary tangles of Alzheimer’s disease brains [15], Lewy bodies in Parkinson’s disease, polyglutamine inclusions associated with Huntington’s disease [21], and more recently, protein aggregates in amyotrophic lateral sclerosis (ALS) [9,19]. p62 was colocalized with ubiquitin in all of these aggregates. Oxidative stress is known to be a major cause of the pathogenesis associated with various neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. In addition, reactive oxygen species generated during reperfusion, including hydrogen peroxide (H2 O2 ) and the hydroxyl radical (OH• ), have been shown to play a crucial role in neuronal cell death after ischemic stroke [4]. p62 expression is rapidly induced by a variety of stress signals, such
Abbreviations: Ad, adenovirus; MOI, multiplicity of infection; MTT, 3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; PDK, 3-phosphoinositidedependent kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C. ∗ Corresponding author. Tel.: +82 2 961 0844; fax: +82 2 966 4497. ∗∗ Corresponding author. Tel.: +82 41 660 1341; fax: +82 41 688 3403. E-mail addresses: [email protected] (Y.K. Kwon), [email protected] (I. Joung). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.11.011
as those known to stimulate cell proliferation and differentiation in several cell types [16]. In addition, a murine p62 homolog, A170, has also been shown to be induced by oxidative stress in macrophages [10] and by kainite-related oxidative stress in the brains of rats [20]. Moreover, the anti-parkinsonian drug deprenyl has been shown to increase the expression of p62 and other anti-oxidative enzymes, implying that p62 is involved in the defense mechanism against oxidative stress [22]. Although a considerable amount of knowledge regarding the roles of p62 in neuronal diseases has been accumulating over the past few years, the interactions between p62 and other signaling proteins under conditions of oxidative stress remains to be clearly elucidated. In our previous study, we reported that p62 exerted cytoprotective effects by modulating the interactions between PKC and Akt during the differentiation of neuronal precursor cells [14]. In this study, we attempted to determine whether p62 protected human neuroblastoma SH-SY5Y cells against H2 O2 -induced cell death and evaluated the function of p62 in the survival signaling pathway activated in response to oxidative insult. Antibodies for Akt, p-Akt (T308), p-PDK1 (S241), and p-PKC (T410) were obtained from Cell Signaling. Anti-Myc, anti- PKC, and anti-14-3-3 were purchased from Santa Cruz. Anti--actin (Sigma), anti-p62, anti-T7 (BD Transduction Laboratories), and anti-galactosidase (Promega) were also used in this study. Human embryonic kidney cell line 293 and SH-SY5Y human neuroblastoma cells were acquired from ATCC. Both cell lines were maintained in
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Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin, and streptomycin at 37 ◦ C. For the SH-SY5Y cells 0.1 mM MEM non-essential amino acids were also added. SH-SY5Y cells were plated at an initial density of 8 × 106 cells per a 100-mm plate for phosphorylation and immunoprecipitation studies, or 5 × 104 cells per well in 96-well plates for the cell survival study. SH-SY5Y cells were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 24 h and subsequently differentiated by incubation in 2% serum-containing media with 10 M of All-transretinoic acid (RA, Tocris) for 3 days prior to H2 O2 treatment. 750 M of H2 O2 (Sigma) was added to the differentiation medium, and the cells were incubated for 72 h unless otherwise stated. Recombinant adenoviruses expressing either T7-tagged p62 (p62Ad) or E. coli -galactosidase (LacZAd) were prepared as previously described [14]. When PDK1 was coexpressed, the PDK1 cDNA was transfected using FuGene 6 (Promega) 24 h prior to viral infection. pcDNA3 vectors harboring Myc-tagged wild-type (PDK1 wt) and the kinaseinactive form (PDK1 KI) of PDK1 were a generous gift from Dr. J. Chung at KAIST, Korea. Cell survival analysis was performed according to the 3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. Briefly, 10 l of 4 mg/ml MTT solution was added to each well of 96-well plates, and the plates were incubated for 3 h after 72 h incubation with H2 O2 . The reaction was terminated by the addition of 60 l of solubilization buffer containing 10% SDS in dimethylformamide (DMF). After overnight solubilization, the absorbance was measured in a microplate reader at 595 nm. Total RNA was extracted from the SH-SY5Y cells using Trizol, reverse-transcribed and amplified with primers specific for p62 and GAPDH. The following primers were used: p62 (5 -GGCCATGTCCTACGTGAAG-3 and 5 -CCTGAACAGTTATCC3 ) and GAPDH (5 -TGGTATCGTGGAAGGACTCATGAC-3 and 5 ATGCCAGTGAGCTTCCCGTTCAGC-3 ). For immunoblotting, SHSY5Y cells were lysed in RIPA buffer (20 mM Tris–Cl, pH 7.5, 137 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 5 mM EDTA, 1 mM Na3 VO4 , 50 mM NaF, and protease inhibitors) and cleared by 15 min of centrifugation. Cell fractions, normalized for total protein, were separated on SDS–PAGE, transferred to a nitrocellulose membrane, and probed with p-Akt, p-PDK1, and p-PKC antibodies by enhanced chemiluminescence (Dongjin Biotech). The blots were
then stripped and re-probed with antibodies against their unphosphorylated counterparts. For immunoprecipitation, the cells were lysed in a buffer containing 20 mM Tris–Cl, pH 7.5, 137 mM NaCl, and 1% Triton X-100, and 300 g of cell lysates were incubated with anti-14-3-3 antibody and precipitated using protein-A agarose beads. Bound proteins were subjected to immunoblot analysis with anti-Myc antibody. The blots were then stripped and re-probed with anti-T7, and the antibodies that were used for immunoprecipitation in sequence. Previous studies have shown that p62 accumulates in several cell types in response to paraquat and kainate, which are associated with glutamate toxicity [11,20]. Therefore, we attempted to determine whether p62 expression is induced by oxidative stress in neuronal cells. Western blot analysis confirmed that p62 expression was markedly increased in differentiated SH-SY5Y cells after treatment with H2 O2 and p62 induction was prolonged for up to 48 h (Fig. 1A). The quantity of p62 transcripts peaked 24 h after H2 O2 exposure and decreased rapidly thereafter (Fig. 1B), suggesting that the p62 gene responds to oxidative stress in neuronal cells. We also investigated the potential role of p62 in neuronal cells under oxidative stress. MTT assay showed that approximately 50% of differentiated SH-SY5Y cells died after treatment with H2 O2 (750 M) for 72 h (Fig. 2A). Overexpression of p62 induced by adenovirus (p62Ad) increased the survival rate of SH-SY5Y cells exposed to H2 O2 , but LacZAd infection had no effect on cell viability. Microscopic observation also showed that the total number of p62Ad-infected SH-SY5Y cells was increased in comparison to the uninfected or LacZAd-infected cells (Fig. 2B), indicating that p62 protects against H2 O2 -induced injury in neuronal cells. The PI3K/PKB pathway was investigated in an attempt to determine the mechanism by which p62 exerts its cytoprotective effects against H2 O2 . One of the principal pathways of neuronal cell survival involves the activation of protein kinase B (PKB)/Akt serine/threonine kinase, which is mediated by the phosphatidylinositol 3-kinase (PI3K) pathway [2,7]. Akt activation via phosphorylation at Thr 308 by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and Ser 473 by another kinase allows the enzyme to facilitate survival by phosphorylating downstream substrates [27]. SH-SY5Y cells were infected with LacZAd or p62Ad and differentiated by incubation with RA 72 h prior to oxidative insult, and
Fig. 1. Induction of p62 by H2 O2 in SH-SY5Y cells. Differentiated SH-SY5Y cells were treated with 750 M of H2 O2 for the indicated times. Endogenous p62 peptide and mRNA were observed by immunoblotting (A) and RT-PCR analysis (B). The blot was also probed with -actin antibody as a loading control. GAPDH was used as a control for RT-PCR. These graphs present the quantitative analysis of p62 protein and mRNA.
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Fig. 2. Protection of SH-SY5Y cells from oxidative stress by p62. (A) Cell viability was measured by the MTT assay. SH-SY5Y cells were either uninfected or infected with adenoviruses, differentiated with RA, and treated with 750 mM of H2 O2 for 72 h. The results were subjected to one-way analysis of variance (ANOVA) for repeated measurements across testing sessions. Differences of means between the groups were investigated using Student’s t-tests. All data are presented as the mean ± S.E.M. and asterisks indicate statistically significant differences (p < 0.05; n = 3). (B) The photomicrograph shows an increase in the number of cells during H2 O2 treatment when infected with p62Ad. Scale bars = 100 m.
the cell lysates were subsequently analyzed (Fig. 3A). Immunoblot analysis with phospho-antibodies indicated that H2 O2 treatment induced rapid Akt phosphorylation in LacZAd-infected cells, which was consistent with previous findings that Akt phosphorylation occurred immediately after H2 O2 treatment in primary cortical neurons and SH-SY5Y cells [5,25]. Akt was phosphorylated as early as 5 min after H2 O2 treatment, returned to the unphosphorylated state within 3 h, and then re-phosphorylated at 24 h. It is likely that Akt is re-phosphorylated at later stage by signaling transmitted through TrkB receptor. It has been shown that the functional TrkB receptor was induced at 3 days of retinoic acid treatment, and peaked after 5 days in SH-SY5Y cells. At this point the binding of BDNF to its cognate receptor TrkB led to Akt phosphorylation accompanied with neurite outgrowth [8]. In LacZAd-infected cells, TrkB receptor might be activated by basal levels of trophic factors in the medium after 24 h treatment of H2 O2 , because cells have been already treated with RA for 4 days. In contrast, Akt phosphorylation was detected in differentiated SH-SY5Y cells, even in the absence of H2 O2 insult when p62 was overexpressed. In addition, the exposure of cells to H2 O2 resulted in decreased Akt phosphorylation during the early stages. However, Akt phosphorylation was
increased after 3 h, and this increase was prolonged for up to 24 h after H2 O2 treatment. We also attempted to determine whether p62 overexpression affects the activity of atypical kinase PKC, which is associated with p62. In differentiated SH-SY5Y cells, PKC was abundantly phosphorylated at Thr 410, which is critical for PKC activity, and this phosphorylation was maintained for 24 h after H2 O2 insult. In contrast, starting 1 h after H2 O2 treatment, PKC phosphorylation was observed for 2 h in p62Ad-infected cells, but it was negligible at most other time points (Fig. 3A). The results also indicated that Akt phosphorylation is associated with PKC dephosphorylation during H2 O2 -induced neuronal cell death. Moreover, these results may support the proposed mechanism by which PKC negatively regulates Akt activity by binding to the pleckstrin homology domain of Akt [1,6,17]. We used a PI3K inhibitor, LY294002 (Sigma), to confirm the involvement of p62 in the PI3K/Akt pathway during H2 O2 -induced neuronal cell death. LY294002 treatment at a concentration of 30 M 1 h prior to H2 O2 insult reduced approximately 80% of the cell survival induced by p62, indicating that PI3K activity participates in cellular activities underlying p62-induced neuronal cell
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Fig. 3. p62 expression promotes cell survival via the PI3K/Akt pathway. (A) Western blot showing increased Akt phosphorylation during H2 O2 treatment in p62expressing cells. SH-SY5Y cells were infected with p62Ad or LacZAd, differentiated with 10 M of RA, and treated with 750 M of H2 O2 for the indicated times. The blots were checked for p-Akt (T308) and p-PKC (T410), stripped and re-probed for unphosphorylated proteins. The expression of transferred p62 or LacZ was also demonstrated via immunoblotting using anti-T7 or -galactosidase antibodies, respectively. (B) MTT assay showing that the inhibition of p62-induced cell survival by LY204002, the PI3K inhibitor. All data are presented as the mean ± S.E.M. and asterisks indicate statistically significant differences (p < 0.05; n = 3).
survival (Fig. 3B). Collectively, these results show that the PI3K/Akt signaling pathway mediates the majority of the effects of p62 on neuronal cell survival under oxidative stress. To obtain further insight into the relative role of p62 in the regulation of Akt activity, myc-tagged PDK1 cDNA was transfected into SH-SY5Y cells along with p62Ad infection. Akt phosphorylation induced by p62 was further augmented in cells transfected with wild-type PDK1, whereas the expression of the kinase-inactive form of PDK1 (PDK KI) reduced Akt phosphorylation in p62Ad-infected cells during H2 O2 treatment (Fig. 4A). Furthermore, the results of the MTT assay indicated that p62 synergistically enhances PDK1mediated cell survival during oxidative insult (Fig. 4B). In contrast, the expression of PDK KI blocked the cytoprotective effects of p62. This result indicates that p62 exerts its protective effects by promoting the activation of Akt in cooperation with PDK1 during oxidative stress-induced neuronal cell death. However, PDK KI did not completely block the effects of p62, which implies that other signaling pathways are affected by p62. We also observed an increase in MAPK activation caused by p62 overexpression during H2 O2 treatment (data not shown). PDK1 is a ubiquitously expressed Ser/Thr kinase that is phosphorylated on five serine residues in vivo. Ser 241, which serves
Fig. 4. Synergistic effects on cell survival by p62 and PDK1 in H2 O2 -treated SH-SY5Y cells. (A) SH-SY5Y cells in a 100-mm plate were transfected with myc-tagged PDK1 cDNA along with adenovirus infection as indicated. Cells were differentiated with RA and then treated with 750 M H2 O2 for 3 h. Blots were checked for p-Akt (T308), stripped and re-probed for unphosphorylated Akt. The expression of transferred PDK1, p62, or LacZ was also confirmed via immunoblotting using anti-Myc, anti-T7 or -galactosidase antibodies, respectively. (B) Cells were treated as similar way as (A) except cells were plated in 96-well plates, and cell viability was measured by MTT assay 72 h after H2 O2 treatment. All data are presented as the mean ± S.E.M. and asterisks indicate statistically significant differences (p < 0.05; n = 3). (C) The dissociation of PDK1 from 14-3-3 in the presence of p62 during H2 O2 treatment. The same cell extracts used in (A) were immunoprecipitated with anti-14-3-3 antibody, and the bound proteins were analyzed via immunoblotting using an antibody against Myc (expressed PDK1 protein). The blot was then stripped and re-probed with antiT7 (expressed p62 protein) and anti-14-3-3 antibodies. 30 g of total cell lysates were also analyzed for the amount of 14-3-3 (total lysate).
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as a PDK1 phosphorylation site, appears to be critical for the activity of PDK1 [3]. It has been reported that the binding of 143-3 to PDK1 through Ser 241 suppresses PDK1 phosphorylation [26]. Of particular interest is the fact that 14-3-3 was identified as a p62-interacting protein in a yeast two-hybrid study (data not shown). This prompted us to hypothesize that p62 modulates PDK1 activity by affecting the association between PDK1 and 14-3-3. Co-immunoprecipitation demonstrated a physical association between PDK1 and 14-3-3 in differentiated SH-SY5Y cells, and this interaction was increased during H2 O2 treatment in the absence of p62 overexpression. However, the association between these proteins was decreased when p62 was expressed (Fig. 4C). Moreover, the reduction in the amount of PDK1 associated with 14-3-3 was correlated with the increase in the amount of 143-3-bound p62 protein in the H2 O2 -treated cells, implying that p62 disrupts the association between 14-3-3 and PDK1 by binding 14-3-3. In this report, we determined that p62 expression was induced by H2 O2 in differentiated neuronal cells. In addition, the results of the MTT assay demonstrated that p62 overexpression increased cell survival during H2 O2 treatment. This result is consistent with the previous finding demonstrating that the expression of A170, murine counterpart of p62 is under the control of Nrf2 transcription factor, a general regulator of reactive oxygen species [11]. This also gives credence to the notion that p62 exerts an anti-apoptotic effect that can be induced by a variety of stimuli [12,14]. Furthermore, we demonstrated that p62 is a mediator that regulates Akt phosphorylation in cooperation with PDK1, a kinase responsible for the phosphorylation of Akt on Thr 308, which is required for Akt activation. In the present study, we present evidence that p62 is involved in a mechanism by which the physical association between 14-3-3 and PDK1 is altered to increase Akt phosphorylation. The 14-3-3 protein has been implicated in both pro- and antiapoptotic pathways. The 14-3-3 protein enhances cell survival by negatively regulating pro-apoptotic proteins, including Bad and Bax, in a phospho-dependent or -independent manner [23,28]. On the other hand, Sato et al. showed that the association between 14-3-3 and PDK1 reduced the activity of PDK1 kinase both in vivo and in vitro, suggesting that 14-3-3 negatively regulates PDK1 by forming a complex [26]. This result is consistent with our data demonstrating a reduction in the interaction between PDK1 and 14-3-3 and increased Akt phosphorylation in p62-expressing cells. Although we did not directly assess the putative role of p62 in the activation of PDK1 upon apoptotic stimuli, the results of our studies collectively showed that p62 overexpression induced the selective release of PDK1 from 14-3-3, which is associated with increased cell survival. The data presented herein strongly indicate that, in addition to its function as a scaffold protein in the formation of aggregates that facilitate the binding between ubiquitin and other neurodegenerative disease-associated proteins to promote cell survival [19,24], p62 exerts a cytoprotective effect by modulating signaling pathways in neuronal cells under oxidative stress. In our previous study, we demonstrated that p62 also induces the dissociation of Akt from PKC, which negatively affects the activity of Akt during the neuronal cell death induced by serum removal [14]. Collectively, the data suggest that p62 releases both PDK1 and Akt by disrupting their binding to 14-3-3 and PKC, respectively, and thus enhances survival signaling. Further studies are needed to delineate the detailed mechanisms by which Akt activity is modulated by p62 in cooperation with other proteins during oxidative stress-induced neuronal cell death. An understanding of the physical interplay between p62, PDK1, 14-3-3, and probably PKC is likely to be of significant value for developing novel strategies to control cell growth, survival, and death.
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Acknowledgements This work was supported by a grant from the Korea Research 276 Foundation to IJ (KRF 2006-C00157) and by the funds from the Korea Health 21R&D Project (A04-0018-AY1204-07M4-00020B) by MOHW and the Brain Research Cen- 277 ter (M103KV01000706K2201-00710) of the 21C Frontier Research 278 program by MOST and to Y.K.K. References [1] N.A. Bourbon, L. Sandirasegarane, M. Kester, Ceramide-induced inhibition of Akt mediated through protein kinase C, J. Biol. Chem. 277 (2002) 3286–3292. [2] A. Brunet, S.R. Datta, M.E. Greenberg, Transcription-dependent and independent control of neuronal survival by the PI3K-Akt signaling pathway, Curr. Opin. Neurobiol. 11 (2001) 297–305. [3] A. Casamayor, N.A. Morrice, D.R. Alessi, Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo, Biochem. J. 342 (1999) 287–292. [4] P.H. 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p62 protects SH-SY5Y neuroblastoma cells against H2 O2 -induced injury through the PDK1/Akt pathway Seong Ryong Heo a , Ah Mi Han b , Yunhee Kim Kwon b,∗ , Insil Joung a,∗∗ a b
Department of Biological Sciences, Hanseo University, Seosan, Chungnam 356-706, Republic of Korea Department of Biology and Department of Life and Nanopharmaceutical Science, Kyunghee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea
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Article history: Received 18 September 2008 Received in revised form 29 October 2008 Accepted 6 November 2008 Keywords: Akt H2 O2 p62 PDK1 Survival 14-3-3
a b s t r a c t The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H2 O2 treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H2 O2 -induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H2 O2 -induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinasedefective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3 protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival. © 2008 Elsevier Ireland Ltd. All rights reserved.
p62 is a ubiquitously expressed phosphoprotein composed of several functional modules that are involved in a variety of cellular signaling pathways, including cell proliferation and survival [13,18]. Several reports have shown that the accumulation of p62 is associated with neurodegenerative disease. More specifically, p62 has been detected in neurofibrillary tangles of Alzheimer’s disease brains [15], Lewy bodies in Parkinson’s disease, polyglutamine inclusions associated with Huntington’s disease [21], and more recently, protein aggregates in amyotrophic lateral sclerosis (ALS) [9,19]. p62 was colocalized with ubiquitin in all of these aggregates. Oxidative stress is known to be a major cause of the pathogenesis associated with various neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. In addition, reactive oxygen species generated during reperfusion, including hydrogen peroxide (H2 O2 ) and the hydroxyl radical (OH• ), have been shown to play a crucial role in neuronal cell death after ischemic stroke [4]. p62 expression is rapidly induced by a variety of stress signals, such
Abbreviations: Ad, adenovirus; MOI, multiplicity of infection; MTT, 3-(4,5dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; PDK, 3-phosphoinositidedependent kinase; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; PKC, protein kinase C. ∗ Corresponding author. Tel.: +82 2 961 0844; fax: +82 2 966 4497. ∗∗ Corresponding author. Tel.: +82 41 660 1341; fax: +82 41 688 3403. E-mail addresses: [email protected] (Y.K. Kwon), [email protected] (I. Joung). 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.11.011
as those known to stimulate cell proliferation and differentiation in several cell types [16]. In addition, a murine p62 homolog, A170, has also been shown to be induced by oxidative stress in macrophages [10] and by kainite-related oxidative stress in the brains of rats [20]. Moreover, the anti-parkinsonian drug deprenyl has been shown to increase the expression of p62 and other anti-oxidative enzymes, implying that p62 is involved in the defense mechanism against oxidative stress [22]. Although a considerable amount of knowledge regarding the roles of p62 in neuronal diseases has been accumulating over the past few years, the interactions between p62 and other signaling proteins under conditions of oxidative stress remains to be clearly elucidated. In our previous study, we reported that p62 exerted cytoprotective effects by modulating the interactions between PKC and Akt during the differentiation of neuronal precursor cells [14]. In this study, we attempted to determine whether p62 protected human neuroblastoma SH-SY5Y cells against H2 O2 -induced cell death and evaluated the function of p62 in the survival signaling pathway activated in response to oxidative insult. Antibodies for Akt, p-Akt (T308), p-PDK1 (S241), and p-PKC (T410) were obtained from Cell Signaling. Anti-Myc, anti- PKC, and anti-14-3-3 were purchased from Santa Cruz. Anti--actin (Sigma), anti-p62, anti-T7 (BD Transduction Laboratories), and anti-galactosidase (Promega) were also used in this study. Human embryonic kidney cell line 293 and SH-SY5Y human neuroblastoma cells were acquired from ATCC. Both cell lines were maintained in
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Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone), penicillin, and streptomycin at 37 ◦ C. For the SH-SY5Y cells 0.1 mM MEM non-essential amino acids were also added. SH-SY5Y cells were plated at an initial density of 8 × 106 cells per a 100-mm plate for phosphorylation and immunoprecipitation studies, or 5 × 104 cells per well in 96-well plates for the cell survival study. SH-SY5Y cells were infected with adenoviruses at a multiplicity of infection (MOI) of 100 for 24 h and subsequently differentiated by incubation in 2% serum-containing media with 10 M of All-transretinoic acid (RA, Tocris) for 3 days prior to H2 O2 treatment. 750 M of H2 O2 (Sigma) was added to the differentiation medium, and the cells were incubated for 72 h unless otherwise stated. Recombinant adenoviruses expressing either T7-tagged p62 (p62Ad) or E. coli -galactosidase (LacZAd) were prepared as previously described [14]. When PDK1 was coexpressed, the PDK1 cDNA was transfected using FuGene 6 (Promega) 24 h prior to viral infection. pcDNA3 vectors harboring Myc-tagged wild-type (PDK1 wt) and the kinaseinactive form (PDK1 KI) of PDK1 were a generous gift from Dr. J. Chung at KAIST, Korea. Cell survival analysis was performed according to the 3(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay method. Briefly, 10 l of 4 mg/ml MTT solution was added to each well of 96-well plates, and the plates were incubated for 3 h after 72 h incubation with H2 O2 . The reaction was terminated by the addition of 60 l of solubilization buffer containing 10% SDS in dimethylformamide (DMF). After overnight solubilization, the absorbance was measured in a microplate reader at 595 nm. Total RNA was extracted from the SH-SY5Y cells using Trizol, reverse-transcribed and amplified with primers specific for p62 and GAPDH. The following primers were used: p62 (5 -GGCCATGTCCTACGTGAAG-3 and 5 -CCTGAACAGTTATCC3 ) and GAPDH (5 -TGGTATCGTGGAAGGACTCATGAC-3 and 5 ATGCCAGTGAGCTTCCCGTTCAGC-3 ). For immunoblotting, SHSY5Y cells were lysed in RIPA buffer (20 mM Tris–Cl, pH 7.5, 137 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, 5 mM EDTA, 1 mM Na3 VO4 , 50 mM NaF, and protease inhibitors) and cleared by 15 min of centrifugation. Cell fractions, normalized for total protein, were separated on SDS–PAGE, transferred to a nitrocellulose membrane, and probed with p-Akt, p-PDK1, and p-PKC antibodies by enhanced chemiluminescence (Dongjin Biotech). The blots were
then stripped and re-probed with antibodies against their unphosphorylated counterparts. For immunoprecipitation, the cells were lysed in a buffer containing 20 mM Tris–Cl, pH 7.5, 137 mM NaCl, and 1% Triton X-100, and 300 g of cell lysates were incubated with anti-14-3-3 antibody and precipitated using protein-A agarose beads. Bound proteins were subjected to immunoblot analysis with anti-Myc antibody. The blots were then stripped and re-probed with anti-T7, and the antibodies that were used for immunoprecipitation in sequence. Previous studies have shown that p62 accumulates in several cell types in response to paraquat and kainate, which are associated with glutamate toxicity [11,20]. Therefore, we attempted to determine whether p62 expression is induced by oxidative stress in neuronal cells. Western blot analysis confirmed that p62 expression was markedly increased in differentiated SH-SY5Y cells after treatment with H2 O2 and p62 induction was prolonged for up to 48 h (Fig. 1A). The quantity of p62 transcripts peaked 24 h after H2 O2 exposure and decreased rapidly thereafter (Fig. 1B), suggesting that the p62 gene responds to oxidative stress in neuronal cells. We also investigated the potential role of p62 in neuronal cells under oxidative stress. MTT assay showed that approximately 50% of differentiated SH-SY5Y cells died after treatment with H2 O2 (750 M) for 72 h (Fig. 2A). Overexpression of p62 induced by adenovirus (p62Ad) increased the survival rate of SH-SY5Y cells exposed to H2 O2 , but LacZAd infection had no effect on cell viability. Microscopic observation also showed that the total number of p62Ad-infected SH-SY5Y cells was increased in comparison to the uninfected or LacZAd-infected cells (Fig. 2B), indicating that p62 protects against H2 O2 -induced injury in neuronal cells. The PI3K/PKB pathway was investigated in an attempt to determine the mechanism by which p62 exerts its cytoprotective effects against H2 O2 . One of the principal pathways of neuronal cell survival involves the activation of protein kinase B (PKB)/Akt serine/threonine kinase, which is mediated by the phosphatidylinositol 3-kinase (PI3K) pathway [2,7]. Akt activation via phosphorylation at Thr 308 by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and Ser 473 by another kinase allows the enzyme to facilitate survival by phosphorylating downstream substrates [27]. SH-SY5Y cells were infected with LacZAd or p62Ad and differentiated by incubation with RA 72 h prior to oxidative insult, and
Fig. 1. Induction of p62 by H2 O2 in SH-SY5Y cells. Differentiated SH-SY5Y cells were treated with 750 M of H2 O2 for the indicated times. Endogenous p62 peptide and mRNA were observed by immunoblotting (A) and RT-PCR analysis (B). The blot was also probed with -actin antibody as a loading control. GAPDH was used as a control for RT-PCR. These graphs present the quantitative analysis of p62 protein and mRNA.
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Fig. 2. Protection of SH-SY5Y cells from oxidative stress by p62. (A) Cell viability was measured by the MTT assay. SH-SY5Y cells were either uninfected or infected with adenoviruses, differentiated with RA, and treated with 750 mM of H2 O2 for 72 h. The results were subjected to one-way analysis of variance (ANOVA) for repeated measurements across testing sessions. Differences of means between the groups were investigated using Student’s t-tests. All data are presented as the mean ± S.E.M. and asterisks indicate statistically significant differences (p < 0.05; n = 3). (B) The photomicrograph shows an increase in the number of cells during H2 O2 treatment when infected with p62Ad. Scale bars = 100 m.
the cell lysates were subsequently analyzed (Fig. 3A). Immunoblot analysis with phospho-antibodies indicated that H2 O2 treatment induced rapid Akt phosphorylation in LacZAd-infected cells, which was consistent with previous findings that Akt phosphorylation occurred immediately after H2 O2 treatment in primary cortical neurons and SH-SY5Y cells [5,25]. Akt was phosphorylated as early as 5 min after H2 O2 treatment, returned to the unphosphorylated state within 3 h, and then re-phosphorylated at 24 h. It is likely that Akt is re-phosphorylated at later stage by signaling transmitted through TrkB receptor. It has been shown that the functional TrkB receptor was induced at 3 days of retinoic acid treatment, and peaked after 5 days in SH-SY5Y cells. At this point the binding of BDNF to its cognate receptor TrkB led to Akt phosphorylation accompanied with neurite outgrowth [8]. In LacZAd-infected cells, TrkB receptor might be activated by basal levels of trophic factors in the medium after 24 h treatment of H2 O2 , because cells have been already treated with RA for 4 days. In contrast, Akt phosphorylation was detected in differentiated SH-SY5Y cells, even in the absence of H2 O2 insult when p62 was overexpressed. In addition, the exposure of cells to H2 O2 resulted in decreased Akt phosphorylation during the early stages. However, Akt phosphorylation was
increased after 3 h, and this increase was prolonged for up to 24 h after H2 O2 treatment. We also attempted to determine whether p62 overexpression affects the activity of atypical kinase PKC, which is associated with p62. In differentiated SH-SY5Y cells, PKC was abundantly phosphorylated at Thr 410, which is critical for PKC activity, and this phosphorylation was maintained for 24 h after H2 O2 insult. In contrast, starting 1 h after H2 O2 treatment, PKC phosphorylation was observed for 2 h in p62Ad-infected cells, but it was negligible at most other time points (Fig. 3A). The results also indicated that Akt phosphorylation is associated with PKC dephosphorylation during H2 O2 -induced neuronal cell death. Moreover, these results may support the proposed mechanism by which PKC negatively regulates Akt activity by binding to the pleckstrin homology domain of Akt [1,6,17]. We used a PI3K inhibitor, LY294002 (Sigma), to confirm the involvement of p62 in the PI3K/Akt pathway during H2 O2 -induced neuronal cell death. LY294002 treatment at a concentration of 30 M 1 h prior to H2 O2 insult reduced approximately 80% of the cell survival induced by p62, indicating that PI3K activity participates in cellular activities underlying p62-induced neuronal cell
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Fig. 3. p62 expression promotes cell survival via the PI3K/Akt pathway. (A) Western blot showing increased Akt phosphorylation during H2 O2 treatment in p62expressing cells. SH-SY5Y cells were infected with p62Ad or LacZAd, differentiated with 10 M of RA, and treated with 750 M of H2 O2 for the indicated times. The blots were checked for p-Akt (T308) and p-PKC (T410), stripped and re-probed for unphosphorylated proteins. The expression of transferred p62 or LacZ was also demonstrated via immunoblotting using anti-T7 or -galactosidase antibodies, respectively. (B) MTT assay showing that the inhibition of p62-induced cell survival by LY204002, the PI3K inhibitor. All data are presented as the mean ± S.E.M. and asterisks indicate statistically significant differences (p < 0.05; n = 3).
survival (Fig. 3B). Collectively, these results show that the PI3K/Akt signaling pathway mediates the majority of the effects of p62 on neuronal cell survival under oxidative stress. To obtain further insight into the relative role of p62 in the regulation of Akt activity, myc-tagged PDK1 cDNA was transfected into SH-SY5Y cells along with p62Ad infection. Akt phosphorylation induced by p62 was further augmented in cells transfected with wild-type PDK1, whereas the expression of the kinase-inactive form of PDK1 (PDK KI) reduced Akt phosphorylation in p62Ad-infected cells during H2 O2 treatment (Fig. 4A). Furthermore, the results of the MTT assay indicated that p62 synergistically enhances PDK1mediated cell survival during oxidative insult (Fig. 4B). In contrast, the expression of PDK KI blocked the cytoprotective effects of p62. This result indicates that p62 exerts its protective effects by promoting the activation of Akt in cooperation with PDK1 during oxidative stress-induced neuronal cell death. However, PDK KI did not completely block the effects of p62, which implies that other signaling pathways are affected by p62. We also observed an increase in MAPK activation caused by p62 overexpression during H2 O2 treatment (data not shown). PDK1 is a ubiquitously expressed Ser/Thr kinase that is phosphorylated on five serine residues in vivo. Ser 241, which serves
Fig. 4. Synergistic effects on cell survival by p62 and PDK1 in H2 O2 -treated SH-SY5Y cells. (A) SH-SY5Y cells in a 100-mm plate were transfected with myc-tagged PDK1 cDNA along with adenovirus infection as indicated. Cells were differentiated with RA and then treated with 750 M H2 O2 for 3 h. Blots were checked for p-Akt (T308), stripped and re-probed for unphosphorylated Akt. The expression of transferred PDK1, p62, or LacZ was also confirmed via immunoblotting using anti-Myc, anti-T7 or -galactosidase antibodies, respectively. (B) Cells were treated as similar way as (A) except cells were plated in 96-well plates, and cell viability was measured by MTT assay 72 h after H2 O2 treatment. All data are presented as the mean ± S.E.M. and asterisks indicate statistically significant differences (p < 0.05; n = 3). (C) The dissociation of PDK1 from 14-3-3 in the presence of p62 during H2 O2 treatment. The same cell extracts used in (A) were immunoprecipitated with anti-14-3-3 antibody, and the bound proteins were analyzed via immunoblotting using an antibody against Myc (expressed PDK1 protein). The blot was then stripped and re-probed with antiT7 (expressed p62 protein) and anti-14-3-3 antibodies. 30 g of total cell lysates were also analyzed for the amount of 14-3-3 (total lysate).
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as a PDK1 phosphorylation site, appears to be critical for the activity of PDK1 [3]. It has been reported that the binding of 143-3 to PDK1 through Ser 241 suppresses PDK1 phosphorylation [26]. Of particular interest is the fact that 14-3-3 was identified as a p62-interacting protein in a yeast two-hybrid study (data not shown). This prompted us to hypothesize that p62 modulates PDK1 activity by affecting the association between PDK1 and 14-3-3. Co-immunoprecipitation demonstrated a physical association between PDK1 and 14-3-3 in differentiated SH-SY5Y cells, and this interaction was increased during H2 O2 treatment in the absence of p62 overexpression. However, the association between these proteins was decreased when p62 was expressed (Fig. 4C). Moreover, the reduction in the amount of PDK1 associated with 14-3-3 was correlated with the increase in the amount of 143-3-bound p62 protein in the H2 O2 -treated cells, implying that p62 disrupts the association between 14-3-3 and PDK1 by binding 14-3-3. In this report, we determined that p62 expression was induced by H2 O2 in differentiated neuronal cells. In addition, the results of the MTT assay demonstrated that p62 overexpression increased cell survival during H2 O2 treatment. This result is consistent with the previous finding demonstrating that the expression of A170, murine counterpart of p62 is under the control of Nrf2 transcription factor, a general regulator of reactive oxygen species [11]. This also gives credence to the notion that p62 exerts an anti-apoptotic effect that can be induced by a variety of stimuli [12,14]. Furthermore, we demonstrated that p62 is a mediator that regulates Akt phosphorylation in cooperation with PDK1, a kinase responsible for the phosphorylation of Akt on Thr 308, which is required for Akt activation. In the present study, we present evidence that p62 is involved in a mechanism by which the physical association between 14-3-3 and PDK1 is altered to increase Akt phosphorylation. The 14-3-3 protein has been implicated in both pro- and antiapoptotic pathways. The 14-3-3 protein enhances cell survival by negatively regulating pro-apoptotic proteins, including Bad and Bax, in a phospho-dependent or -independent manner [23,28]. On the other hand, Sato et al. showed that the association between 14-3-3 and PDK1 reduced the activity of PDK1 kinase both in vivo and in vitro, suggesting that 14-3-3 negatively regulates PDK1 by forming a complex [26]. This result is consistent with our data demonstrating a reduction in the interaction between PDK1 and 14-3-3 and increased Akt phosphorylation in p62-expressing cells. Although we did not directly assess the putative role of p62 in the activation of PDK1 upon apoptotic stimuli, the results of our studies collectively showed that p62 overexpression induced the selective release of PDK1 from 14-3-3, which is associated with increased cell survival. The data presented herein strongly indicate that, in addition to its function as a scaffold protein in the formation of aggregates that facilitate the binding between ubiquitin and other neurodegenerative disease-associated proteins to promote cell survival [19,24], p62 exerts a cytoprotective effect by modulating signaling pathways in neuronal cells under oxidative stress. In our previous study, we demonstrated that p62 also induces the dissociation of Akt from PKC, which negatively affects the activity of Akt during the neuronal cell death induced by serum removal [14]. Collectively, the data suggest that p62 releases both PDK1 and Akt by disrupting their binding to 14-3-3 and PKC, respectively, and thus enhances survival signaling. Further studies are needed to delineate the detailed mechanisms by which Akt activity is modulated by p62 in cooperation with other proteins during oxidative stress-induced neuronal cell death. An understanding of the physical interplay between p62, PDK1, 14-3-3, and probably PKC is likely to be of significant value for developing novel strategies to control cell growth, survival, and death.
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Acknowledgements This work was supported by a grant from the Korea Research 276 Foundation to IJ (KRF 2006-C00157) and by the funds from the Korea Health 21R&D Project (A04-0018-AY1204-07M4-00020B) by MOHW and the Brain Research Cen- 277 ter (M103KV01000706K2201-00710) of the 21C Frontier Research 278 program by MOST and to Y.K.K. References [1] N.A. Bourbon, L. Sandirasegarane, M. Kester, Ceramide-induced inhibition of Akt mediated through protein kinase C, J. Biol. Chem. 277 (2002) 3286–3292. [2] A. Brunet, S.R. Datta, M.E. Greenberg, Transcription-dependent and independent control of neuronal survival by the PI3K-Akt signaling pathway, Curr. Opin. Neurobiol. 11 (2001) 297–305. [3] A. Casamayor, N.A. Morrice, D.R. Alessi, Phosphorylation of Ser-241 is essential for the activity of 3-phosphoinositide-dependent protein kinase-1: identification of five sites of phosphorylation in vivo, Biochem. J. 342 (1999) 287–292. [4] P.H. 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