Cellular Signalling 26 (2014) 697–704
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Protein kinase C-β mediates neuronal activation of Na+/H+ exchanger-1 during glutamate excitotoxicity Bo Kyung Lee a, Jae Seok Yoon c,1, Min Goo Lee c, Yi-Sook Jung a,b,⁎ a b c
College of Pharmacy, Ajou University, 206, Worldcup-ro, Yeongtong-gu, Suwon 443-749, Republic of Korea Research Institute of Pharmaceutical Sciences and Technology, Ajou University, 206, Worldcup-ro, Yeongtong-gu, Suwon 443-749, Republic of Korea Department of Pharmacology, Yonsei University, College of Medicine, 50, Yonsei-ro, Seodaemun-gu, Seoul 120-752, Republic of Korea
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Article history: Received 22 August 2013 Received in revised form 5 December 2013 Accepted 22 December 2013 Available online 27 December 2013 Keywords: Na+/H+ exchanger Glutamate Protein Kinase C-β Neuroprotection Phosphorylation
a b s t r a c t Na+/H+ exchanger-1 (NHE-1) activity is known to play a critical role in the neuronal injury caused by glutamate. However, the underlying mechanism is not clear. This study shows that NHE-1 activation and its phosphorylation during glutamate exposure were attenuated by the inhibition of protein kinase C (PKC)-βI and -βII, leading to reduced neuronal death. In addition, activations of PKC-βI and -βII by PKC-βI and -βII CAT plasmid or by PMA, PKC-β pharmacological activator have stimulated the activity and phosphorylation of NHE-1, which were abolished by inhibition of PKC-β in neuronal cells. Furthermore, the inhibition of PKC-β has mediated neuroprotective effect on glutamate-induced cells, which is similar to neuroprotective efficacy of siRNA NHE-1 transfection. Taken together, these results suggest that activation of the PKC-βI and -βII pathway by glutamate increases the activity and phosphorylation of NHE-1, and that these increases contribute to neuronal cell death. In this study, we demonstrate that PKC-βI and -βII are involved in the regulation of NHE-1 activation following glutamate exposure in neuron. © 2013 Elsevier Inc. All rights reserved.
1. Introduction The Na+/H+ exchanger-1 (NHE-1) is a pH-regulatory protein that is distributed almost universally in mammalian tissues. NHE-1 extrudes one H+ and takes up one Na+ ion when a decrease in intracellular pH occurs [1], and has been implicated in a number of cellular functions, which include cell volume regulation, cell migration, and cell proliferation. Increased NHE-1 activity during ischemic conditions is a key step in cell volume regulation and acid–base homeostasis in neuronal cells [2,3]. During brain ischemia, increases in intracellular Na+ concentration due to NHE-1 activation result in an intracellular Ca2 + overload by Na+/Ca2+ exchanger and subsequent neuronal damage [4]. These inter-relationships underlie the importance of understanding the regulatory mechanisms that control NHE-1 during ischemic injury. A great deal of effort has gone into determining the regulatory
mechanisms of NHE-1 [5], though NHE-1 has generally been studied by observing its direct phosphorylation. It was recently found that the activations of serine/threonine kinases, such as, extracellular signalrelated kinase1/2 (ERK1/2) and 90-kDa ribosomal S6 kinase (p90RSK), are required for the phosphorylation of NHE-1 induced by ischemia [6]. Protein kinase C (PKC) has been reported to be able to regulate NHE1 in various cells, such as, cardiomyocytes and fibroblasts [7,8], but no study has been undertaken to investigate the relationship between NHE-1 regulation and PKC in neuronal cells. Furthermore, nothing is known regarding the possible roles of major PKC isoforms in the regulation of NHE-1 activation. In the present study, we investigated whether the activation of PKC affects the stimulation of NHE-1 activity by glutamate excitotoxicity, and in addition, we sought to identify the PKC isoforms responsible for the regulation of NHE-1 in neuronal cells. 2. Methods
Abbreviations: NHE-1, Na+/H+ exchanger-1; PKC, protein kinase C; PMA, 12-myristate 13-acetate; ERK1/2, extracellular signal-related kinase1/2; p90RSK, 90-kDa ribosomal S6 kinase; Gö6976, 12-(2-Cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3a]pyrrolo[3,4-c]carbazole; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene; SL0101, Kaempferol-3-O–(3′,4′-di-O–acetyl-α-L-rhamnopyranoside); SK-N-MC, human neuroblastoma cell line. ⁎ Corresponding author at: College of Pharmacy, Ajou University, 206, Worldcup-ro, Yeongtong-gu, Suwon 443-749, Republic of Korea. Tel.: +82 31 219 3444; fax: +82 31 219 3435. E-mail addresses: pfi
[email protected] (B.K. Lee),
[email protected] (J.S. Yoon),
[email protected] (M.G. Lee),
[email protected] (Y.-S. Jung). 1 Present address: Department of Pharmacology, Kwandong University College of Medicine, Gangneung 210-701, Republic of Korea. 0898-6568/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellsig.2013.12.011
2.1. Chemicals reagents Cariporide was synthesized at the Bio-organic Division of the Korea Research Institute of Chemical Technology (Daejon, Korea). Glutamate and 12-myristate 13-acetate (PMA) were purchased from Sigma (St. Louis, MO). Gö6976 was purchased from Biomol Research Labs Inc. (Plymouth Meeting, PA). MK-801 ((+)-5-Methyl-10,11-dihydro-5Hdibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate) and U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene) were from Tocris (Ballwin, MO), and PKC-β inhibitor (3-(1-(3-Imidazol-1-
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ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione) and SL0101 (Kaempferol-3-O-(3′,4′-di-O-acetyl-α-L-rhamnopyrano-side)) were purchased from Calbiochem (Germany). 2.2. Primary cultures of cortical neurons All experimental procedures were performed in accordance with the guidelines on the use and care of laboratory animals issued by the Animal Care Committee at Ajou University. Primary mouse cortical neurons were cultured as described previously [9]. Briefly, cerebral cortices were removed from the brains of fetal ICR mice on gestation day 14, gently titrated 3–4 times using a large-bore Pasteur pipette, dissociated into individual cells using a small-bore Pasteur pipette, and plated on 6- or 24-well plates precoated with 100 μg/ml poly-Dlysine (Sigma) and 4 μg/ml natural mouse laminin (Gibco-BRL, Gaithersburg, MD). Cells (approximately 2.5 × 105 cells/10 ml) were maintained in culture media, consisting of Eagle's minimum essential medium (MEM) (Earle's salts, JBI, Korea) supplemented with 21 mM glucose, 5% fetal bovine serum (Gibco-BRL), 5% horse serum (GibcoBRL), and 2 mM L-glutamine. Cytosine arabinofuranoside (10 μM AraC, Sigma) was added to cultures on culture days 3–4 in vitro (DIV 3–4) to prevent glial cell overgrowth. Cells were maintained in 5% CO2 atmosphere at 37 °C for 7–8 days, and then used for experiments. More than 80% of the cell population at this stage was neuronal cells, as determined by NeuN (neuronal nuclei, specific neuronal markers) and GFAP (glial fibrillary acidic protein, glial cell markers) staining (data not shown).
perfusion chamber. The chamber was placed on an inverted microscope and intralobular ducts were identified by morphology. BCECF-AM fluorescence was recorded at excitation wavelengths of 440 and 490 nm using a recording setup (Delta Ram; PTI Inc., Brunswick, NJ). NHE activities were measured by estimating Na+-dependent pHi recovery in acidified cells as follows. Cells were first acidified by a + NH+ 4 (20 mM) pulse, and then perfused with a Na -free solution + prepared by replacing Na in the standard HEPES-buffered solution. Maximal Na+-dependent pHi recovery was measured in cells acidified to a pH of 6.3–6.4. Buffer capacity was calculated by measuring pHi in response to 5–20 mM NH4Cl pulses. During the experiment, the intrinsic buffer capacity was found to show a negative linear relationship with pHi between pH values of 6.2 and 7.6. 2.6. Subcellular fractionation for the isolation of PKC and Immunoblotting
SK-N-MC neuroblastoma cells were purchased from the ATCC (Manassas, VA) and grown in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL) containing 10% fetal bovine serum and 20 mM glucose, and maintained in a humidified 5% CO2 incubator at 37 °C. The cells were used for experiments after reaching ~80% confluence.
Subcellular fractionation for PKC was performed as described previously [11]. Briefly, cells were harvested in homogenization buffer (20 mM Tris–HCl, 2 mM EDTA, 5 mM EGTA, 5 mM DTT, 6 mM βmercaptoethanol, 1 mM PMSF, 20 μM leupeptin, and 10 μg/ml aprotinin, pH 7.4) and centrifuged at 100,000 g for 1 h at 4 °C. Supernatants were retained as cytosolic fractions. Pellets were resuspended in 1% Triton X-100-containing homogenization buffer, and centrifuged at 10,000 g for 10 min at 4 °C. Supernatants are referred to as membrane fractions. Protein contents were determined using the Bradford protein assay (Biorad, Hercules, CA). The samples were resolved on 8% SDSpolyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA). Blots were incubated in 5% non-fat dry milk for 1 h at room temperature, and then incubated overnight at 4 °C with a polyclonal antibody against PKC isoform (Santa Cruz, CA). The blots were then rinsed with Tris-buffered saline and incubated with horse-radish peroxidase-conjugated secondary IgG (Cell Signaling Technologies, Beverly, MA) for 1 h. Bound antibody was detected with an ECL kit (Intron), and bands analyzed using a LAS1000 (Fuji Photo Film, Japan).
2.4. RNA preparation and RT-PCR
2.7. Isolation of ERK1/2 and p90RSK from cell lysates
Cultured cells were washed with ice-cold PBS and total RNA was extracted using the easy-blue® kit (Intron, Korea). Reverse transcription (RT) was carried out using AMV reverse transcriptase (Takara, Japan) according to the manufacturer's instructions. The primer sequences used in this study for NHE-1 were; 5′-TCTGCCGTCTCAACTG TCTTA-3′ (forward) and 5′-CCCTTCAACTCC TCATTCACCA-3′ (reverse). PCR amplification was performed over 30 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s, and extension at 72 °C for 35 s. PCR products were separated by electrophoresis on 1% agarose gels, which were then stained with ethidium bromide and photographed. The optical densities of NHE-1 bands were measured using a Gel doc system (GEL DOC 2000, Bio-Rad, Hercules, CA). Optical density measurements were normalized versus glyceraldehyde 3phosphate dehydrogenase (GAPDH).
Cells were harvested in RIPA buffer (150 mM NaCl, 20 mM Tris–HCl, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, and protease inhibitors at pH 7.4), homogenized, and nuclei and cell debris were removed by centrifugation at 10,000 g for 15 min at 4 °C. Supernatants were collected for immunoblotting. Protein contents were determined using the BCA™ protein assay (Pierce Rockford, IL). Protein samples were denatured in Laemli buffer (1:4 by volume), and total ERK1/2 and pERK1/2 levels were quantified by immunoblotting using polyclonal antibody against ERK1/2 and monoclonal antibody against p-ERK1/2, respectively (both from Cell Signaling Technologies). Polyclonal antibodies against RSK and phosphorylated p90RSK (both from Cell Signaling Technologies) were used to detect total p90RSK and pp90RSK, respectively.
2.3. SK-N-MC cell cultures
2.8. Analysis of NHE-1 phosphorylation by immunoprecipitation 2.5. Measurements of pHi and NHE activity NHE activity was measured using as previously described method with a few modifications [10]. Briefly, cells were loaded with a pHsensitive fluorescent dye BCECF-AM (acetoxymethyl esters of 2′,7′bis(2-carboxyethyl)-5,6-carboxyfluorescein, Invitrogen, Carlsbad, CA) and pHi changes were measured. For primary cultured neuronal cells, cells grown on poly-D-lysine-coated glass coverslips were loaded with 5 μM BCECF-AM by incubation for 15 min at room temperature in standard HEPES-buffered solution. The standard HEPES-buffered solution contained in mM: 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES (pH 7.4 with NaOH). Cells were then washed with standard HEPES-buffered solution, and assembled in the bottom of a
Phosphorylation levels of NHE-1 were measure as described by Snabaitis et al. [12]. Cells were lysed in ice-cold RIPA buffer as described above and centrifuged at 10,000 g for 15 min at 4 °C. Supernatants containing proteins were collected and incubated overnight at 4 °C with mouse monoclonal antibody against the phosphor-Ser 14-3-3β protein binding motif (Cell Signaling Technologies) or with goat monoclonal NHE-1 antibody (Santa Cruz). The immunocomplexes obtained were mixed with protein A and G (Merck, Germany) for 4 h at 4 °C and then washed three times with ice-cold modified RIPA buffer. Immunocomplexes were dissociated from beads by heating at 100 °C for 5 min. Protein samples from immunocomplexes were resolved on 8% SDS-PAGE and analyzed by immunoblotting using goat polyclonal
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NHE-1 antibody (BD Bioscience, San Jose, CA) or rabbit monoclonal phospho-serine antibody (Invitrogen).
(CAT) were generously provided by Dr. Soh at Inha University. pcDNA3 plasmid was used as a negative control.
2.9. Small interfering RNA treatment
2.12. Statistical analysis
Predesigned, single-stranded small interfering RNA (siRNA) oligonucleotides of mouse NHE-1, PKC-β and control oligonucleotides were purchased from Santa Cruz. Double-stranded RNA oligo (1 μg) was added to serum-free medium containing transfection reagent (Santa Cruz), incubated for 30 min at room temperature, and placed in 6 well plates in 1 ml of serum-free medium, following the manufacturer's suggestions (Santa Cruz). After 6 h, the medium was changed to medium containing serum, and cells were incubated for a further 16–24 h at 37 °C before further experimental manipulation.
All data are presented as the means ± SEM. Statistical analysis was performed using Student's t-test, and p b 0.05 was considered statically significant. The data are representative of means of at least three independent experiments.
2.10. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
There was a rapid and significant decrease in pHi following the application of 100 μM glutamate. After 5 min glutamate treatment, pHi increased from 6.77 ± 0.03 to 6.94 ± 0.04 in neurons, but pHi did not increase from 6.72 ± 0.02 in neurons treated with buffer to 6.75 ± 0.06 in neurons treated with cariporide NHE-1 inhibitor (Fig. 1A). To investigate NHE-1 kinetics in neurons following glutamate exposure, we first investigated whether the intrinsic buffer capacity of neurons is altered by glutamate. Changes in pHi were induced by applying progressively lower concentrations of NH3/NH+ 4 . NHE activation was greater in neurons exposed to glutamate than in untreated controls (Fig. 1B). Cariporide, a potent and selective pharmacological inhibitor of NHE-1, was used to confirm that glutamate increased NHE isoform 1 activity. Pretreatment with cariporide decreased the ability to recover pHi to normal ranges in neurons exposed to glutamate to a level significantly below acid-stimulated recovery (basal condition). To determine changes in NHE-1 expression induced by glutamate, we
Glutamate excitotoxicity was evaluated colorimetrically using an MTT assay (Sigma). MTT (5 mg/ml) solution was added to cells grown in 24-well plates. After incubation with MTT solution for 2 h at 37 °C, the culture medium was removed and cells were incubated in 100% dimethyl sulfoxide (DMSO) for 15 min at room temperature. Optical densities of wells were measured at 540 nm. 2.11. Transfection and reagents On day 3 after plating, naive SK-N-MC cells were transiently transfected in serum-free medium with DNA using Lipofectamine ™2000 Reagent (Invitrogen), according to the manufacturer's instructions. PKC expression vectors encoding PKC-βI or -βII activated types
3. Results 3.1. Changes in NHE-1 activity in neuronal cells following exposure to glutamate
Fig. 1. The activation and phosphorylation of NHE-1 in neurons following glutamate exposure. (A) Representative recordings pHi of in primary cortical neuron cultures to intracellular acidosis by 1 min exposure to 100 μM glutamate. pHi levels were measured under vehicle (black) and 0.1 μM cariporide (light gray). (B) Representative recordings pHi to intracellular acidosis by transient (1 min) exposure to 20 mM NH4Cl. pHi levels were measured under control (black) and 100 μM glutamate loaded conditions (dark gray). Note that the dotted line represents treatment with 0.1 μM cariporide (light gray). (C) Neurons were exposed to 100 μM glutamate (Glut) for ~30 min to assess levels of NHE-1 at the mRNA and protein levels. mRNA (top) and protein (bottom) levels were determined as described in Methods. Loading levels were normalized using GAPDH and actin as controls, respectively. (D) Quantitative determination of NHE-1 for each group (mRNA, ●; protein, ○; n = 3, values are mean ± SEM). (E) Cells were treated with glutamate for 5 min in the presence or absence (Veh; vehicle) of 10 μM MK-801 (MK). Cells were immunoprecipitated with NHE-1 antibody, and immunoblotted for phospho-serine (p-Ser) (upper) or NHE-1 (lower) (n = 3). (F) Cells were immunoprecipitated with 14-3-3β antibody and immunoblotted for NHE-1 (upper) or 14-3-3β (lower), as described in Methods (n = 4, values are mean ± SEM, *p b 0.05 vs. CTL, #p b 0.05 vs. Veh).
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measured its mRNA and protein levels, but levels of NHE-1 at the mRNA and protein levels were found to be unaffected by glutamate (Fig. 1C and D). We then investigated whether glutamate stimulation leads to the phosphorylation of NHE-1 in neurons. Immunoprecipitation of NHE-1 followed by immunoblotting for phospho-serine (p-Ser) was performed to assay their interaction (Fig. 1E). NHE-1 binding to p-Ser was found to increase in neurons following glutamate stimulation. We then used phospho-Ser 14-3-3 binding motif antibody to detect the phosphorylation of NHE-1 in neurons, as described by Luo et al. [6]. As illustrated in Fig. 1F, the intensity of the protein band representing the phosphorylation level of NHE-1 was increased in neurons exposed to glutamate. Furthermore, in the presence of MK-801 (a NMDA receptor antagonist), this increase in NHE-1 phosphorylation was abolished.
3.2. Changes in NHE-1 activity following glutamate exposure depended on the activation of PKC-β-mediated pathways We determined the expressions of the conventional PKC isoforms (α, βI, βII, and γ) in neurons following glutamate exposure as a measure of the translocations of PKC isoforms from the cytosolic to the membrane fraction. As shown in Fig. 2A (left), whereas the translocations of the PKC-α and -γ isoforms remained unaltered after treatment with glutamate, both PKC-βI and -βII levels began to increase and decrease in membrane and cytosolic fractions, respectively, from 3 min after treatment. After 5 min of glutamate exposure, the activations of both PKC-βI and -βII increased maximally, and were returned to untreated control levels by MK-801, Gö6976 (conventional PKC inhibitor) or specific PKC-β inhibitor (PKC-βI and II inhibitor) (Fig. 2A, right). We then investigated whether phosphorylation of NHE-1 induced by glutamate was mediated by PKC-β. After glutamate
treatment, NHE-1 phosphorylation was detected in neurons, and this was blocked by Gö6976 or PKC-β inhibitor but not by cariporide (Fig. 2B), which suggested that the phosphorylation of NHE-1 depends on the PKC-β pathway and is not affected by direct blockage of NHE-1. To obtain further evidence of the importance of the PKC-β-mediated phosphorylation of NHE-1, we examined the effect of PKC-β gene deletion on glutamate-induced phosphorylation of NHE-1. The inhibition of PKC-β by siRNA PKC-β transfection was found to attenuate glutamate-mediated phosphorylation of NHE-1 significantly (298.9 ± 69.9 to 91.5 ± 8.4%) (Fig. 2C). In response to 100 μM glutamate, NHE1 activity increased about 3-fold in neuronal cells (from 0.2 ± 0.04 to 0.6 ± 0.05 pHi/min), and this was decreased by PKC-β inhibitor (to 0.3 ± 0.04 pHi/min) and by cariporide (to 0.1 ± 0.04 pHi/min), respectively (Fig. 2D). NHE-1 activity is also calculated by H+ efflux, because the phosphorylation of NHE-1 changes its affinity for H+. A dramatic decrease in H+ efflux rate was observed from neurons treated with PKC-β inhibitor, suggesting that the phosphorylation of NHE-1 induced by PKC-β activation has a primary effect on the affinity of NHE-1 for H+ (Fig. 2E).
3.3. NHE-1 activation induced by PKC-β activation To investigate the role of PKC-β activation on NHE-1 activation in neuronal cells, we first examined whether treatment with PMA (which stimulates conventional PKC isoforms translocation) regulates the phosphorylation of NHE-1 in cortical neurons. Treatment with 0.1 μM PMA for 30 min led to the translocations of PKC-βI and -βII to the membrane in a manner resembling that induced by glutamate. In addition, the translocations of PKC-βI and -βII mediated by PMA were abolished by PKC-β inhibitor (Fig. 3A). We then investigated whether
Fig. 2. NHE-1 activation was mediated by PKC-β activation in neurons treated with glutamate. (A) Representative immunoblots of PKC isoforms (α, βI, βII, and γ) detected in the cytosol (C) and membrane (M) fractions of cortical neurons treated for different times (from 0 to 30 min) with 100 μM glutamate (left). On the right, cells were treated with 100 μM glutamate for 5 min in the presence or absence (Veh) of 10 μM MK-801 (MK), 1 μM Gö6976 (Gö), or 0.1 μM PKC-β inhibitor (βi). (B) Samples were lysed and immunoprecipitated with NHE-1 antibody and immunoblotted for phosphor-Ser (upper) or NHE-1 (lower) (n = 4, values are mean ± SEM, *p b 0.05 vs. CTL, #p b 0.05 vs. Veh). (C) Top, neuronal cells was transfected with siRNA control (siCTL) or siRNA PKC-β (siPKC-β, 0.75 or 1 μg), as described in Methods. Bottom, 1 μg siCTL or 1 μg siPKC-β transfected neurons were incubated in the presence or absence of glutamate. Samples were lysed and immunoprecipitated with NHE-1 antibody and immunoblotted for phosphor-Ser (upper) or NHE-1 (lower) (n = 5, *p b 0.05 vs. siCTL, #p b 0.05 vs. siCTL + glutamate). (D) pHi recovery rates were calculated using the first 60 s of each recovery curve. Cells were treated with or without glutamate in the presence or absence of 0.1 μM PKC-β inhibitor (PKC-βi) or 0.1 μM cariporide (n = 5, values are mean ± SEM, *p b 0.05 vs. untreated vehicle, #p b 0.05 vs. vehicle with glutamate). (E) Rate of H+ efflux during + pHi recovery was determined in neurons following NH3/NH+ 4 prepulse ± glutamate. H efflux rate was plotted against pHi. Cells were treated with (glutamate, ●) or without glutamate (control, ○) in the presence of 0.1 μM PKC-β inhibitor (glutamate + PKC-βi, ▼), or in the presence of 0.1 μM cariporide (glutamate + cariporide, △) (n = 5).
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Fig. 3. The regulation of NHE-1 during PKC-β activation in neurons treated with glutamate. (A) Primary cortical neuronal cells were pretreated in the presence or absence of 0.1 μM PKC-β inhibitor and then treated with 0.1 μM PMA for 30 min. The figure shows representative immunoblots for PKC-βI and -βII in the cytosol (C) and membrane (M) fractions of cortical neurons. Loading was normalized using actin and COX IV in cytosol and membrane fractions, respectively. (B) Samples were lysed and immunoprecipitated with NHE-1 antibody and immunoblotted for phosphor-Ser (upper) or NHE-1 (lower) (n = 3, values are mean ± SEM, *p b 0.05 vs. CTL, #p b 0.05 vs. Veh). (C) siCTL or siPKC-β transfected neurons were incubated in the presence or absence of 0.1 μM PMA. Samples were lysed and immunoprecipitated with NHE-1 antibody and immunoblotted for phosphor-Ser (upper) or NHE-1 (lower) (n = 4, *p b 0.05 vs. siCTL, #p b 0.05 vs. siCTL + PMA). (D) pHi recovery rates in primary neuronal cells (n = 5, *p b 0.05 vs. CTL, #p b 0.05 vs. Veh). (E) The activation of NHE-1 in SK-N-MC cells transfected with activated PKC-βI or -βII (CAT). Representative immunoblots of PKC-βI and -βII in cytosol (cyto) and membrane (memb) fractions after transfecting SK-N-MC cells with PKC-β-CAT. Loading was normalized versus actin. (F) Samples were lysed and immunoprecipitated with NHE-1 antibody and immunoblotted for phosphor-Ser (top) or NHE-1 (bottom) (n = 3, values are mean ± SEM, *p b 0.05 vs. Mock). G, pHi recovery rate in SK-N-MC cells (n = 5, values are mean ± SEM, *p b 0.05 vs. Mock).
the phosphorylation of NHE-1 was mediated by PMA. NHE-1 phosphorylation was detected by PMA and was blocked by pharmacological PKC-β inhibitor and by siRNA PKC-β transfection, respectively (Fig. 3B and C). PMA treatment also increased NHE-1 activity (0.25 ± 0.02 pHi/min) versus untreated controls (0.11 ± 0.01 pHi/min), and this increase was abolished by PKC-β inhibitor pretreatment (0.15 ± 0.03 pHi/min) (Fig. 3D). To confirm the essential role of PKC-β as a regulator of NHE-1 activity, SK-N-MC neuroblastoma cells were transduced with HA-tagged PKC-βI and -βII-CAT plasmids (the activated forms of PKC-βI and -βII). In SK-N-MC cells, PKC-βI and -βII were expressed at N70 kDa in the mock transduced control and cleaved to b50 kDa in CAT transfected cells. As shown in Fig. 3E, the activations of PKC-βI and -βII were increased by transducing SK-N-MC cells with PKCβI or -βII-CAT plasmids, respectively. We also found that both the phosphorylation and activation of NHE-1 were induced by transfection with PKC-βI and -βII-CAT (Fig. 3F and G, G; 0.12 ± 0.01 to 0.24 ± 0.02 and 0.27 ± 0.03 pHi/min, respectively). To obtain further evidence of the importance of the PKC-β-mediated activation of NHE-1, we examined the effect of PKC-β inhibition on glutamate-induced neuronal cell death. The inhibition of PKC-β by PKC-β inhibitor and the inhibition of NHE-1 activity by siRNA NHE-1 transfection were found to attenuate glutamate-mediated cell death significantly (54.3 ± 2.3 to 68.3 ± 3.9% and to 72.1 ± 4.3%). However, no additional neuroprotective effect was found when siRNA NHE-1 transfected neurons were exposed to PKC-β inhibitor or Gö6976, respectively (73.1 ± 2.8% or 70.6 ± 3.2%) (Fig. 4). This finding suggests
Fig. 4. Role of NHE-1 phosphorylation in neurons following glutamate exposure. Top, neuronal cells was transfected with siRNA control (siCTL) or siRNA NHE-1 (siNHE-1, 0.75 or 1 μg), as described in Methods. Bottom, cell viabilities were assessed in neuronal cultures after exposure to glutamate for 20 h. siCTL or siNHE-1 transfected neurons were incubated in the presence or absence of PKC-β inhibitor or Gö6976 (n = 7,values are mean ± SEM, *p b 0.05 vs. CTL, #p b 0.05 vs. siCTL, †p b 0.05 vs. siCTL + glutamate).
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that activation of PKC-β contributes to neuronal death by NHE-1 phosphorylation after glutamate exposure. 3.4. PKC-β activation mediated the stimulation of NHE-1 activity through an ERK1/2-p90RSK pathway in neurons treated with glutamate An ERK1/2-p90RSK pathway has been shown to phosphorylate the regulatory domain of NHE-1 and possibly to mediate the ischemia/reperfusion-induced stimulations of NHE-1 activity in cultured myocytes [13] and neuronal cells [6]. We investigated whether an ERK1/2-p90RSK pathway is also involved in the activation of the PKC-β-induced stimulation of NHE-1 activity in neuronal cells treated with glutamate. As shown in Fig. 5A, glutamate treatment increased the phosphorylations of ERK1/2 and p90RSK, and these glutamateinduced phosphorylation were abolished by U0126 (a MEK1/2 inhibitor) or PKC-β inhibitor, but U0126 did not inhibit the activations of PKC-βI and -βII, whereas SL0101 (a p90RSK specific inhibitor) inhibited only the phosphorylation of p90RSK. Additionally, the inhibition of PKC-β by siRNA PKC-β transfection attenuates glutamateinduced phosphorylation of ERK1/2 and p90RSK significantly (Fig. 5B). The phosphorylation and activation of NHE-1 were also blocked by U0126 or SL0101 (Fig. 5C and D). These finding suggests that the stimulation of NHE-1 activity after glutamate exposure occurs via PKC-β-ERK1/2-p90RSK signaling pathways in neuronal cells. To investigate the effect of PKC-β activation on the regulation of NHE-1 by ERK1/2-p90RSK activation, we examined whether transfection with PKC-βI or -βII-CAT regulated the phosphorylations of ERK1/2 and p90RSK in SK-N-MC neuroblastoma cells. As shown in Fig. 5E, the activations of PKC-βI or -βII induced an increase in the phosphorylations of ERK1/2 and p90RSK. Furthermore, increased
ERK1/2 phosphorylation was inhibited by U0126 but not by SL0101, whereas p90RSK phosphorylation was inhibited by both, which suggests that p90RSK activation occur downstream of ERK1/2 activation, and that ERK1/2 activation is modulated by PKC-β. In addition, the phosphorylation and activation of NHE-1 by PKC-β CAT transfection were also blocked by U0126 or SL0101 in SK-N-MC neuroblastoma cells (Fig. 5F and G). 4. Discussion In this study, we identified two neuronal PKC isoforms, PKC-βI and -βII, that are important for the regulation of NHE-1 activity, which is associated with ERK1/2-p90RSK signaling pathways as a kinase of NHE-1 in cortical neuronal cells exposed to glutamate. Furthermore, we found that NHE-1 activity increased in neuronal cells exposed to glutamate, which might be due to the phosphorylation of NHE-1. It is known that ischemic injury reliably stimulates NHE-1 activity by phosphorylation in neurons and cardiomyocytes. The phosphorylation of NHE-1 plays an essential role in its activation. In fact, NHE-1 is constitutively phosphorylated in resting cells, and mitogenic stimulation leads to a gradual increase in the phosphorylation of its serine residues in parallel with increases in intracellular pH [14]. These results suggest that the phosphorylation of NHE-1 directly triggers its activation. Furthermore, it has been suggested that various protein kinases can directly phosphorylate NHE-1 at its amino acid serine residues in the distal C terminus phosphorylation domain and activate NHE-1 in ischemic neurons and myocardium [6,15]. Members of the PKC family are important regulators of neuronal cellular mechanism, and have been implicated in cerebral ischemia and glutamate excitotoxicity [16,17]. Neuronal cell stimulation by
Fig. 5. The relationship between PKC-β and ERK1/2-p90RSK during glutamate exposure in neurons. (A and B) Primary cortical neuronal cells were treated with 100 μM glutamate (Glut) for 5 min in the presence or absence of 0.1 μM PKC-β inhibitor (βi), 10 μM U0126 (U), or 1 μM SL0101 (SL). (A) Representative immunoblots for PKC-βI, -βII, p-ERK1/2, and p-p90RSK. pERK1/2 and p-p90RSK loadings were normalized versus ERK1/2 and p90RSK, respectively. (B) siCTL or siPKC-β transfected neurons were incubated in the presence or absence of glutamate. Samples were lysed and immunoblotted for p-ERK1/2 and p-p90RSK. (C) Samples were lysed and immunoprecipitated with NHE-1 antibody and immunoblotted for phosphor-Ser (upper) or NHE-1 (lower) (n = 3, values are mean ± SEM, *p b 0.05 vs. CTL, #p b 0.05 vs. Veh). (D) pHi recovery rates were calculated from the first 60 s of each recovery curve (n = 6, *p b 0.05 vs. CTL, #p b 0.05 vs. Veh). (E–G) The regulation of NHE-1 in activated PKC-β achieved by activating ERK1/2-p90RSK. (E) Representative immunoblots for p-ERK1/2 and pp90RSK detected in SK-N-MC cells transfected with PKC-β-CAT. Cells were treated in the presence or absence of 10 μM U0126 (U) or 1 μM SL0101 (SL) in PKC-β-CAT-transfected SKN-MC cells. (F) Samples were lysed and immunoprecipitated with NHE-1 antibody and immunoblotted for phosphor-Ser (upper) or NHE-1 (lower) (n = 3, values are mean ± SEM, *p b 0.05 vs. Mock, #p b 0.05 vs. Veh). (G) pHi recovery rates in SK-N-MC cells (n = 5, *p b 0.05 vs. Mock, #p b 0.05 vs. Veh).
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glutamate receptor activation leads to an increase in intracellular Ca2+, and consequently could activate Ca2 +-dependent conventional PKC isoforms (α, βI, βII, and γ) [18]. In addition, these conventional PKC isoforms can cause dysfunctions of some ion channels or ion exchangers in neuronal cells, for example, PKC-βI has been reported to regulate VRAC (volume-regulated anion channel) activity in glial cells during glutamate excitotoxicity [19]. Furthermore, PKC-βII has been reported to regulate voltage-gated Na+ channels during diabetic neuronal damage [20]. In addition, it has been reported that conventional PKC isoforms can activate NHE isoforms in various cell type, for example, PKC-α is implicated in the growth factor-induced activation of the NHE-1 in NIH3T3 cells [21]. Furthermore, the activations of conventional PKC isoforms have been reported to increase the exchange of Na+ and H+ via NHE in goldfish somatotropes [22]. Consistent with these results, our study shows that glutamate-induced PKC-βI and -βII activations lead to NHE-1 activation by increasing the affinity of NHE-1 for H+ in neuronal cells. Furthermore, in the present study, the activation of PKC-β was found to increase NHE-1 activity in several experiments. First, we found that Gö6976 and specific PKC-β inhibitor as well as siRNA PKC-β prevented the phosphorylation and activation of NHE-1. Second, the phosphorylation and activation of NHE-1 were found to be induced by PMA (a pharmacological activator of PKC-β) and by PKC-β-CAT transfection. Third, deletion of the NHE-1 gene was found to inhibit glutamate-induced cell death, in a manner similar to the neuroprotective efficacy exhibited by PKC-β inhibition in cortical neurons. However, we are unable to conclude based on these experiments that PKC-β directly phosphorylates and activates NHE-1, because it is possible that some other kinase is influenced by PKC-β, and that it is responsible for the actual phosphorylation. In fact, several protein kinases including ERK1/2, p90RSK, protein kinase B (PKB) [12], Ca 2 +/calmodulin-dependent protein kinase (CaMK)II, p38MAPK [14], and Rho-associated kinases [23] have been shown to activate NHE-1 by directly phosphorylating its carboxyl-terminal regulatory domain. PKB binds to the phosphorylation site at Ser648 in NHE-1, which inhibits binding of Ca2+-activated CaMK to the NHE-1 regulatory domain in cardiac myocytes [12]. In addition, activated p38MAPK directly phosphorylates the four phosphorylation sites at NHE-1, Thr717, Ser722, Ser725, and Ser728, and causes intracellular alkalinization [14]. In fibroblasts, the phosphorylation of NHE-1 induced by the directly binding of Rho-associated kinases to NHE-1 is necessary for the regulation of cytoskeletal functions, which may be an important determinant in pathophysiological conditions associated with abnormal cytoskeletal organization [23]. In particular, the functional importance of ERK1/2 and of the p90RSK-mediated phosphorylation of the serine residues of NHE-1 have been established [24,15]. With specific regard to neuro-physiology, recent studies conducted by Sun et al. confirmed the functional importance of the ERK1/2-p90RSK pathwaymediated phosphorylation of NHE-1 in ischemic neuronal cells [6,25]. In addition, ERK1/2-p90RSK has been reported to phosphorylate NHE1 directly at amino acids Ser770 and Ser771 in AP-1 cells [24]. A serine/threonine kinase p90RSK, a downstream substrate of ERK1/2, has also been reported to directly phosphorylate NHE-1 at Ser703, which creates a binding motif for protein 14-3-3β in ischemic cardiomyocytes [12]. In line with these results, we suggest that glutamate induces the phosphorylation of Ser770/Ser771/703 within the regulatory domain of NHE-1 protein, which can be immunoprecipitated by phosphor-Ser14-3-3 binding motif antibody in neuronal cells (Fig. 1F, [6]), because NHE-1 binds to phosphor-Ser14-3-3. Furthermore, NHE-1 phosphorylation by ERK1/2-p90RSK stimulates NHE-1 activity, which is prevented by inhibition of PKC-β in response to glutamate in neurons. Consequently, we determined that the present study identifies PKC-β as a major regulator of NHE-1, although there is no evidence that NHE-1 is site phosphorylated by PKC-β rather than another kinase or downstream kinase such as ERK1/2 and p90RSK. These findings are supported by a previous report in which the activation of
703
endogenous p90RSK as a known NHE-1 kinase was found to be regulated by PKC-β, which induces the phosphorylation of cardiac troponin I in the presence of H2O2 [26]. Thus, it may be that the activation of PKC-β is responsible for NHE-1 serine residue phosphorylation in cardiomyocytes. Although PKC-β has multiple cellular functions, it appears that PKC-β is a critical stimulator of NHE-1, at least in neuronal cells treated with glutamate. We also found that the inhibition of PKC-β was neuroprotective in neurons treated with glutamate, which is similar to the neuroprotective efficacy of NHE-1 inhibition by genetic ablation (Fig. 4). Furthermore, the inhibition of NHE-1 phosphorylation achieved by blocking PKC-β decreased glutamate-activated NHE-1 activity without inhibiting basal, homeostatic NHE-1 function (Fig. 2D and E). This implies that the phosphorylation of NHE-1 mediated by PKC-β plays a critical role in the stimulation of NHE-1 activity-mediated neuronal cell death following glutamate exposure, which in turn, suggests that the inhibition of PKC-β may provide a better therapeutic strategy than treatment with potent NHE-1 inhibitors which completely block ion transport in neuronal cells. 5. Conclusions We postulate that the regulation of NHE-1 activity mediated by PKCβ activation involves NHE-1 phosphorylation, which implies that the phosphorylation of NHE-1 directly affects glutamate-induced neuronal cell death. These results suggest that PKC-β plays important roles in pathological conditions such as ischemia. Acknowledgments This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology [Grant 2012R1A1A2044497], by the Bio & Medical Technology Development Program of the National Research Foundation (MRF) funded by the Korean government (MEST) [2011-0019397], and by the Bio Medicine of the Chungcheong Leading Industry Office of the Korean Ministry of Knowledge Economy (MKE). References [1] I.S. Jung, S.H. Lee, M.K. Yang, J.W. Park, K.Y. Yi, S.E. Yoo, S.H. Kwon, H.J. Chung, W.S. Choi, H.S. Shin, Arch. Pharm. Res. 33 (2010) 1241–1251. [2] Y. Matsumoto, S. Yamamoto, Y. Suzuki, T. Tsuboi, S. Terakawa, N. Ohashi, K. Umemura, Stroke 35 (2004) 185–190. [3] J. Luo, H. Chen, D.B. Kintner, G.E. Shull, D. Sun, J. Neurosci. 25 (2005) 11256–11268. [4] M. Karmazyn, in: L. Fliegel (Ed.), The Na+/H+ Exchanger, Springer/R. G. Landes Company, Austin, Texas, 1996, pp. 189–216. [5] H. Wang, N.L. Silva, P.A. Lucchesi, R. Haworth, K. Wang, M. Michalak, S. Pelech, L. Fliegel, Biochemistry 36 (1997) 9151–9158. [6] J. Luo, D.B. Kintner, G.E. Shull, D. Sun, J. Biol. Chem. 282 (2007) 28274–28284. [7] T.F. Rehring, J.I. Shapiro, B.S. Cain, D.R. Meldrum, J.C. Cleveland, A.H. Harken, A. Banerjee, Am. J. Physiol. 275 (1998) H805–H813. [8] M.R. Wiederkehr, H. Zhao, O.W. Moe, Am. J. Physiol. 276 (1999) C1205–C1217. [9] B.K. Lee, S. Lee, K.Y. Yi, S.E. Yoo, Y.S. Jung, Biomol. Ther. 19 (2011) 445–450. [10] J. Kim, Y.S. Jung, W. Han, M.Y. Kim, W. Namkung, B.H. Lee, K.Y. Yi, S.E. Yoo, M.G. Lee, K.H. Kim, Eur. J. Pharmacol. 567 (2007) 131–138. [11] Y.S. Jung, B.R. Ryu, B.K. Lee, I. Mook-Jung, S.U. Kim, S.H. Lee, E.J. Baik, C.H. Moon, Biochem. Biophys. Res. Commun. 320 (2004) 789–794. [12] A.K. Snabaitis, F. Cuello, M. Avkiran, Circ. Res. 103 (2008) 881–890. [13] R.S. Haworth, C. McCann, A.K. Snabaitis, N.A. Roberts, M. Avkiran, J. Biol. Chem. 278 (2003) 31676–31684. [14] A. Khaled, A.N. Moor, A. Li, K. Kim, D.K. Ferris, K. Muegge, R.J. Fisher, L. Fliegel, S.K. Durum, Mol. Cell. Biol. 21 (2001) 7545–7557. [15] N. Maekawa, J. Abe, T. Shishido, S. Itoh, B. Ding, V.K. Sharma, S.S. Sheu, B.C. Blaxall, B.C. Berk, Circulation 113 (2006) 2516–2523. [16] K. Buchner, E. Adamec, M.L. Beermann, R.A. Nixon, Mol. Brain Res. 64 (1999) 222–235. [17] R. Selvatici, S. Marino, C. Piubello, D. Rodi, L. Beani, E. Gandini, A. Siniscalchi, J. Neurosci. Res. 71 (2003) 64–71. [18] F. Vaccarino, A. Guidotti, E. Costa, Proc. Natl. Acad. Sci. U. S. A. 64 (1987) 8707–8711. [19] A. Rudkouskaya, A. Chernoguz, R.E. Haskew-Layton, A.A. Mongin, J. Neurochem. 105 (2008) 2260–2270.
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