Introduction

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The mood-stabilizing drug sodium valproate (VPA) is a branched-chain saturated fatty acid (2-propylpentanoic acid) that possesses anticonvulsant effects in the treatment of epilepsy (Penry and Dean, 1989). Recent evidence has established that VPA has a mood-stabilizing effect; this drug is quickly becoming a first line treatment for bipolar disorder (BD) because it has a broad spectrum of efficacy in patients with this illness (McElroy et al., 1992; Bowden, 1996). Long-term prophylactic treatment with mood-stabilizing drugs is currently recommended for BD. It is believed that chronic treatment with these drugs regulates gene expression (Post, 1992; Hyman and Nestler, 1996). The specific targets of these drugs are, however, poorly understood. Increased Na⁺ channel subunit mRNA levels have been reported after VPA treatment (Yamamoto et al., 1997). VPA has also been shown to decrease myristoylated alanine-rich C kinase substrate expression (Lenox et al., 1996). VPA increases activator protein-1 binding in cultured cells (Chen et al., 1997; Asghari et al., 1998), suggesting that genes containing this consensus sequence in their promoter may be targets of this drug. The available evidence is far from explaining the mechanism of this drug. Therefore, isolation of specific valproate-regulated genes (VRGs) is important, particularly if the products of these genes are found to be relevant to the pathophysiology of BD.

Differential display polymerase chain reaction (PCR) was developed to identify differentially expressed genes and to detect individual mRNA differences in various sets of mammalian cells (Liang et al., 1992). Side by side comparison of RNA samples from different cells allows the identification of differentially expressed genes. Amplified cDNAs can then be used as probes to isolate genes from genomic and cDNA libraries for further molecular characterization. To date, these methods have been successfully applied to isolate differentially expressed genes in cancer (Liang et al., 1992), heart disease (Utans et al., 1994), and diabetes (Nishio et al., 1994). We used this method to isolate differentially expressed genes in rat cerebral cortex after administering VPA for 3 weeks. Cerebral cortex was chosen because this is the region in which abnormalities have been reported in BD patients and where mood stabilizers have been shown to have numerous effects (Song and Jope, 1992; Li et al., 1993). One of these genes is the 78-kDa glucose-regulated protein (GRP78), a molecular chaperone that participates in protein trafficking and endoplasmic reticulum (ER) calcium homeostasis. We have confirmed the specificity and dose-response relationship of GRP78 regulation by VPA. Additionally, VPA treatment resulted in increased GRP78 protein levels.

	Materials and Methods

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Animal Treatment and Cell Culture. Male Sprague-Dawley rats (200-250 g) were injected i.p. with VPA (300 mg/kg) or 0.9% saline (control) once daily for 21 days. Rats were weighed weekly to control for nutritional status, sacrificed by decapitation, and their brains were dissected rapidly in 0.32 M sucrose at 4°C. All areas of cerebral cortex were dissected, pooled for each animal, and used immediately for RNA extraction. Rat C6 glioma cells were grown in a medium containing Dulbecco's modified Eagle's medium and 10% fetal calf serum. Cells were treated at various times in the presence or absence of different concentrations of VPA. Cell viability was greater than 98% as confirmed by tryptan blue exclusion and was not different across drug treatments (data not shown).

Differential Display PCR. Total RNA from tissues and cell lines was isolated using Trizol reagent (Gibco Life Technologies, Gaithersburg, MD) according to the manufacturer's protocol. Chromosomal DNA was digested with RNase-free DNase I (Boehringer Mannheim, Laval, Quebec) for 30 min at 37°C. The concentration of RNA was determined by measuring the absorbance at 260 nm and the purity determined by the 260:280 ratio. Three micrograms of total RNA was subjected to electrophoresis on a denaturing 1% agarose gel to check the integrity of the RNA (18S and 28S rRNA bands).

DNA Cloning and Sequence Analysis. Differentially displayed PCR products were ligated into a pNoTA/T7 vector at 25°C for 30 min with T4 DNA ligase (5 Prime-3 Prime, Inc., Boulder, CO). Competent bacterial cells were transformed with the ligated construct. Plasmid DNA from subclones containing inserts were then purified using a Wizard Miniprep DNA purification system (Promega Biotec, Madison, WI). Inserts were sequenced by the dideoxy chain-termination method (Sanger et al., 1977). The sequencing products were run by electrophoresis on a 6% polyacrylamide-urea gel and identified by autoradiography. Sequences thus derived were compared for homology to the sequences present in the current GenBank database through the National Center for Biotechnology Information with the BLASTN program (Altschul et al., 1990).

Northern Hybridization and Slot Blot Analysis. Total RNA from rat cerebral cortex or C6 glioma cells was applied to nylon membranes using either a slot blot apparatus or by capillary transfer after electrophoresis in 1% agarose/formaldehyde gel, and ultraviolet-cross-linked. Membranes were then prehybridized at 42°C for 3 h in 5× sodium chloride/sodium dihydrogen phosphate/EDTA (SSPE), 50% formamide, 5× Denhardt's, 1.0% SDS, 5% dextran sulfate, and salmon sperm DNA (100 µg/ml) and then hybridized at 42°C overnight in this buffer with differentially displayed cDNA probes derived as described above. cDNA probes were labeled with [-³²P]dCTP by using a random prime labeling method (Feinberg and Vogelstein, 1983). Membranes were then washed at room temperature for 45 min in 2× SSPE and 0.1% SDS twice and at 58°C for 15 min in 0.1× SSPE and 0.1% SDS twice. Membranes were exposed overnight at 80°C to Kodak X-Omat film with an intensifying screen.

Nuclear Runoff. Nuclear runoff assays were performed as previously described (Lofquist et al., 1995). Briefly, C6 cells were treated or untreated with VPA at 1 mM for 1 week. The cells were resuspended in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, and 0.5% Nonidet P-40). Several microliters of cell lysate were examined with a hemacytometer on a phase-contrast microscope to determine whether the cells were free of membrane components. Nuclei were collected by centrifugation. The nuclei were resuspended in 100 µl of glycerol storage buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl₂, and 0.1 mM EDTA). Runoff transcription was initiated by adding 100 µl of reaction buffer [(10 mM Tris-Cl, pH 8.0, 5 mM MgCl₂, 0.3 M KCl, 1 mM dithiothreitol, 40 units/ml RNasin, 1 mM ATP, 1 mM GTP, 1 mM CTP, and 25 µl of [-³²P]UTP (3000 Ci/mmol)] at 30°C for 30 min. DNA was digested by adding 1 µl of 20,000 U/ml RNase-free DNase, whereas protein was digested by adding 10 µl of proteinase K (20 mg/ml). Newly transcribed RNA was purified by 25:24:1 buffered phenol/chloroform/isoamyl alcohol, followed by precipitation with isopropyl alcohol. The membrane with cDNAs applied by slot blotting was then prehybridized at 42°C for 3 h in 5× SSPE, 50% formamide, 5× Denhardt's, 1.0% SDS, and salmon sperm DNA (100 µg/ml), and was hybridized at 42°C overnight in this buffer with the ³²P-labeled newly transcribed RNA. After hybridization, membranes were then washed at room temperature for 45 min in 2× SSPE and 0.1% SDS twice and at 58°C for 15 min in 0.1× SSPE and 0.1% SDS twice. Membranes were exposed overnight at 80°C to Kodak X-Omat film with an intensifying screen.

Immunoblotting Analysis Cells were washed in PBS (100 mM NaCl, 100 mM Na₂HPO₄/NaH₂PO₄, pH 7.5) and then suspended in cell lysis buffer (150 mM NaCl, 15 mM Mg₂Cl, 1 mM EGTA, 50 mM HEPES-KOH, 10% glycerol, 1% Triton X-100, pH 7.5) containing 1 mM phenylmethylsulfonyl fluoride and 2 µM benzamidine. After gentle agitation for 30 min at 4°C, cell lysates were centrifuged for 10 min at 12,000g. Supernatants were used for immunoblotting analysis. The protein concentration was determined according to the method of Bradford (1976).

Analysis and Interpretation of Data. All sequence data were aligned with sequences available in GenBank. Data from slot blot, nuclear runoff, and immunoblotting studies were obtained by densitometric analysis of autoradiograms using the Northern Exposure program from ImagExperts, Inc. (Oakville, Ontario, Canada). Results were expressed as a percentage of control. Changes in gene expression after drug treatment were expressed as the mean ± S.E.M. from three separate experiments. Statistical significance of differences between means was determined by Student's t tests or one-way ANOVA.

	Results

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Identification of Candidate VRGs by Differential Display PCR. With differential display PCR, six cDNA PCR products were differentially expressed in the rat cerebral cortex between controls and rats treated for 3 weeks with VPA at 300 mg/kg i.p.. The six differentially expressed cDNA PCR products were then excised from the 6% denaturing polyacrylamide gel and reamplified with the same primers used in differential display PCR. These six candidate VRGs were purified and ligated into a pNo-TA/T7 cloning vector. The differentially expressed candidate VRG cDNA inserts were sequenced and named: VRG1 [112 base pairs (bp)], VRG2 (106 bp), VRG3 (146 bp), VRG4 (105 bp), VRG5 (62 bp), and VRG6 (386 bp). VRG1 and VRG3-6 are previously unidentified cDNA fragments and showed no significant homology (<85%) to any entry in existing nucleic acid databases available through the National Center for Biotechnology Information. VRG2, however, displayed 100% homology to the GRP78, which is a stress-induced molecular chaperone. Figure 1 shows an autoradiograph of VRG2 demonstrating increased expression after VPA treatment. The cDNA fragments of candidate VRGs were radioactively labeled for Northern blot hybridization. Total RNA from cerebral cortex of rats treated for 3 weeks with VPA at 300 mg/kg i.p. and from control animals were used for Northern blot analysis. VRG1, VRG2, VRG4, and VRG6 were verified to be differentially expressed by Northern blot analysis (Fig. 2), whereas no expression of VRG3 or VRG5 was detected in rat brain.

View larger version (73K): [in this window] [in a new window]   Fig. 1.   Differential display of mRNA in control (Ctl) and sodium VPA (300 mg/kg i.p. for 3 weeks)-treated rats. Each lane represents tissue from one animal. A sample of 0.2 µg of total RNA from each animal was used for differential display PCR. Arrows point to the differentially displayed product VRG2.

View larger version (67K): [in this window] [in a new window]   Fig. 2.   Northern blot analysis of candidate VRGs in rat cerebral cortex and C6 glioma cells. Total RNA was isolated from rat cerebral cortex after 3 weeks of sodium VPA (300 mg/kg i.p.) treatment or from rat C6 glioma cells after 1 week of 1-mM VPA compared with untreated control (Ctl) tissues. Twenty-five micrograms of RNA was used for Northern blot analysis using probes prepared from products of differential display (VRG1, VRG2, VRG4, and VRG6) or the housekeeping gene GAPDH.

Dose-Response Relationship of VRG Expression. To further investigate the effect of VPA on the differentially expressed candidate VRGs (VRG1, VRG2, VRG4, and VRG6), we determined VPA dose-response curves for these genes. First, VRG1, VRG2, VRG4, and VRG6 were confirmed to be differentially expressed by Northern blot analysis in C6 glioma cells treated with 1 mM VPA for 1 week. We used C6 cells to study the dose-response effect on GRP78 because 1) C6 cells are commonly used to study the mechanism of mood stabilizers including VPA, and 2) GRP78 is known to be expressed in this cell line (Brostrom et al., 1991; Chen et al., 1996, 1997). As shown in Fig. 2, all four genes were increased by VPA in C6 glioma cells as in rat cerebral cortex. Next, C6 glioma cells were exposed to VPA at different concentrations of 0, 0.25, 0.5, and 1 mM for 1 week and VRG1, VRG2, VRG4, and VRG6 mRNAs were quantitated by slot blot hybridization. These concentrations represent serum levels commonly used to treat patients (Bowden et al., 1996). The results indicated that VPA increased VRGs' mRNA expressions in a dose-dependent manner and that VPA dose-response curves for these genes showed similar patterns (Fig. 3).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   The effects of different doses of VPA on candidate VRG expression. Rat C6 glioma cells were grown for 1 week with 0.25, 0.5, and 1 mM VPA. Control cells (Ctl) were treated identically but without addition of drug. Total RNA was analyzed by slot blot hybridization using [-32P]dCTP-labeled probes prepared from products of differential display (VRG1, VRG2, VRG4, and VRG6) or the housekeeping gene GAPDH. Autoradiograms were quantitated by densitometry. Results are the mean ± S.E.M. of triplicate determinations. *p < .05 and **p < .01 significantly different from controls by ANOVA.

Effects of VPA on GRP78 and Heat Shock Protein 70 (HSP70) mRNA Levels. HSP70 is a related stress-induced molecular chaperone. To study the specificity of VPA on GRP78 mRNA expression and to further verify the effect of VPA on GRP78, HSP70 and GRP78 mRNA levels were measured with Northern blot analysis using HSP70 and GRP78 coding region sequences as probes in rat cerebral cortex and C6 glioma cells. Seven hundred sixty-seven-bp GRP78 and 495-bp HSP70 cDNA probes were generated by reverse transcription-PCR. The GRP78 upstream primer (5'-CCACCGTAACAATCAAGGTC-3') and downstream primer (5'-CACGTGAGCAACTGCTAATG-3'), which are located in regions +276 to +295 and +1024 to +1043 of the GRP78 gene, and HSP70 upstream primer (5'-GGTGCTGACCAAGATGAAG-3') and downstream primer (5'-CAGAGAGTCGATCTCCAGG-3'), which are located in regions +543 to +561 and +1020 to +1038 of HSP70 gene were used, respectively, for PCR amplification. GRP78 and HSP70 cDNA probes from PCR were confirmed by sequencing analysis. As shown in Fig. 4, VPA increased GRP78, but not HSP70, mRNA in both rat cerebral cortex and C6 glioma cells. VPA at 1 mM also showed time-dependent increases of GRP78 mRNA after treatment for 1 and 5 h, and 1, 3, 5, and 7 days. After 7 days of treatment with 1 mM VPA, the drug was subsequently withdrawn from cells to determine the reversibility of GRP78 induction and rule out cell death as a result of VPA treatment. As shown in Fig. 5, the GRP78 mRNA level was decreased from 65 ± 9% to 16 ± 8% at 6 h, and to 6 ± 8% at 24 h after VPA withdrawal.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis of GRP78 and HSP70 in the rat cerebral cortex and C6 glioma cells. Total RNA was isolated from rat cerebral cortex after 3 weeks of VPA (300 mg/kg i.p.) and from C6 glioma cells grown for 1 week with 1 mM VPA and untreated control (Ctl) tissues. Total RNA was used by Northern blot analysis using probes prepared from GRP78 cDNA, HSP70 cDNA, or the housekeeping gene GAPDH.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Time course and withdrawal of VPA on GRP78 gene expression. C6 glioma cells were treated with 1 mM VPA for 1 and 5 h (h), and 1, 3, 5, and 7 days (d). VPA was withdrawn at 2 h, 6 h, 1 day, and 2 days after a 1-week treatment with VPA at 1 mM. Control cells were treated identically but without addition of drug. Autoradiograms were quantitated by densitometry. Results are the mean ± S.E.M. of triplicate determinations. *p < .05 and **p < .01 significantly different from control by ANOVA.

Effect of Other Mood Stabilizers such as Lithium and Carbamazepine on GRP78 mRNA Abundance. To further investigate possible similarities between VPA and the two other mood stabilizers commonly used to treat BD, we determined the effect of therapeutically relevant concentrations of lithium chloride (LiCl) and carbamazepine (CBM) on GRP78 gene expression by slot blot analysis. As shown in Fig. 6, 0.5 to 2 mM LiCl and 0.025 and 0.05 mM CBM had no effect on GRP78 gene expression. VPA at 0.5 mM and 1.0 mM significantly increased GRP78 gene expression by 31.6 ± 6.2% (p < .05) and 50.5 ± 8.9% (p < .05,) respectively. CBM at 0.1 mM also increased GRP78 gene expression by 25.2 ± 5.3% (p < .05). There was no difference in the level of hybridization of the housekeeping gene glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) compared to control cells after any of these treatments.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effects of sodium VPA, LiCl, and CBM on GRP78 gene expression. C6 glioma cells were grown for 1 week with VPA at concentrations of 0.25, 0.5, and 1 mM; LiCl at 0.5, 1, and 2 mM; CBM at 0.025, 0.05, and 0.1 mM. Control cells were treated identically but without addition of drug. Total RNA was analyzed by slot blot hybridization using [-32P]dCTP-labeled probe of GRP78 or the housekeeping gene GAPDH. Autoradiograms were quantitated by densitometry. Results are the mean ± S.E.M. of triplicate determinations. *p < .05 significantly different from control by ANOVA.

Effect of VPA on GRP78 Gene Transcription. To determine whether the changes in the GRP78 mRNA level is contributed to by the induction of GRP78 transcription, we used nuclear runoff analysis to measure the effect of VPA on transcription of the GRP78 gene. Five micrograms of empty plasmid DNA or plasmid DNA with GRP78 cDNA insert was blotted to nylon membranes. C6 glioma cells were treated with and without VPA at 1 mM for 1 week. Nuclei were isolated from control and VPA-treated cells. Newly transcribed RNAs from nuclei of control and VPA-treated cells were hybridized with GRP78 cDNA and empty plasmid DNA blots. As shown in Fig. 7, VPA treatment induced a significant increase of GRP78 gene transcription in C6 glioma cells.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Effect of sodium VPA on GRP78 transcription. Nuclei were isolated from C6 glioma cells after treatment without (Ctl) or with 1 mM VPA. Equal amounts (cpm) of 32P-labeled nuclear runoff RNA were hybridized to slot blots containing 5 µg of GRP78 plasmid cDNA and the empty plasmid vector pNoTA/T7 DNA (TA/T7). Results are the mean ± S.E.M. of triplicate determinations. *p < .05 significantly different from control by ANOVA.

Effect of VPA on GRP78 Protein Level. To determine whether the changes in the GRP78 mRNA level contribute to GRP78 translation, we measured the effect of VPA on the GRP78 protein level using immunoblotting analysis. Figure 8 shows that VPA treatment of C6 glioma cells for 1 week at therapeutically relevant concentrations (0.25-1.0 mM) (Bowden et al., 1996; Chen et al., 1996, 1997; Asghari et al., 1998) increased the GRP78 protein level in a dose-dependent manner.

View larger version (49K): [in this window] [in a new window]   Fig. 8.   Effects of VPA on GRP78 protein levels. C6 glioma cells were treated with VPA at 1 mM for 1 week. Control cells (Ctl) were treated identically but without addition of drugs. Five micrograms of protein was analyzed by immunoblotting analysis using a GRP78 antisera. Autoradiograms were quantitated by densitometry. Results are the mean ± S.E.M.. *p < .05 significantly different from control by ANOVA.

	Discussion

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Differential display PCR revealed four differentially expressed gene products in rat cerebral cortex after i.p. injection with VPA (300 mg/kg) for 3 weeks. Although the identity of three of these gene products is presently unknown, the expression of a cDNA with 100% homology to the gene coding for the molecular chaperone GRP78 was markedly increased after VPA treatment. The expression of this cDNA in rat cerebral cortex was confirmed by Northern blot hybridization. Quantitative slot blot analysis demonstrated that VPA increased the expression of these candidate genes in a dose-dependent manner at therapeutically relevant concentrations in rat C6 glioma cells. These results strongly suggest that specific genes, including the molecular chaperone GRP78, are regulated by VPA treatment. Furthermore, GRP78 mRNA expression was also increased by treatment with the closely related mood-stabilizing anticonvulsant carbamazepine, but not lithium chloride. These results are consistent with the current notion that psychotropic drug treatment may act through long-term adaptational changes in the central nervous system (CNS) to bring about their therapeutic effects (Post, 1992; Hyman and Nestler, 1996).

GRP78 is induced as part of the stress response in eukaryotic cells (Brostrom and Brostrom, 1998). This gene product functions in endoplasmic reticulum (ER) glycoprotein trafficking, possesses molecular chaperone activity, and protects cells from the deleterious effects of damaged proteins. GRP78 is constitutively expressed in the ER, and transcription of the GRP78 gene is elevated in response to malfolded proteins and treatments that interfere with protein glycosylation, protein trafficking, and ER storage of calcium (Kim et al., 1987; Wooden et al., 1991; Little et al., 1994). The molecular chaperone activities of GRP78 make it critical to ER functioning. GRP78 assists in the folding of the newly synthesized proteins and the acquisition of their correct tertiary and quaternary structure (Bole et al., 1986; Lee, 1987). Malfolded protein binds to GRP78 ultimately to be disposed of by nonlysosomal proteolytic process (Bonifacino and Lippincott-Schwartz, 1991; Werner et al., 1996).

Our results indicate that chronic VPA treatment increases both GRP78 mRNA and protein levels at therapeutically relevant concentrations in rat C6 glioma cells. Although the functional relevance of these changes is not yet known, the fact that VPA increased GRP78 protein levels suggests that these changes may be relevant to the pharmacological action of VPA. It has been previously reported that classical inducers of stress proteins such as thapsigargin lead to 6-fold or higher induction of GRP78 in various cell lines (Miles et al., 1994; Hsieh et al., 1996). In the present study, VPA induced GRP78 more modestly than thapsigargin, suggesting that the effect of VPA on GRP78 may be due to a different mechanism. Thapsigargin depletes ER Ca²⁺, which in turn inhibits ER protein processing and cellular protein synthesis. These changes after thapsigargin treatment secondarily lead to GRP78 induction, which may protect the cell against damage or cell death that would ultimately occur after ER Ca²⁺ depletion. Our results suggest that GRP78 induction by VPA is not the result of ER damage or insult. Indeed, withdrawal of VPA after 1 week of treatment resulted in a return to baseline of GRP78 mRNA levels, which occurs as early as 6 h.

There is increasing interest in the role of GRP78 in CNS disease and injury. GRP78 expression is induced after CNS injuries such as seizures, global ischemia, and acute trauma (Lowenstein et al., 1994), and in neurodegenerative diseases like Alzheimer's (Hamos et al., 1991). GRP78 induction by chronic VPA treatment may play a neuroprotective role by clearing malfolded proteins. Recently, chronic lithium treatment at therapeutically relevant concentrations was found to protect cultured rat cerebellar, cerebral cortical, and hippocampal neurons against glutamate-induced excitotoxicity (Nonaka et al., 1998). In our study, chronic lithium treatment did not alter GRP78 gene expression, suggesting that these drugs may both have neuroprotective effects, but these effects occur through different pathways.

It has recently been shown that GRP78 not only participates in protein trafficking but is an important Ca²⁺-binding protein in the ER (Lièvremont et al., 1997). Overexpression of exogenous hamster GRP78 in HeLa cells induced increases of the ER Ca²⁺ storage capacity (Lièvremont et al., 1997). Therefore, GRP78 may play an important role in the control of the ER luminal Ca²⁺ homeostasis. Increased expression of the molecular chaperone GRP78 after VPA suggests that this drug may regulate Ca²⁺ homeostasis. Consistent with this hypothesis is the fact that VPA has been shown to inhibit 5-hydroxytryptamine-induced increase of intracellular free calcium in C6 glioma cells (Yamaji et al., 1996). Lithium was also found to inhibit N-methyl-D-aspartate receptor-mediated calcium influx (Nonaka et al., 1998). Because increased intracellular calcium levels have consistently been reported in blood cells obtained from patients with BD (Dubovsky et al., 1989; Emamghoreishi et al., 1997), a novel target for VPA that could regulate intracellular Ca²⁺ levels is of potential importance.

HSP70 is another molecular chaperone that is induced by stress. HSP70 mRNA levels were not regulated by VPA treatment, suggesting specific regulation of GRP78 by VPA. It has been shown that the stress response is associated with the independent induction of two groups of evolutionarily conserved proteins, HSPs and glucose-regulated proteins (GRPs). HSP70 induction is fostered by damaged or aberrant cytosolic, nuclear, and mitochondrial proteins caused by thermal or chemical insult, whereas the accumulation of abnormally or poorly processed proteins within the lumen of the ER causes GRP78 to be synthesized (Brostrom and Brostrom, 1998). Increased GRP78, but not HSP70, expression after VPA treatment suggests that this drug may specifically regulate protein processing linked to GRP78 induction.

The increase of GRP78 mRNA expression by VPA appears to occur in part at the level of transcription based on the results from the nuclear runoff assay. The dose-response curves for GRP78 and the other candidate VRGs are highly similar, suggesting that VPA could regulate these genes through a common mechanism. Indeed, it is possible that there is a VPA-responsive sequence in the promoter of these genes similar to that found for ethanol (Hsieh et al., 1996). Although VPA has been found to increase AP-1 DNA binding activity (Chen et al., 1997; Asghari et al., 1998), there is no known AP-1 site in the GRP78 promoter, suggesting the potential importance of other elements (Resendez et al., 1988). The identity of a VPA responsive cis-acting element and isolation of trans-acting factors need to be further investigated. The role of the other unidentified candidate VRGs induced by VPA remains to be determined. Although all of the VRGs isolated here show induction at therapeutically relevant VPA concentrations, further studies are needed to characterize the therapeutic importance of these changes in gene expression.

In conclusion, chronic VPA treatment resulted in the increased expression of several mRNAs in rat cerebral cortex and C6 glioma cells, one of which has been identified as the molecular chaperone GRP78. The results of the present study demonstrate the potential utility of differential display PCR to identify the targets of psychotropic drugs and suggest potentially important and previously unidentified targets for VPA.