Cultured Embryonic Hippocampal Neurons Deficient in Glucocorticoid (GC) Receptor: A Novel Model for Studying Nongenomic Effects of GC in the
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《内分泌学杂志》
Institute of Neuroscience, Department of Neurobiology (L.X., Y.C.), and Department of Physiology (A.Q.), Second Military Medical University, Shanghai, 200433, People’s Republic of China
Abstract
Glucocorticoid (GC) acts through both genomic and nongenomic mechanisms. It affects the structure and function of the central nervous system, especially the hippocampus. Here we report an in vitro culture system that can yield embryonic hippocampal neurons deficient in the expression of GC receptor as demonstrated by immunoblotting, immunocytochemistry, and RT-PCR. Owing to this unique feature, those neuron preparations can serve as an ideal model for studying the nongenomic actions of GC on neural cells. In this study, we found that the Erk1/2, c-Jun N-terminal kinase (JNK), and p38 MAPKs were activated in these neurons by BSA-conjugated corticosterone within 15 min of treatment. This activation was not blocked by RU38486, spironolactone, or cycloheximide. Therefore, it is concluded that the activation of MAPKs observed here was due to the nongenomic action of GC. Furthermore, a 24-h incubation with corticosterone at concentrations ranged from 10–11–10–5 M did not have an effect on the viability of GC receptor-deficient neurons.
Introduction
GLUCOCORTICOID (GC) PLAYS critical roles in a variety of physiological and pathophysiologic processes. The underlying mechanisms of GC action are complex, which include both genomic and nongenomic pathways. Recently, much attention has been paid to the nongenomic mechanism (1).
GC exerts a series of important effects on the central nervous system, and many of them seem to work through nongenomic pathways, though the detail mechanisms are still to be elucidated (2). The hippocampus expresses a high level of GC receptor (GR) and is the main target tissue of GC in the central nervous system. RU38486 has been often used to demonstrate the nongenomic effects of GC because it blocks the activity of classic GR. However, the effect of RU38486 is not specific, and the exclusion of genomic effect is not fully warranted (3). Here we reported a serum-free culture system that can make embryonic hippocampal neurons deficient in GR expression as demonstrated by Western blot analysis, immunocytochemistry, and RT-PCR analysis. This model provided us a valuable tool to study the nongenomic effects of GC in neurons.
We have previously reported that GC could rapidly activate Erk1/2, c-Jun N-terminal kinase (JNK), and p38 MAPKs in PC12 cells and neonatal hippocampal cells (4, 5, 6) through nongenomic mechanisms. In this study, we verified these observations on GR-deficient neurons. We found that Erk1/2, JNK, and p38 were still activated in GR-deficient neurons after stimulated by BSA-conjugated corticosterone (B)-BSA. Furthermore, these activation effects were not blocked by RU38486, spironolactone, and cycloheximide. Finally, the effects of B on neuronal viability of those GR-deficient neurons were also investigated.
Materials and Methods
Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), B 21-sulfate potassium salt, B 21-hemisuccinate:BSA (B-BSA), Hochest33342, spironolactone, RU38486, cycloheximide, poly-L-lysine, fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG antibody, and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated antimouse IgG antibody were purchased from Sigma (St. Louis, MO). Antibodies against phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, phospho-p38 (Thr180/Tyr182), p38, phospho-JNK (Thr183/Tyr185), JNK, and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Trypsin, horse serum (HS), fetal bovine serum (FBS), DMEM, Neurobasal medium, and B27 were purchased from Life Technologies, Inc. (Grand Island, NY). All other chemicals used were of analytical grade.
Preparation of hippocampal neuron cultures and PC12 cell cultures
Hippocampal neuron cultures were prepared according to the protocols from the laboratories of Nelson (7), with some modifications. All animal procedures were approved by the Institutional Animal Care and Use Committee of Second Military Medical University. Procedures were designed to minimize the number of animals used and their suffering. Briefly, hippocampi were dissected from embryonic d 18 (E18) Sprague-Dawley rat fetuses in ice-cold dissection solution containing sucrose/glucose/HEPES (136 mM NaCl, 5.4 mM KCl, 0.2 mM Na2HPO4, 2 mM KH2PO4, 16.7 mM glucose, 20.8 mM saccharose, 0.0012% phenol red, and 10 mM HEPES, pH 7.4) (8). Isolated hippocampi were mechanically triturated and then digested in solution containing 0.25% trypsin and 1 mM EDTA at 37 C for 15 min. Single cell suspension was obtained by repeatedly passaging dissociated tissues through a fire-polished pipette in DMEM supplemented with 10% heat-inactivated FBS and HS. Cells were finally plated on poly-L-lysine (0.1 mg/ml)-coated plates, dishes, or glass coverslips for different experiments at optimal cell densities. The serum containing plating medium was replaced by a serum-free Neurobasal medium supplemented with 2% B27 (culture medium) 24 h after plating. Half the culture medium was changed every 3 d thereafter. More than 95% cells were neurons after they were cultured for 10 d in vitro (10DIV), verified by positive staining for microtubule-associated protein-2 (MAP2), a neuron-specific marker. All experiments were carried out on 10DIV unless indicated otherwise. PC12 cells were the kind gift of Dr. K. Kuba (Department of Physiology, Saga Medical School, Saga, Japan) and were grown in DMEM supplemented with 5% heat-inactivated HS and FBS. All cultures were kept at 37 C in a humidified 5% CO2-containing atmosphere.
Western blot analysis of GR expression and Erk1/2, p38, JNK phosphorylation
For detection of GR expression, PC12 cells and cultured embryonic (E18) hippocampal neurons of 1DIV (within 24 h after plating) or 10DIV were washed twice with ice-cold 0.1 M PBS, and the cell lysates were obtained by incubating cells in ice-cold lysis buffer (0.1% sodium dodecyl sulfate, 1% Igepal, 0.2 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonylfluoride) for 20 min. For detection of GC-induced MAPKs activation, neurons of 10DIV were starved in DMEM without any supplements for 10 h and then subjected to different treatments. All cell lysates were cleared by centrifugation at 12,000 x g for 10 min at 4 C, and protein concentration in the supernatant was determined by the BCA Protein Assay (Sigma). After being boiled for 5 min, 15 μg total protein from whole-cell lysates was separated by 15% SDS-PAGE denaturing gel and were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Inc., Dassel, Germany). Membranes were blocked with 10% nonfat dry milk in Tris-buffered saline with Tween 20 (50 mM Tris, 150 mM NaCl, and 0.1% Tween 20 vol/vol, pH 7.4) for 1 h and immunoblotted with anti-GR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with antiphosphorylated Erk1/2, JNK, p38 primary antibodies at a dilution of 1:1000 overnight at 4 C. Horseradish peroxidase-conjugated secondary antibody was used together with ECL detection system (Pierce Biotechnology, Inc., Rockford, IL) to detect the final signal. For detection of multiple protein on the same blot, membranes were stripped and reprobed with different antibodies as needed.
Indirect double immunofluorescence staining
At 1DIV or 10DIV, the cultured embryonic (E18) hippocampal neurons grown on glass coverslips were washed three times for 5 min each with PBS. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.3% Triton X-100 in PBS for 5 min. Rabbit anti-GR and mouse anti-MAP2 (NeoMarkers, Fremont, CA), at a dilution of 1:200 in PBS containing 5% normal goat serum, were used, respectively, overnight, at 4 C to detect the expression of GR and MAP2. The final detection was made by incubating with FITC-conjugated antirabbit IgG antibody and TRITC-conjugated antimouse IgG antibody (1:400 diluted in PBS containing 5% normal goat serum) for 1 h at 37 C. An Olympus IX70 inverted microscope (Olympus Optical Co. Ltd., Tokyo, Japan) fitted for fluorescence detection, connected to a personal computer via a charge-coupled device camera, was used to visualize the slides; and the photographs were analyzed by an image analysis software (MetaMorph).
RT-PCR analysis of GR mRNA
Total RNAs from cultured embryonic (E18) hippocampal neurons (1DIV or 10DIV) were extracted using RNAfast200 purification kit (Fastagen Biotech, Shanghai, China) and were reversely transcribed with RevertAid First Strand cDNA Synthesis Kits (Fermentas). Thirty-two cycles were used to amply the cDNA of rat GR and rat -actin simultaneously. The oligonucleotide primer pairs were as follows: rat GR (Gene bank access no. M14053) forward: 5'-AGGCAGTGTGAA ATTGTATCCCAC-3', reverse: 5'-GAGGCTTACAATCCTCATTCGTGT-3'; rat -actin (Gene bank access no. V01217) forward: 5'-CTATCGGCAATGAGCGGTTC-3', reverse: 5'-GATCTTGATCTTCATGGTGCTAGG-3'.
Neuronal viability and cytotoxicity assay
MTT assay (9) and lactate dehydrogenase (LDH) release assay (10) were used to assess the total number of neurons damaged or that survived after 24 h exposure to B. Briefly, hippocampal neurons were plated in 96-well plates at about 40,000 cells per well. The culture medium was completely changed at d 9. B was added directly to the 10DIV cells, with the final concentrations ranging from 10–11–10–5 M. Twenty-four hours after drug treatment, 50 ml cell supernatant was taken out from each well and subjected to LDH assay using a Cytotoxicity assay Kit (Promega Corp., Madison, WI). The amount of neuronal cell damage was measured by the absorbance at 490 nm with a microplate reader 680 (Bio-Rad Laboratories, Inc., Hercules, CA), and the percentage of neuronal damage was calculated by normalizing to the cells with vehicle treatment (control). The medium remaining in the wells was replaced by 100 ml fresh MTT solution (0.5 mg/ml) dissolved in DMEM. After another 3 h incubation at 37 C, the supernatant was removed, and the formazan crystals were dissolved in 100 ml acidic isopropanol (0.04 N HCL in isopropanol), and the viability of neuron was measured by the absorbance at 570 nm. The ratio of neuronal survival was calculated by normalizing to control. Addition of N-methyl-D-aspartic acid (NMDA) (100 mM) to the culture served as a positive control. Neurons were viewed and photographed with an Olympus microscope before assays were performed.
Statistical analysis
The statistical significance between treatment groups was analyzed by Student’s t test. Differences were considered significant at P < 0.05.
Results
GR protein and GR mRNA were undetectable in embryonic hippocampal neurons of 10DIV grown in serum-free condition
To examine whether serum-free condition has any effects on GR expression in neurons, we determined the GR expression in 1DIV and 10DIV neurons generated as described in Materials and Methods. As shown in Fig. 1, GR was detected in 1DIV neurons but not detected in 10DIV neurons by Western blot analysis. To further verify this observation, cells were costained by antibodies against GR and MAP2 simultaneously. GR was found in 1DIV neurons but not in 10DIV neurons (Fig. 2), which was inconsistent with the result by Western blot. When the expression of GR mRNA was examined using RT-PCR, GR mRNA was found in 1DIV, but not 10DIV, neurons (Fig. 3), suggesting that the expression of GR in 10DIV neurons grown in serum-free condition was blocked at the level of transcription.
Rapid phosphorylation of Erk1/2, JNK, and p38 MAPKs by B-BSA still occurred in the GR-deficient neurons
Because it has been previously reported that B and B-BSA could rapidly activate MAPKs in PC12 cells and in postnatal hippocampal cells (4, 5, 6), we questioned whether this is also true in the GR-deficient embryonic neurons established above. As shown in Fig. 4, B-BSA activated Erk1/2, JNK, and p38 within 15 min in a dose-dependent manner. A significant increase of phosphorylation forms of MAPKs was seen when the neurons were treated by B-BSA between 10–10–10–7 M. The doses were well within the range of GC present in cerebrospinal fluid in rat at normal state or at stressed state (11). Antibodies that detect total Erk1/2, JNK, and p38 were used to assure equal loading of samples. Similar results were obtained when B was used to stimulate the cells (data not shown). To examine whether the activation by B-BSA is neuronal specific, the effect of B-BSA on glia cells was also studied. There was no detectable activation of p38 when treated identically by B-BSA (data not shown).
RU38486, spironolactone, and cycloheximide failed to block the B-BSA-induced activation of MAPKs
GC is traditionally believed to primarily affect gene transcription and, subsequently, protein synthesis through classic GR (12). Furthermore, GC is known to possess a high binding affinity with mineralocorticoid receptor (MR) (11). To exclude the possibility that the activation of Erk1/2, JNK, and p38 MAPKs observed here was a result of classic genomic action through nuclear GR or nuclear MR, the effects of GR antagonist, RU38486; MR antagonist, spironolactone; and translation inhibitor, cycloheximide on the activation were investigated. Neurons were pretreated with those drugs for 30 min before B-BSA was administrated. As shown in Fig. 5, none of these pretreatments had any significant blocking effects on the B-BSA-induced Erk1/2, JNK, and p38 MAPKs activation.
The survival of GR-deficient neurons were not significantly affected by B treatment
It has been reported that B might affect the survival of neurons (13); we examined whether B affects the survival of GR-deficient neurons. The neurocytotoxicity was determined by measuring LDH release, and the cell viability by measuring MTT reduction. As shown in Fig. 6, B did not significantly affect the viability of GR-deficient neurons at the dose range tested (from 10–11–10–5 M), whereas the 100 mM NMDA decreased the viability of GR-deficient neurons significantly. Hochest staining revealed that B did not change the morphologic features of the nucleus of GR-deficient neurons compared with vehicle treatment (data not shown) either. Thus, the viability of GR-deficient neurons was not affected by B in the concentration ranges indicated.
Discussion
In normal adult brain, the expression of GR in the hippocampus is abundant and relatively stable. But in the hippocampus during development or in the cultured hippocampal neurons, the expression of GR seemed to be sensitive to regulations (14, 15). For example, neonatal treatment with thyroid hormone resulted in significantly increased GR binding capacity in the hippocampus in animals examined as adults (16). Long-term exposure to serotonin could dramatically increase the binding capacity of GR in cultured embryonic hippocampal neurons from E19–E20 Long-Evans rat fetus or E18–E19 Wistar rat fetus (17, 18, 19). Moreover, the GR mRNA in cultured E18 CD1 mice hippocampal neurons was also increased by serotonin treatment and decreased by GC treatment (20). Conflicting results have been seen on the effects of GC on GR expression in hippocampal neurons (21, 22, 23, 24, 25). It seemed that the regulation of GR expression in neurons was complicated and might be dependent on maturational stage and culture condition. It is worth mentioning here that the in vitro studies cited above used serum-containing medium to grow hippocampal neurons. In our culture system, hippocampal neurons isolated from E18 Sprague-Dawley rat fetuses were initially cultured in the serum-containing medium for 24 h, which was then changed into serum-free medium for as long as cells were in the culture. We did see the expression of GR in neurons when initially isolated from E18 embryonic hippocampus (1DIV neurons); but interestingly, it was no longer detectable in neurons of 10DIV, as was demonstrated by Western blot analysis and immunofluorescence staining. In addition, RT-PCR study found that the GR mRNA was not expressed in those neurons, though the underlying mechanism was not known. This finding was inconsistent with the result of a 3H-B binding assay in embryonic hippocampal cells (26) and hippocampal slices (27), implying that serum factors might be critical for the maintenance of GR in embryonic hippocampal neurons. We noticed that a different result was obtained by Wang et al. (25), that GR was detectable by Western blot in primary hippocampal neurons from E17 rat grown in serum-free medium (Neurobasal/DMEM/F12, 3:1:1 and supplemented with some other nutrition factors). But in their case, the neurons were subjected to treatment of dexamethasone, and the details of the cultured neurons used in their study were not given.
RU38486, an antagonist of GR, has been widely used in previous studies for the exclusion of the involvement of classic GR in characterizing and studying the nongenomic effects of GC (1, 2). Despite the great benefit that such pharmacological manipulation has brought to this field, it had its limitations, as many other pharmacological tools did (4). Here, we obtained neurons that are deficient in GR; and thus, any effects from GC in those neurons are not likely mediated through the classic GR-dependent genomic pathway. This unique feature makes it much more convenient to study GC’s nongenomic actions in neurons. Hence, for the first time, we acquired an ideal cell model for the study of the nongenomic effects of GC in the neural system.
We have previously reported our work on various nongenomic effects of GC (5, 6, 7, 28, 29), including the rapid activation of MAPKs in PC12 cells and postnatal hippocampal cells induced both by B and B-BSA (5, 6, 7). Taking advantage of the present GR-deficient neuron model, we examined whether the same effects occur in those embryonic neurons. We found that GC was still able to rapidly phosphorylate Erk1/2, JNK, and p38 MAPKs in GR-deficient neurons. To further exclude the involvement of GR and MR, the antagonists of GR and MR, i.e. RU38486 and spironolactone, were applied to the GR-deficient neurons when they were stimulated by B-BSA. Results showed that neither of the antagonists was able to block the B-BSA-induced activation of MAPKs. Furthermore, we showed that cycloheximde, a protein translation inhibitor, also failed to block the B-BSA-induced activation. This could help to exclude the involvement of any new protein synthesis on which most genomic pathways often depend. Because B-BSA is generally believed to be impermeable to cell membrane, the result of B-BSA-induced MAPKs activation in the present model suggested that the activation was initiated from the cell membrane. What’s more, the time frame of the activation was only 15 min, which seemed to be too short to guarantee a synthesis of any new proteins as a result of steroid nuclear receptor-mediated transcriptional activation. Taken together, we conclude that the activation of Erk1/2, JNK, and p38 MAPKs observed here was unequivocally due to a nongenomic GC action. This result justified our previous hypothesis that GC activated MAPKs through a classic GR-independent nongenomic pathway (5, 6, 7); and, for the first time, we demonstrated that this also occurred in the embryonic hippocampal neurons.
Erk1/2, JNK, and p38 MAPKs are important intracellular signaling molecules in mediating a variety of cellular and systematic functions in the neural system, like synaptic plasticity and memory formation (30, 31). These two important biological processes can also be affected greatly by GC (32). Whether there exists any relationship between the activation of MAPKs here and the GC’s regulatory effects on synaptic plasticity and memory formation remains to be investigated. On the other hand, Erk1/2, JNK, and p38 MAPKs are also associated with the survival or death of neuron cells. Most evidence so far suggested that Erk1/2 activation contributed to neuroprotection against various damaging insults (33), whereas JNK and p38 activation seemed to be involved in the mediation of neuronal cell apoptosis or death (34). GC was reported to have regulatory functions on excitotoxic neuronal damage (13). It is not known whether the nongenomic activation of the MAPKs by GC reported here would have the same effects on the process and on regulation of neuronal survival or death during the development of neurons.
The hippocampus is one of the principle GC targets and also one of the most vulnerable regions in the brain to neuronal loss in response to various neurotoxic factors. GC has been well documented as an enhancer of multiple neurotoxicity insults (13), and the action was supposed to be mediated through GR (35). However, it is rather controversial whether GC itself can have any effects on neuronal survival. Kimonides et al. (36) and Lu et al. (37) reported that GC had neurotoxic effects on primary hippocampal neurons measured by -tubulin III assay or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay after B treatment. In contrast, Behl et al. (38) and Mulholland et al. (39) showed that there was no detectable damage of neurons when they were exposed to GC alone, as shown by MTT reduction assay or propidium iodide uptake assay. As revealed by both LDH release and MTT reduction, our results showed that 24 h of exposure of B at concentrations ranging from 10–11–10–5 M had no significant toxic effects on GR-deficient neurons. We guess that this result might be partially attributed to the lack of classic GR in those neurons.
Taken together, our results of cultured GR-deficient embryonic hippocampal neurons provided an ideal cellular model for the study of GC’s nongenomic effects in the neural system. We also demonstrated that GC rapidly and nongenomically activated Erk1/2, JNK, and p38 MAPKs in those GR-deficient neurons but had no detectable effects on their neuronal viability.
Acknowledgments
The authors thank Professor Xiao Ping Chen of Tong Ji University for her kind editorial help.
Footnotes
This work was supported by grants from the National Natural Science Foundation of China (nos. 39330100, 39840019) and the Major State Basic Research Development Program of China (G1999054003).
Abbreviations: B, Corticosterone; B-BSA, BSA-conjugated corticosterone; DIV, days in vitro; E18, embryonic day 18; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GC, glucocorticoid; GR, GC receptor; HS, horse serum; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAP2, microtubule-associated protein-2; MR, mineralocorticoid receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; NMDA, N-methyl-D-aspartic acid; TRITC, tetramethyl rhodamine isothiocyanate.
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Abstract
Glucocorticoid (GC) acts through both genomic and nongenomic mechanisms. It affects the structure and function of the central nervous system, especially the hippocampus. Here we report an in vitro culture system that can yield embryonic hippocampal neurons deficient in the expression of GC receptor as demonstrated by immunoblotting, immunocytochemistry, and RT-PCR. Owing to this unique feature, those neuron preparations can serve as an ideal model for studying the nongenomic actions of GC on neural cells. In this study, we found that the Erk1/2, c-Jun N-terminal kinase (JNK), and p38 MAPKs were activated in these neurons by BSA-conjugated corticosterone within 15 min of treatment. This activation was not blocked by RU38486, spironolactone, or cycloheximide. Therefore, it is concluded that the activation of MAPKs observed here was due to the nongenomic action of GC. Furthermore, a 24-h incubation with corticosterone at concentrations ranged from 10–11–10–5 M did not have an effect on the viability of GC receptor-deficient neurons.
Introduction
GLUCOCORTICOID (GC) PLAYS critical roles in a variety of physiological and pathophysiologic processes. The underlying mechanisms of GC action are complex, which include both genomic and nongenomic pathways. Recently, much attention has been paid to the nongenomic mechanism (1).
GC exerts a series of important effects on the central nervous system, and many of them seem to work through nongenomic pathways, though the detail mechanisms are still to be elucidated (2). The hippocampus expresses a high level of GC receptor (GR) and is the main target tissue of GC in the central nervous system. RU38486 has been often used to demonstrate the nongenomic effects of GC because it blocks the activity of classic GR. However, the effect of RU38486 is not specific, and the exclusion of genomic effect is not fully warranted (3). Here we reported a serum-free culture system that can make embryonic hippocampal neurons deficient in GR expression as demonstrated by Western blot analysis, immunocytochemistry, and RT-PCR analysis. This model provided us a valuable tool to study the nongenomic effects of GC in neurons.
We have previously reported that GC could rapidly activate Erk1/2, c-Jun N-terminal kinase (JNK), and p38 MAPKs in PC12 cells and neonatal hippocampal cells (4, 5, 6) through nongenomic mechanisms. In this study, we verified these observations on GR-deficient neurons. We found that Erk1/2, JNK, and p38 were still activated in GR-deficient neurons after stimulated by BSA-conjugated corticosterone (B)-BSA. Furthermore, these activation effects were not blocked by RU38486, spironolactone, and cycloheximide. Finally, the effects of B on neuronal viability of those GR-deficient neurons were also investigated.
Materials and Methods
Materials
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), B 21-sulfate potassium salt, B 21-hemisuccinate:BSA (B-BSA), Hochest33342, spironolactone, RU38486, cycloheximide, poly-L-lysine, fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG antibody, and tetramethyl rhodamine isothiocyanate (TRITC)-conjugated antimouse IgG antibody were purchased from Sigma (St. Louis, MO). Antibodies against phospho-p44/42 MAPK (Thr202/Tyr204), p44/42 MAPK, phospho-p38 (Thr180/Tyr182), p38, phospho-JNK (Thr183/Tyr185), JNK, and horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Trypsin, horse serum (HS), fetal bovine serum (FBS), DMEM, Neurobasal medium, and B27 were purchased from Life Technologies, Inc. (Grand Island, NY). All other chemicals used were of analytical grade.
Preparation of hippocampal neuron cultures and PC12 cell cultures
Hippocampal neuron cultures were prepared according to the protocols from the laboratories of Nelson (7), with some modifications. All animal procedures were approved by the Institutional Animal Care and Use Committee of Second Military Medical University. Procedures were designed to minimize the number of animals used and their suffering. Briefly, hippocampi were dissected from embryonic d 18 (E18) Sprague-Dawley rat fetuses in ice-cold dissection solution containing sucrose/glucose/HEPES (136 mM NaCl, 5.4 mM KCl, 0.2 mM Na2HPO4, 2 mM KH2PO4, 16.7 mM glucose, 20.8 mM saccharose, 0.0012% phenol red, and 10 mM HEPES, pH 7.4) (8). Isolated hippocampi were mechanically triturated and then digested in solution containing 0.25% trypsin and 1 mM EDTA at 37 C for 15 min. Single cell suspension was obtained by repeatedly passaging dissociated tissues through a fire-polished pipette in DMEM supplemented with 10% heat-inactivated FBS and HS. Cells were finally plated on poly-L-lysine (0.1 mg/ml)-coated plates, dishes, or glass coverslips for different experiments at optimal cell densities. The serum containing plating medium was replaced by a serum-free Neurobasal medium supplemented with 2% B27 (culture medium) 24 h after plating. Half the culture medium was changed every 3 d thereafter. More than 95% cells were neurons after they were cultured for 10 d in vitro (10DIV), verified by positive staining for microtubule-associated protein-2 (MAP2), a neuron-specific marker. All experiments were carried out on 10DIV unless indicated otherwise. PC12 cells were the kind gift of Dr. K. Kuba (Department of Physiology, Saga Medical School, Saga, Japan) and were grown in DMEM supplemented with 5% heat-inactivated HS and FBS. All cultures were kept at 37 C in a humidified 5% CO2-containing atmosphere.
Western blot analysis of GR expression and Erk1/2, p38, JNK phosphorylation
For detection of GR expression, PC12 cells and cultured embryonic (E18) hippocampal neurons of 1DIV (within 24 h after plating) or 10DIV were washed twice with ice-cold 0.1 M PBS, and the cell lysates were obtained by incubating cells in ice-cold lysis buffer (0.1% sodium dodecyl sulfate, 1% Igepal, 0.2 mM sodium orthovanadate, 0.2 mM phenylmethylsulfonylfluoride) for 20 min. For detection of GC-induced MAPKs activation, neurons of 10DIV were starved in DMEM without any supplements for 10 h and then subjected to different treatments. All cell lysates were cleared by centrifugation at 12,000 x g for 10 min at 4 C, and protein concentration in the supernatant was determined by the BCA Protein Assay (Sigma). After being boiled for 5 min, 15 μg total protein from whole-cell lysates was separated by 15% SDS-PAGE denaturing gel and were electrotransferred onto nitrocellulose membranes (Schleicher & Schuell, Inc., Dassel, Germany). Membranes were blocked with 10% nonfat dry milk in Tris-buffered saline with Tween 20 (50 mM Tris, 150 mM NaCl, and 0.1% Tween 20 vol/vol, pH 7.4) for 1 h and immunoblotted with anti-GR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with antiphosphorylated Erk1/2, JNK, p38 primary antibodies at a dilution of 1:1000 overnight at 4 C. Horseradish peroxidase-conjugated secondary antibody was used together with ECL detection system (Pierce Biotechnology, Inc., Rockford, IL) to detect the final signal. For detection of multiple protein on the same blot, membranes were stripped and reprobed with different antibodies as needed.
Indirect double immunofluorescence staining
At 1DIV or 10DIV, the cultured embryonic (E18) hippocampal neurons grown on glass coverslips were washed three times for 5 min each with PBS. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature and then permeabilized with 0.3% Triton X-100 in PBS for 5 min. Rabbit anti-GR and mouse anti-MAP2 (NeoMarkers, Fremont, CA), at a dilution of 1:200 in PBS containing 5% normal goat serum, were used, respectively, overnight, at 4 C to detect the expression of GR and MAP2. The final detection was made by incubating with FITC-conjugated antirabbit IgG antibody and TRITC-conjugated antimouse IgG antibody (1:400 diluted in PBS containing 5% normal goat serum) for 1 h at 37 C. An Olympus IX70 inverted microscope (Olympus Optical Co. Ltd., Tokyo, Japan) fitted for fluorescence detection, connected to a personal computer via a charge-coupled device camera, was used to visualize the slides; and the photographs were analyzed by an image analysis software (MetaMorph).
RT-PCR analysis of GR mRNA
Total RNAs from cultured embryonic (E18) hippocampal neurons (1DIV or 10DIV) were extracted using RNAfast200 purification kit (Fastagen Biotech, Shanghai, China) and were reversely transcribed with RevertAid First Strand cDNA Synthesis Kits (Fermentas). Thirty-two cycles were used to amply the cDNA of rat GR and rat -actin simultaneously. The oligonucleotide primer pairs were as follows: rat GR (Gene bank access no. M14053) forward: 5'-AGGCAGTGTGAA ATTGTATCCCAC-3', reverse: 5'-GAGGCTTACAATCCTCATTCGTGT-3'; rat -actin (Gene bank access no. V01217) forward: 5'-CTATCGGCAATGAGCGGTTC-3', reverse: 5'-GATCTTGATCTTCATGGTGCTAGG-3'.
Neuronal viability and cytotoxicity assay
MTT assay (9) and lactate dehydrogenase (LDH) release assay (10) were used to assess the total number of neurons damaged or that survived after 24 h exposure to B. Briefly, hippocampal neurons were plated in 96-well plates at about 40,000 cells per well. The culture medium was completely changed at d 9. B was added directly to the 10DIV cells, with the final concentrations ranging from 10–11–10–5 M. Twenty-four hours after drug treatment, 50 ml cell supernatant was taken out from each well and subjected to LDH assay using a Cytotoxicity assay Kit (Promega Corp., Madison, WI). The amount of neuronal cell damage was measured by the absorbance at 490 nm with a microplate reader 680 (Bio-Rad Laboratories, Inc., Hercules, CA), and the percentage of neuronal damage was calculated by normalizing to the cells with vehicle treatment (control). The medium remaining in the wells was replaced by 100 ml fresh MTT solution (0.5 mg/ml) dissolved in DMEM. After another 3 h incubation at 37 C, the supernatant was removed, and the formazan crystals were dissolved in 100 ml acidic isopropanol (0.04 N HCL in isopropanol), and the viability of neuron was measured by the absorbance at 570 nm. The ratio of neuronal survival was calculated by normalizing to control. Addition of N-methyl-D-aspartic acid (NMDA) (100 mM) to the culture served as a positive control. Neurons were viewed and photographed with an Olympus microscope before assays were performed.
Statistical analysis
The statistical significance between treatment groups was analyzed by Student’s t test. Differences were considered significant at P < 0.05.
Results
GR protein and GR mRNA were undetectable in embryonic hippocampal neurons of 10DIV grown in serum-free condition
To examine whether serum-free condition has any effects on GR expression in neurons, we determined the GR expression in 1DIV and 10DIV neurons generated as described in Materials and Methods. As shown in Fig. 1, GR was detected in 1DIV neurons but not detected in 10DIV neurons by Western blot analysis. To further verify this observation, cells were costained by antibodies against GR and MAP2 simultaneously. GR was found in 1DIV neurons but not in 10DIV neurons (Fig. 2), which was inconsistent with the result by Western blot. When the expression of GR mRNA was examined using RT-PCR, GR mRNA was found in 1DIV, but not 10DIV, neurons (Fig. 3), suggesting that the expression of GR in 10DIV neurons grown in serum-free condition was blocked at the level of transcription.
Rapid phosphorylation of Erk1/2, JNK, and p38 MAPKs by B-BSA still occurred in the GR-deficient neurons
Because it has been previously reported that B and B-BSA could rapidly activate MAPKs in PC12 cells and in postnatal hippocampal cells (4, 5, 6), we questioned whether this is also true in the GR-deficient embryonic neurons established above. As shown in Fig. 4, B-BSA activated Erk1/2, JNK, and p38 within 15 min in a dose-dependent manner. A significant increase of phosphorylation forms of MAPKs was seen when the neurons were treated by B-BSA between 10–10–10–7 M. The doses were well within the range of GC present in cerebrospinal fluid in rat at normal state or at stressed state (11). Antibodies that detect total Erk1/2, JNK, and p38 were used to assure equal loading of samples. Similar results were obtained when B was used to stimulate the cells (data not shown). To examine whether the activation by B-BSA is neuronal specific, the effect of B-BSA on glia cells was also studied. There was no detectable activation of p38 when treated identically by B-BSA (data not shown).
RU38486, spironolactone, and cycloheximide failed to block the B-BSA-induced activation of MAPKs
GC is traditionally believed to primarily affect gene transcription and, subsequently, protein synthesis through classic GR (12). Furthermore, GC is known to possess a high binding affinity with mineralocorticoid receptor (MR) (11). To exclude the possibility that the activation of Erk1/2, JNK, and p38 MAPKs observed here was a result of classic genomic action through nuclear GR or nuclear MR, the effects of GR antagonist, RU38486; MR antagonist, spironolactone; and translation inhibitor, cycloheximide on the activation were investigated. Neurons were pretreated with those drugs for 30 min before B-BSA was administrated. As shown in Fig. 5, none of these pretreatments had any significant blocking effects on the B-BSA-induced Erk1/2, JNK, and p38 MAPKs activation.
The survival of GR-deficient neurons were not significantly affected by B treatment
It has been reported that B might affect the survival of neurons (13); we examined whether B affects the survival of GR-deficient neurons. The neurocytotoxicity was determined by measuring LDH release, and the cell viability by measuring MTT reduction. As shown in Fig. 6, B did not significantly affect the viability of GR-deficient neurons at the dose range tested (from 10–11–10–5 M), whereas the 100 mM NMDA decreased the viability of GR-deficient neurons significantly. Hochest staining revealed that B did not change the morphologic features of the nucleus of GR-deficient neurons compared with vehicle treatment (data not shown) either. Thus, the viability of GR-deficient neurons was not affected by B in the concentration ranges indicated.
Discussion
In normal adult brain, the expression of GR in the hippocampus is abundant and relatively stable. But in the hippocampus during development or in the cultured hippocampal neurons, the expression of GR seemed to be sensitive to regulations (14, 15). For example, neonatal treatment with thyroid hormone resulted in significantly increased GR binding capacity in the hippocampus in animals examined as adults (16). Long-term exposure to serotonin could dramatically increase the binding capacity of GR in cultured embryonic hippocampal neurons from E19–E20 Long-Evans rat fetus or E18–E19 Wistar rat fetus (17, 18, 19). Moreover, the GR mRNA in cultured E18 CD1 mice hippocampal neurons was also increased by serotonin treatment and decreased by GC treatment (20). Conflicting results have been seen on the effects of GC on GR expression in hippocampal neurons (21, 22, 23, 24, 25). It seemed that the regulation of GR expression in neurons was complicated and might be dependent on maturational stage and culture condition. It is worth mentioning here that the in vitro studies cited above used serum-containing medium to grow hippocampal neurons. In our culture system, hippocampal neurons isolated from E18 Sprague-Dawley rat fetuses were initially cultured in the serum-containing medium for 24 h, which was then changed into serum-free medium for as long as cells were in the culture. We did see the expression of GR in neurons when initially isolated from E18 embryonic hippocampus (1DIV neurons); but interestingly, it was no longer detectable in neurons of 10DIV, as was demonstrated by Western blot analysis and immunofluorescence staining. In addition, RT-PCR study found that the GR mRNA was not expressed in those neurons, though the underlying mechanism was not known. This finding was inconsistent with the result of a 3H-B binding assay in embryonic hippocampal cells (26) and hippocampal slices (27), implying that serum factors might be critical for the maintenance of GR in embryonic hippocampal neurons. We noticed that a different result was obtained by Wang et al. (25), that GR was detectable by Western blot in primary hippocampal neurons from E17 rat grown in serum-free medium (Neurobasal/DMEM/F12, 3:1:1 and supplemented with some other nutrition factors). But in their case, the neurons were subjected to treatment of dexamethasone, and the details of the cultured neurons used in their study were not given.
RU38486, an antagonist of GR, has been widely used in previous studies for the exclusion of the involvement of classic GR in characterizing and studying the nongenomic effects of GC (1, 2). Despite the great benefit that such pharmacological manipulation has brought to this field, it had its limitations, as many other pharmacological tools did (4). Here, we obtained neurons that are deficient in GR; and thus, any effects from GC in those neurons are not likely mediated through the classic GR-dependent genomic pathway. This unique feature makes it much more convenient to study GC’s nongenomic actions in neurons. Hence, for the first time, we acquired an ideal cell model for the study of the nongenomic effects of GC in the neural system.
We have previously reported our work on various nongenomic effects of GC (5, 6, 7, 28, 29), including the rapid activation of MAPKs in PC12 cells and postnatal hippocampal cells induced both by B and B-BSA (5, 6, 7). Taking advantage of the present GR-deficient neuron model, we examined whether the same effects occur in those embryonic neurons. We found that GC was still able to rapidly phosphorylate Erk1/2, JNK, and p38 MAPKs in GR-deficient neurons. To further exclude the involvement of GR and MR, the antagonists of GR and MR, i.e. RU38486 and spironolactone, were applied to the GR-deficient neurons when they were stimulated by B-BSA. Results showed that neither of the antagonists was able to block the B-BSA-induced activation of MAPKs. Furthermore, we showed that cycloheximde, a protein translation inhibitor, also failed to block the B-BSA-induced activation. This could help to exclude the involvement of any new protein synthesis on which most genomic pathways often depend. Because B-BSA is generally believed to be impermeable to cell membrane, the result of B-BSA-induced MAPKs activation in the present model suggested that the activation was initiated from the cell membrane. What’s more, the time frame of the activation was only 15 min, which seemed to be too short to guarantee a synthesis of any new proteins as a result of steroid nuclear receptor-mediated transcriptional activation. Taken together, we conclude that the activation of Erk1/2, JNK, and p38 MAPKs observed here was unequivocally due to a nongenomic GC action. This result justified our previous hypothesis that GC activated MAPKs through a classic GR-independent nongenomic pathway (5, 6, 7); and, for the first time, we demonstrated that this also occurred in the embryonic hippocampal neurons.
Erk1/2, JNK, and p38 MAPKs are important intracellular signaling molecules in mediating a variety of cellular and systematic functions in the neural system, like synaptic plasticity and memory formation (30, 31). These two important biological processes can also be affected greatly by GC (32). Whether there exists any relationship between the activation of MAPKs here and the GC’s regulatory effects on synaptic plasticity and memory formation remains to be investigated. On the other hand, Erk1/2, JNK, and p38 MAPKs are also associated with the survival or death of neuron cells. Most evidence so far suggested that Erk1/2 activation contributed to neuroprotection against various damaging insults (33), whereas JNK and p38 activation seemed to be involved in the mediation of neuronal cell apoptosis or death (34). GC was reported to have regulatory functions on excitotoxic neuronal damage (13). It is not known whether the nongenomic activation of the MAPKs by GC reported here would have the same effects on the process and on regulation of neuronal survival or death during the development of neurons.
The hippocampus is one of the principle GC targets and also one of the most vulnerable regions in the brain to neuronal loss in response to various neurotoxic factors. GC has been well documented as an enhancer of multiple neurotoxicity insults (13), and the action was supposed to be mediated through GR (35). However, it is rather controversial whether GC itself can have any effects on neuronal survival. Kimonides et al. (36) and Lu et al. (37) reported that GC had neurotoxic effects on primary hippocampal neurons measured by -tubulin III assay or terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay after B treatment. In contrast, Behl et al. (38) and Mulholland et al. (39) showed that there was no detectable damage of neurons when they were exposed to GC alone, as shown by MTT reduction assay or propidium iodide uptake assay. As revealed by both LDH release and MTT reduction, our results showed that 24 h of exposure of B at concentrations ranging from 10–11–10–5 M had no significant toxic effects on GR-deficient neurons. We guess that this result might be partially attributed to the lack of classic GR in those neurons.
Taken together, our results of cultured GR-deficient embryonic hippocampal neurons provided an ideal cellular model for the study of GC’s nongenomic effects in the neural system. We also demonstrated that GC rapidly and nongenomically activated Erk1/2, JNK, and p38 MAPKs in those GR-deficient neurons but had no detectable effects on their neuronal viability.
Acknowledgments
The authors thank Professor Xiao Ping Chen of Tong Ji University for her kind editorial help.
Footnotes
This work was supported by grants from the National Natural Science Foundation of China (nos. 39330100, 39840019) and the Major State Basic Research Development Program of China (G1999054003).
Abbreviations: B, Corticosterone; B-BSA, BSA-conjugated corticosterone; DIV, days in vitro; E18, embryonic day 18; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; GC, glucocorticoid; GR, GC receptor; HS, horse serum; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAP2, microtubule-associated protein-2; MR, mineralocorticoid receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; NMDA, N-methyl-D-aspartic acid; TRITC, tetramethyl rhodamine isothiocyanate.
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