Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3β signaling

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Zinc induces cell death in immortalized embryonic hippocampal cells via activation of Akt-GSK-3β signaling Young Kyu Min b , Jong Eun Lee c , Kwang Chul Chung a,⁎ a

Department of Biology, College of Sciences, Yonsei University, Seoul 120-749, Republic of Korea Department of Medical Science, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea c Anatomy, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Zinc is an essential catalytic and structural element of many proteins and a signaling

Received 21 August 2006

messenger that is released by neuronal activity at many central excitatory synapses.

Revised version received

Excessive synaptic release of zinc followed by entry into vulnerable neurons contributes

4 October 2006

severe neuronal cell death. We have previously observed that zinc-induced neuronal cell

Accepted 16 October 2006

death is accompanied by Akt activation in embryonic hippocampal progenitor (H19-7) cells.

Available online 25 October 2006

In the present study, we examined the role of Akt activation and its downstream signaling events during extracellular zinc-induced neuronal cell death. Treatment of H19-7 cells with

Keywords:

10 μM of zinc plus zinc ionophore, pyrithione, led to increased phosphorylation of Akt at Ser-

Zinc

473/Thr-308 and increased Akt kinase activity. Zinc-induced Akt activation was

Neuronal cell death

accompanied by increased Tyr-phosphorylated GSK-3β as well as increased GSK-3β

Akt

kinase activity. Transient overexpression of a kinase-deficient Akt mutant remarkably

Protein kinase B

suppressed GSK-3β activation and cell death. Furthermore, tau phosphorylation, but not the

Phosphatidylinositol-3-kinase

degradation of β-catenin, was dependent upon zinc-induced GSK-3β activation and

Glycogen synthase kinase-3β

contributed to cell death. The current data suggest that, following exposure to zinc, the

Tau

sequential activation of Akt and GSK-3β plays an important role directing hippocampal

β-catenin

neural precursor cell death. © 2006 Elsevier Inc. All rights reserved.

Introduction Zinc is one of the most abundant transition metals in the brain [1,2]. Most brain zinc is tightly bound to or sequestered in cellular compartments. Upon stimulation of Zn2+-containing pathways, zinc is released from presynaptic terminals [3]. Zinc is potentially neurotoxic in vitro. Furthermore, it appears likely that zinc movement from pre- to postsynaptic neurons contributes to the selective nerve cell injury observed in cerebral ischemia, epilepsy, and brain trauma [2]. Zn2+ accumulation in neurons strongly corresponds with neuronal

damage, suggesting a role for zinc in cellular injury. Nevertheless, the mechanisms through which intracellular Zn2+ promotes cell death are not well understood. H19-7 cell line was generated from early E17 rat hippocampal cells, which provide a neuronal differentiation model for central nervous system neurons [4,5]. These cells have been conditionally immortalized by expression of a temperature-sensitive simian virus 40 large T antigen. Thus, the H197 cells offer the advantage of temporary immortalization which can be effectively reversed to yield cells in a more normal state. Furthermore, H19-7 cells respond differentially

⁎ Corresponding author. Fax: +82 2 312 5657. E-mail address: [email protected] (K. Chul Chung). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.10.013

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to EGF and FGF. At the permissive temperature (33°C), EGF treatment induces proliferation; at the nonpermissive temperature (39°C), addition of FGF or phorbol 12,13-dibutyrate but not EGF induces differentiation. The differentiated hippocampal cells do not respond mitogenically to serum, but they express neuronal markers such as neurofilaments and brain type II sodium channels and display action potentials [4,6]. Thus, H19-7 cells provide a good model system for testing the role of a variety of signaling molecules in neuronal differentiation. The serine–threonine kinase Akt, which is also referred to as protein kinase B (PKB), promotes viability in various cell types [7,8]. Activation of Akt/PKB involves the binding of phosphoinositide 3-kinase (PI-3K)-generated phosphoinositides to Akt via its pleckstrin homology (PH) domain (amino acids 1–114) [9]. PI-3K-dependent activation of Akt also involves 3′-phosphoinositide-dependent kinase-1-mediated phosphorylation of Thr-308, leading to the auto-phosphorylation of Ser-473 [10]. Akt prevents apoptosis by inactivating several targets, including BAD [9], glycogen synthase kinase-3 (GSK-3) [11,12], forkhead transcription factors, and caspase-9 [13]. Additionally, Akt has been reported to promote cell survival via NF-κB-dependent expression of anti-apoptotic genes such as FLIP, as well as other inhibitors of apoptosis. Furthermore, Akt has been shown to suppress activation of mitochondrial apoptotic pathways [14–16]. GSK-3β, a kinase that is abundant in brain and widely distributed in neurons in vivo, has been identified a target for Akt [11]. GSK-3β has also been directly implicated in the regulation of apoptosis. Overexpression of catalytically active GSK-3β induces apoptosis in NGF-differentiated PC12 cells [17]. Activated GSK-3β leads to hyper-phosphorylation of tau [18] and degradation of βcatenin during neuronal cell death [19]. Contrary to the well-known function of Akt in cell survival, there were several reports demonstrating its involvement mediating cytotoxicity and cell death signals. For example, ligation of Fas receptor to Fas ligand induces tyrosine phosphorylation and activates PI-3K/Akt, which is required for Fas-mediated cell death [20]. In addition, induced expression of Akt increases Fas transcription [21], suggesting that Akt positively regulates Fas death signaling. We have previously reported that pyrrolidine dithiocarbamate (PDTC)-mediated accumulation of intracellular zinc causes cell death by modulating several intracellular signaling pathways in neuronal hippocampal progenitor (H19-7) cells [22]. Many protein kinases, including PI-3K, Akt, casein kinase 2 (CK2), c-Jun N-terminal kinase (JNK), and IκB kinase (IKK), are activated by PDTC [22]. The mechanism by which Akt activation may promote cell death is unclear. Here, we demonstrate that activation of Akt and its downstream substrate, GSK-3β, plays an important role mediating zinc-induced death in H19-7 cells.

Materials and methods Materials Fetal bovine serum, Dulbecco's modified Eagle's medium, and geneticin were from Invitrogen (Grand Island, NY) and

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[γ-32P]ATP from Perkin Elmer Life Sciences (Boston, MA). LY294002 and wortmannin were purchased from Calbiochem (La Jolla, CA). Anti-Akt, anti-GSK-3β, anti-phospho-Akt, and anti-phospho-GSK-3β antibodies were purchased from Cell Signaling Biotechnology (Beverly, MA). Anti-sera against tau, β-catenin, phospho-tau, and phospho-β-catenin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Hemaglutinin (HA)-tagged kinase-dead Akt with a K179M mutation cloned into pCMV5 (pHA-KD-Akt) was a kind gift from T.F. Franke (Columbia University College of Physicians and Surgeons, New York, NY). The HA-tagged non-myristoylated Akt mutant lacking a PH domain (Δ4–129), which was cloned into pCMV (pHA-A2-Myr-Akt), was generously provided by R.A. Roth (Stanford University, Stanford, CA). Plasmids encoding kinase-dead GSK-3β with amino acid substitutions at the ATP binding sites (K85M and K86I; KDGSK-3β) and its dominant-negative mutant with S9E mutation (GSK-3β-S9E) were provided by J. Sadoshima (Pennsylvania State University College of Medicine, Danville, PA) and E. Krebs (University of Washington, Seattle, WA), respectively. Plasmids encoding wild-type tau and its C-terminal deletion mutant were provided by Y.K. Jung (Seoul National University). DNA constructs to express wild-type (WT-cat) and dephosphorylated β-catenin (DP-cat) were kind gifts from K.Y. Choi (Yonsei University). DP-cat is characterized by the mutation of the five serine residues in the N-terminal 132 amino acids to alanines. All other chemicals used were of analytical grade and were obtained from Sigma.

Cell culture and DNA transfection Immortalized hippocampal progenitor H19-7 cells were grown and maintained as described previously [22]. Transient transfections were performed using LipofectAMINE reagents (Invitrogen) according to the manufacturer's protocol.

Assessment of cell death For the tetrazolium extraction assay, 62.5 μl of a 5 mg/ml 3, (4,5-dimethyldiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) stock solution was added to each well in 24-well plate, which contained 250 μl of media. After 2 h of incubation at 37°C, 250 μl of extraction buffer containing 20% SDS and 50% N,N-dimethylformamide, pH 7.4 was added. After an overnight incubation at 37°C, the optical density at 570 nm was measured using a VERSA MAX enzyme-linked immunosorbent assay reader (Molecular Devices), employing extraction buffer as the blank. For the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay, cells were seeded overnight at 70% confluence onto coverslips in six-well dishes. Cultures were then transfected with the appropriate plasmids the following day for 24 h and treated with zinc for 1 h. Apoptosis was analyzed using an in situ cell death detection kit, TMR-red, based on TUNEL technology (Roche). Statistical analyses were performed with the aid of StatView II program (Abacus Concepts). Data were analyzed by one-way analysis of variance. Preplanned comparisons with controls were performed using the Dunnett's t-test.

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Western blot analysis Western blot analysis was performed by using antibodies against either phosphorylated proteins, such as phospho-Akt (New England Biolab), phospho-GSK-3β, and phospho-Tau (Thr-212 and Ser-400) (Cell Signaling, Beverly, MA), or against non-phosphorylated proteins, such as Akt (Promega, Madison, WI), GSK-3β, Tau, and β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA), as described elsewhere [22]. Endogenous total and modified proteins were visualized by enhanced chemiluminescence (Amersham, Buckinghamshire, United Kingdom).

Akt activity assay The activity of Akt kinase was measured in vitro as described previously [23]. Confluent cells were harvested using buffer A containing 20 mM Tris, pH 7.9, 137 mM NaCl, 5 mM Na2EDTA, 10% glycerol, 1.0% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 2 μg/ml leupeptin, 1 mM Na3VO4, 1 mM EGTA, 10 mM NaF, 1 mM tetrasodium pyrophosphate, and 1 mM β-glycerophosphate. The soluble cell lysate fraction was incubated for 2 h at 4°C with antibodies against Akt. The reaction mixture was incubated for an additional 2 h at 4°C in the presence of protein A– Sepharose beads (Pharmacia, Piscataway, NJ) and then rinsed with buffer A. Immunocomplex kinase assays were performed by incubating the immunopellets for 30 min at 30°C with recombinant GST-CREB fusion proteins in the reaction buffer containing 0.2 mM sodium orthovanadate, 2 mM DTT, 10 mM MgCl2, 2 μCi [γ-32P]ATP, and 20 mM HEPES, pH 7.4. After reaction termination, the mixtures were subjected to 12.5% SDS-PAGE. The phosphorylated substrates were visualized by autoradiography. Bacterial GST-fusion proteins were prepared with a GST purification module (Pharmacia) according to the manufacturer's protocols.

and 380 nm, and the emission fluorescence was recorded at 510 nm. The values of [Zn2+]i were calculated using a modification of the equation described by Grynkiewicz et al. [25].

Results Zinc/pyrithione triggers death in embryonic hippocampal progenitor cells via activation of PI-3K and Akt signaling We have previously observed that the sequential activation of PI-3K and Akt occurs during PDTC-induced neuronal cell death in H19-7 cells [22]. To confirm this phenomenon, cells were treated with zinc plus a zinc-specific ionophore, pyrithione, which has been shown to increase the intracellular transport of zinc ions [26]. As shown in Fig. 1A, the combined application of zinc plus pyrithione caused the cell death in a dose dependent manner, reaching maximum level at 10 μM of zinc. However, when the cells were treated with zinc or

Glycogen synthase kinase-3β assay The activity of GSK-3β was measured by using a primed substrate, phosphoglycogen synthase peptide-2 (Upstate Biotechnology), as described elsewhere [17].

Measurement of intracellular Zn2+ Intracellular Zn2+ concentration ([Zn2+]i) was measured by using the method described previously in detail [24]. Briefly, H19-7 were loaded with magfura-2 at 33°C in a 5% CO2 incubator by including 3 μM magfura-2AM for 20 min in a HCO−3-buffered solution containing 110 mM NaCl, 4.5 mM KCl, 1 mM NaH2PO4, 1 mM MgSO4, 1.5 mM CaCl2, 5 mM HEPES–Na, 5 mM HEPES free acid, 25 mM NaHCO3, and 10 mM D-glucose (pH 7.4). Cells were then rinsed twice and incubated in the HCO−3-buffered solution for at least 20 min before use. The [Zn2+]i was measured on the stage of an inverted microscope (Nikon, Tokyo, Japan) by spectrofluorometry (Photon Technology International, Brunswick, NJ, USA), while cells were superfused at a constant perfusion rate of 2 ml/min with the HCO−3-buffered solution equilibrated with 95% O2, 5% CO2 to maintain a pH of 7.4. All experiments were performed at 33°C. The excitation wavelength was alternated between 340

Fig. 1 – Induction of cell death by zinc plus pyrithione in embryonic hippocampal progenitor H19-7 cells. (A) Where specified, H19-7 cells were cultured either with the indicated concentrations of zinc sulfate in DMEM containing 10% FBS and 10 μM pyrithione for 24 h. Cell viability was measured by MTT extraction assay. Viability of the cells in regular serum media was defined as 100%, and values are means ± SD (n = 3). (B) Changes in [Zn2+]i were measured in magfura-2 loaded cells using ratiometric fluorescence recording techniques. Cells were exposed to 10 μM pyrithione, 10 μM Zn2+, or 10 μM pyrithione in the presence of 10 μM Zn2+. The TPEN (10 μM) was added at the indicated time to chelate Zn2+ when cells were exposed to Zn2+ followed by pyrithione. The result is representative of three independent experiments.

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pyrithione alone, the significant occurrence of cell death has not been observed. Furthermore, stimulation with 10 μM Zn2+ plus 10 μM pyrithione (Zn2+ pyrithione) caused the increase of [Zn2+]i levels without depolarizing the cells (Fig. 1B). The fluorescence ratio began to increase immediately after the addition of Zn2+ pyrithione and reached a plateau within 20 min. Application of 10 μM TPEN, a membrane permeable Zn2+-specific chelator, promptly decreased the fluorescence ratio to the basal level (Fig. 1B). However, 10 μM Zn2+ or 10 μM pyrithione alone did not affect the fluorescence ratio in magfura-2-loaded H19-7 cells (Fig. 1B). This result indicated that, although magfura-2 could detect Ca2+ and Mg2+, the increased fluorescence ratio caused by combined application of Zn2+ pyrithione resulted solely from the influx of Zn2+. The elevated [Zn2+]i achieved by applying Zn2+ pyrithione was calculated to be 165 ± 12 nm (mean ± SEM, n = 3). However, we do not rule out the possibility that the value may be slightly underestimated because the high-affinity Zn2+ probe magfura-2 (Kd = 20 nM) was used [24]. Then, the activation of Akt was assessed by detecting Akt phosphorylation at Thr-308 and Ser-473. As shown in Fig. 2A, the phosphorylation of Akt at both of Ser-308 and Thr-473 residues increased after the addition of Zn2+ pyrithione and maintained for at least 6 h. In addition, we measured the kinase activity of Akt in response to intracellular zinc

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accumulation. Based on a previous report that Akt overexpression potently induces Ser-133 phosphorylation of CREB and promotes the recruitment of CBP [27], cells were transiently transfected with Akt followed by the stimulation with zinc plus pyrithione, and Akt activity was assayed. As shown in Fig. 2B, the phosphorylation of GST-CREB by anti-Akt IgG-complexes increased slightly 10 min after zinc addition. Thereafter, GST-CREB phosphorylation was significantly enhanced, reaching its maximum level at 2 h. This phosphorylation was maintained for at least another hour. As a negative control, GST was used as a substrate. GST was not phosphorylated at all by anti-Akt immunoprecipitates. To achieve Akt activation, the PI-3K phosphoinositide products are thought to recruit Akt and 3-phosphoinositidedependent protein kinase-1 (PDK1) to the plasma membrane. This recruitment occurs because both Akt and PDK1 contain PH domains, which have a high affinity for phosphoinositide-3,4-biphosphate and phosphoinositide3,4,5-triphosphate. At the membrane, PDK1 and an unidentified PDK2 phosphorylate two residues on Akt (Thr-308 and Ser-473), fully activating it [28]. Based on these findings, the dominant-negative Akt mutants were constructed by either point mutation of two Ser-308/Thr-473 residues to alanines (KD-Akt) or deletion of Akt PH domain (A2-myr-Akt). Transfection of H19-7 cells with the kinase-

Fig. 2 – Zinc-induced Akt activation promotes apoptosis in H19-7 cells. Immortalized hippocampal progenitor H19-7 cells were incubated with zinc sulfate (10 μM) plus pyrithione (10 μM) for the indicated times. (A) Western blot analysis of total and Thr-308 and Ser-473 phosphorylated Akt was performed, as indicated. (B) Alternatively, Akt activity was assayed using either GST-CREB or GST as a substrate. (C) Where indicated, cells were mock-transfected or transfected with HA-tagged kinase-inactive (HA-KD-Akt) or non-myristoylated Akt mutant (HA-A2-Myr-Akt) for 24 h, treated with zinc plus pyrithione for 2 h, and Akt activity was assayed. The proper expression of endogenous Akt and its transfected mutants was confirmed by western blotting with anti-HA and Akt IgG. (D) Where indicated, cells were mock-transfected or transfected with either KD-Akt or A2-myr-Akt for 24 h and treated with zinc and pyrithione for 12 h. Cell viability and apoptosis were measured by the MTT extraction method and TUNEL assay, respectively. All values are expressed as means ± SD. (E) Cells were stimulated with zinc sulfate plus pyrithione for 12 h in the absence or presence of LY294002 (25 μM) or wortmannin (100 nM), and cell viability was measured.

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deficient or myristoylation-defective Akt mutant led to significantly reduced CREB phosphorylation by anti-Akt immunocomplexes (Fig. 2C). To further assess the functional role of zinc-induced Akt activation, cells were mock-transfected or transfected with one of the dominant-negative Akt plasmids. H19-7 cell viability was measured following stimulation with zinc plus pyrithione and compared in the absence or presence of Akt mutants. As shown in Fig. 2D, the addition of zinc and pyrithione resulted in a time-dependent decrease in cell viability, with a maximal loss in viability (50%) occurring after 24 h. In contrast, overexpression of kinase-dead or nonmyristoylated Akt mutant significantly preserved cell viability following zinc stimulation, when compared with mocktransfected control cells (Fig. 2D). Using TUNEL-positive quantitation of apoptotic cell number, we observed the same extent of the rescue from apoptotic cell death in cells transfected with Akt mutants (Fig. 2D). The addition of the specific PI-3K inhibitors, LY294002 or wortmannin, also suppressed zinc-induced cell death to a similar extent (Fig. 2E). LY294002 or wortmannin alone did not result in significant cell death (data not shown). These results demonstrate that zinc promotes pro-apoptotic Akt activation. Furthermore, this occurs in a PI-3K-dependent manner.

Zinc/pyrithione induces GSK-3β phosphorylation and activation To determine whether signaling cascades downstream of Akt are modulated by zinc in H19-7 cells, we examined activation of GSK-3α/β. GSK-3α and β become inactivated by phosphorylation of Ser-21 and Ser-9, respectively. Conversely, dephosphorylation or mutation of these sites results in activation of the kinase [29]. Several kinases, including Akt, can phosphorylate those serine residues and thus negatively regulate GSK3β activity. However, phosphorylation of GSK-3β at tyrosine 216 by an unknown tyrosine kinase can increase its activity [30,31]. As shown in Fig. 3A, western blot analysis revealed that zinc/pyrithione stimulation induced phosphorylation of Ser-21 in GSK-3α and Ser-9 in GSK-3β. Paradoxically, zinc-induced Ser-9 GSK-3β phosphorylation was associated with remarkably enhanced GSK-3β activity (Fig. 3B). To begin the delineation of the differences between conventional anti-apoptotic Akt signaling and novel proapoptotic Akt signaling, we then measured the levels of tyrosine-phosphorylated GSK-3β. To do this, GSK-3β immunoprecipitates were subjected to western blot analysis using anti-phosphotyrosine antibodies. As shown in Fig. 3C, GSK-3β tyrosine phosphorylation significantly increased within 5 min of zinc/pyrithione stimulation and was sustained for at least 30 min. To assess the effect of GSK-3β phosphorylation on H19-7 cell viability, the effects of kinase-dead or dominant-negative GSK-3β mutant were examined. When compared with mocktransfected cells, the overexpression of these two functionally defective GSK-3β mutants significantly inhibited the occurrence of zinc-induced cell death (Fig. 4). This suggests that the zinc-induced cytotoxic signals are relayed through Akt and GSK-3β. Consequently, the activation of these proteins promotes the cell death.

Fig. 3 – Zinc induces Ser and Tyr phosphorylation of GSK-3α/β and enhances GSK-3α/β activity. (A) Where specified, H19-7 cells were stimulated with Zn2+ plus pyrithione for the indicated times. Western blot analysis of whole cell extracts was performed with anti-phospho-GSK-3α/β (Ser-21/Ser-9) or anti-GSK-3β antibodies. (B) Cells were treated with zinc and pyrithione for the indicated times, and GSK-3β activity was measured using synthetic phosphoglycogen synthase peptide-2 as a substrate. (C) Where specified, total cell lysates were immunoprecipitated with anti-GSK-3β antibody, and immunoprecipitates were subjected to western blot analysis of phosphotyrosine (PY20) and GSK-3β.

Akt stimulates the activation of GSK-3β during zinc/pyrithione-induced cell death Akt may mediate the phosphorylation of GSK-3β at critical serine and tyrosine residues. To test this, H19-7 cells were transfected with plasmids carrying kinase-dead or myristoylation-resistant Akt mutations. As shown in Fig. 5A, the overexpression of these two dominant-negative Akt mutants significantly blocked the phosphorylation of Ser-21 and Ser-9 in GSK-3α and GSK-3β, respectively. Similarly, GSK-3β tyrosine phosphorylation and GSK-3β activity were significantly blocked by the transient expression of the kinase-dead or nonmyristoylated Akt mutant proteins (Figs. 5B and C). LY294002 also suppressed GSK-3β phosphorylation at Ser and Tyr residues (Figs. 5A and B). These data indicate that the zincinduced pro-apoptotic signals transmit through Akt and GSK3β, which then lead to their activation.

Zinc/pyrithione-induced tau phosphorylation is mediated by GSK-3β and contributes to cell death The microtubule-associated protein tau is known to be phosphorylated by GSK-3β [32]. Tau hyper-phosphorylation by GSK-3β is followed by increased formation of neurofibrillary tangles. In addition, the chronic phosphorylation of tau

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Fig. 4 – GSK-3β activation contributes zinc-induced cell death. Where specified, cells were mock-transfected or transfected with kinase-dead GSK-3β (KD-G3b) or GSK-3β-S9E (G3b-S9E) for 24 h. Cells were then treated with zinc and pyrithione for 12 h, and apoptosis was measured by TUNEL assay. All values are expressed as means ± SD.

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transfected with wild-type GSK-3β (Fig. 7B). To further assess the contribution of β-catenin processing to zinc-induced cell death, cells were transfected with plasmid encoding either wild-type β-catenin or a non-cleavable mutant protein (Fig. 7C). As shown in Fig. 7D, when compared with mocktransfected control cells, transfection with the dephosphorylated β-catenin mutant (DP-cat) did not significantly affect cell death. In addition, overexpression of wild-type β-catenin only slightly blocked cell death, although this effect was not statistically significant (Fig. 7D). Taken together, a possible role of Akt activity during extracellular zinc ion-induced neuronal cell death could be hypothesized to modulate several signal transduction pathways involving the activity of GSK-3β and phosphorylation of tau, but not degradation of β-catenin.

induces the enhanced formation of neurotoxic tau aggregates [33]. Furthermore, autopsies of Alzheimer's patients reveal that the neurofibillary tangles contain a high percentage of phosphorylated tau. Based on the finding that GSK-3β is activated during zinc-induced neuronal cell death, we examined whether zinc stimulation is associated with tau phosphorylation in H19-7 cells. Western blot analysis revealed that the addition of zinc results in a time-dependent increase in Thr-212 and Ser-400 phosphorylated tau. This increase was maintained for at least 6 h (Fig. 6A). To determine whether this tau phosphorylation was mediated by active GSK-3β, cells were transiently transfected with either kinase-deficient GSK3β or its dominant-negative mutant. Cells transfected with the mutant proteins exhibited significantly reduced zinc-induced tau phosphorylation, compared with mock-transfected control cells. This indicates that zinc-induced tau hyper-phosphorylation is actually mediated by GSK-3β (Fig. 6C). The effect of tau phosphorylation on cell viability was also examined after cells were transfected with either wild-type tau or a deletion mutant. The deletion mutant is lacking the Cterminal 20 amino acids, which includes an additional GSK3β target, Ser-404, and a caspase-3 cleavage site. Cultures transfected with wild-type tau exhibited 20% increase in apoptosis in comparison to mock-transfected cultures (Fig. 6D). In contrast, when cells were transfected with the tau deletion mutant, the occurrence of zinc-induced apoptosis decreased considerably (Fig. 6D).

Zinc induces the degradation of β-catenin, which occurs independent of GSK-3β activity β-catenin is one of multiple GSK-3β downstream targets [19]. Upon the phosphorylation by GSK-3β, β-catenin becomes cleaved and processed via proteasomal machinery [34]. We examined whether the processing of β-catenin occurs during zinc-induced H19-7 cell death. As shown in Fig. 7A, the cleavage of β-catenin and the formation of its fragments were apparent 12 h after zinc stimulation. However, ectopic expression of kinase-defective GSK-3β or dominant-negative mutant did not affect zinc-induced cleavage of β-catenin, when compared with mock-transfected control cells or cells

Fig. 5 – Zinc-induced GSK-3β activation occurs through a PI-3K/Akt-dependent pathway. (A) Cells were pretreated with LY294002 for 2 h (left panel). Alternatively, cells were mock-transfected or transfected with KD-Akt or A2-myr-Akt for 24 h (right panel). The cells were then left untreated or added with zinc plus pyrithione for 2 h. The levels of total and Ser-9 phosphorylated GSK-3β were measured by western blotting, as indicated. (B) Cells were treated as above, and lysates were subjected to western blot analysis of tyrosine-phosphorylated GSK-3β. (C) Where specified, cells were either mock-transfected (●) or transfected with KD-Akt (○) or A2-myr-Akt (▼) for 24 h. The cells were treated with zinc and pyrithione for the indicated times, and GSK-3β activity was measured.

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Discussion Zinc is a signaling messenger that is released by neuronal activity at many central excitatory synapses. Excessive

Fig. 6 – Zinc treatment leads to GSK-3β-mediated tau phosphorylation in H19-7 cells. (A) H19-7 cells were stimulated with Zn2+ plus pyrithione for the indicated times. Whole cell extracts were subjected to western blot analysis of total, Thr-212-, and Ser-400-phosphorylated tau. (B) Cells were mock-transfected or transfected with kinase-dead-GSK-3β or S9E-GSK-3β for 24 h, as indicated. Cells were then left untreated or stimulated with zinc plus pyrithione for the indicated times. Whole cell extracts were analyzed for total, Thr-212-, and Ser-400-phosphorylated tau. (C) Schematic diagram showing Tau constructs: wt, wild-type Tau protein containing 441 amino acids; Tau-1, Tau proteins lacking the C-terminus spanning residues 421–441. MT binding repeats is denoted as four black blocks near the carboxyl terminal of tau. (D) Cells were mock-transfected or transfected with wild-type tau or Tau-1 for 24 h, as indicated. The cells were then treated with zinc plus pyrithione for 12 h, and apoptosis was measured by TUNEL assay.

Fig. 7 – Zinc induces the degradation of β-catenin, which occurs independently of GSK-3β activity. (A) H19-7 cells were incubated with zinc sulfate plus pyrithione for the indicated times. β-catenin degradation was monitored by western blotting. (B) Where specified, cells were mock-transfected or transfected with wild-type GSK-3β or its kinase-dead or KDS9E mutant for 24 h. Cells were then left untreated or stimulated with zinc plus pyrithione for 12 h. Whole cell lysates were subjected to western blot analysis of β-catenin. (C) Schematic diagram of the structure of wild-type and mutant forms of β-catenin with individual domains highlighted. The numbers indicate the amino acid positions in β-catenin. In the DP mutant form of β-catenin, the Ser/Thr residues in the amino terminus, which are phosphorylated and regulate protein stability, have been substituted. The amino acid changes, indicated by asterisks, are S33A, S37A, T40A, S45A, and S47A. (D) Where specified, cells were mock-transfected or transfected with wild-type β-catenin or DP-β-catenin for 24 h. Cells were then treated with zinc plus pyrithione for 12 h, and apoptosis was measured by TUNEL assay.

synaptic release of zinc and subsequent uptake into vulnerable neurons contribute to severe neuronal cell death. We have previously shown that the intracellular transport of zinc ions into hippocampal progenitor cells leads to cell death via zinc-mediated activation of several intracellular signaling

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pathways, including NF-κB [35]. We have also demonstrated that the addition of zinc triggers activation of IKK, CK2, PI-3K, Akt, and JNK [22]. Further examination of possible ‘cross-talk’ between these protein kinases revealed that Akt and CK2, which are present upstream of JNK, act independently and directly regulate cell viability in response to zinc [23]. In the present study, we have extended these findings regarding the role of Akt in zinc-induced H19-7 cell death. Contrary to its well-known, anti-apoptotic role, Akt promoted the cytotoxicity of zinc in H19-7 cells, as confirmed by overexpression of kinase-dead or non-myristoylated Akt mutants. Consistent with our findings, Kim et al. have shown that PI-3K and Akt are activated by zinc in Swiss 3T3 cells [36]. In addition, PI-3K has been shown to be involved in zinc-induced JNK pathway in primary cortical neurons and HNN neuroblastoma cells [37]. Although Akt is generally known to act as an important mediator of metabolic, as well as survival responses to growth factors [38], there were several reports of Akt activation during cell death and of a pro-death role for Akt. For example, peroxynitrite, a potent oxidizing and nitrating species, induces apoptosis through unknown mechanisms and is believed to interfere with receptor tyrosine kinase signaling through nitration of tyrosine residues. Klotz and co-workers demonstrated that administration of peroxynitrite to primary human skin fibroblasts results in a dose- and time-dependent activation of Akt followed by the phosphorylation of GSK-3 [39]. These findings suggest that Akt activation occurs as an acute response to peroxynitrite treatment and could play an important role in influencing cell survival and/or in altering cellular responses to other growth regulatory signals. PI-3K/ Akt has also been implicated in the regulation of pro-apoptotic signals in the death receptor pathways. Ligation of Fas receptor in Jurkat T lymphocytes induces tyrosine phosphorylation and activates PI-3K/Akt, which is required for Fasmediated cell death [20]. Moreover, induced expression of Akt or p110, the catalytic subunit of PI-3K, increases Fas transcription [21], suggesting that PI-3K/Akt positively regulates Fas death signaling. Lu et al. recently reported that Akt positively regulates Fas-mediated apoptosis in epidermal CI41 cells through a mechanism that involves transcriptional activation of Fas receptor [40]. Akt is also activated in response to oxidative stress or other stressors known to exert their cytotoxic effects through generation of reactive oxygen species or perturbation in cellular redox status [41,42]. GSK-3β was initially identified as an enzyme that inhibits glycogen synthesis through phosphorylation of glycogen synthase. Many studies have shown that GSK-3β regulates a wide range of cellular functions, including development, gene expression, cytoskeletal organization, protein translation, cell cycle regulation, and apoptosis [43]. The activation of GSK-3β has been shown to promote apoptosis in a remarkably wide variety of conditions, such as trophic factor withdrawal, PI-3K inhibition, and toxicity induced by ceramide, platelet activating factor, heat shock, and mitochondrial toxins [44]. Our results reveal that GSK-3β activation plays a critical role in zinc-induced neurotoxicity. In contrast to many protein kinases, GSK-3β is catalytically active even in unstimulated cells and becomes inactivated by phosphorylation at Ser-9. Dephosphorylation of this site or mutations that prevent phosphorylation result in activation of the kinase. Several

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protein kinases including Akt, protein kinase A, protein kinase C, integrin-linked kinase, and p90Rsk kinase, negatively regulate GSK-3β activity through serine phosphorylation [29]. Conversely, phosphorylation of GSK-3β at Tyr-216 enhances GSK-3β activity; however, the identity of the tyrosine kinase mediating Tyr-216 phosphorylation is unknown [30,31]. Our data demonstrate that GSK-3β is activated via the phosphorylation of its tyrosine residue. However, Akt appears to inactivate GSK-3α/β by simultaneous phosphorylation at Ser-21 and Ser-9. This zinc-induced non-conventional AktGSK-3β signaling has also been reported by others. For example, the addition of zinc at 100 μM to serum-deprived SH-SY5Y neuroblastoma cells activates Akt and GSK-3β, leading to tau modification [45]. Moreover, these zinc-induced double phosphorylations at Ser-9 and Tyr-216 in GSK-3β were also blocked by pretreatment of cells with wortmannin and LY294002 [45]. A number of GSK-3β substrates have been identified. These include β-catenin, CREB, NFAT, cyclin D1, c-Myc, E-cadherin, and eIF2B [29]. Tau itself is a widely recognized substrate of GSK-3β. Furthermore, GSK-3β is prime candidate for contributing to Alzheimer's disease-associated hyper-phosphorylation of tau [18,32]. GSK-3 phosphorylation of tau results in decreased microtubule binding, perhaps enhancing paired helical filament formation. This represents a possible prelude to neurofibrillary tangle formation [33]. The current study supports this model and shows that tau phosphorylation by GSK-3β plays an important role in the transmission of zincinduced cytotoxic signals in H19-7 cells. Although GSK-3β also phosphorylates β-catenin, targeting it for ubiquitination and degradation by proteasomal degradation [19,34], β-catenin does not appear to be the critical substrate by which GSK-3β triggers neuronal cell death. In summary, Akt promotes zinc-induced neuronal apoptosis. Furthermore, this novel, pro-apoptotic Akt activity appears to be mediated, in part, by non-conventional activation of GSK-3β and subsequent tau phosphorylation in H19-7 cells. Additional studies will be required to identify the tyrosine kinase(s) required for GSK-3β activation and to clarify how this kinase and Akt are mutually and differentially coupled during cell death and survival.

Acknowledgments The authors are grateful to T.F. Franke, R.A. Roth, J. Sadoshima, E. Krebs, I. Mook-Jung, and Choi K.Y. for providing plasmids and to J.T. Seo for technical assistance for the measurement of intracellular zinc transport. This study was supported by a grant from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology of Republic of Korea (M103KV010011-06K2201-01110 to K.C.C.), by Basic Science Research Grant from the Korea Research Foundation (KRF2003-015-C00527 to K.C.C.), by grants from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A050181 and A060440 to K.C.C.), and by Basic Research Grant from the Korea Science and Engineering Foundation (R01-2004-000-10673-0 to K.C.C.). It was also partly supported by the Korea Research Foundation Grant funded by

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the Korean Government (MOEHRD) (KRF-2004-005-E00017 to K.C.C.).

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