Elsevier

Neuropharmacology

Volume 39, Issue 11, October 2000, Pages 2162-2169
Neuropharmacology

Increased expression of endoplasmic reticulum stress proteins following chronic valproate treatment of rat C6 glioma cells

https://doi.org/10.1016/S0028-3908(00)00029-0Get rights and content

Abstract

The anticonvulsant sodium valproate has been shown to be an effective treatment for bipolar disorder, however, its precise mechanism of action has yet to be determined. It has been suggested that adaptational changes in gene expression are critical for valproate's prophylactic effects. Previous studies in our lab have shown that one gene that may be regulated by valproate is the 78-kilodalton glucose-regulated protein (GRP78). We report that treatment of rat C6 glioma cells with valproate can also increase the expression of additional endoplasmic reticulum stress proteins, GRP94 and calreticulin. All three proteins showed similar concentration-dependent increases in messenger RNA abundance. Chronic (seven days) treatment significantly increased GRP78 and GRP94 messenger RNA expression, whereas calreticulin expression increased after both acute and chronic treatment. Increases in mRNA expression corresponded to a similar increase in protein expression. The roles of GRP78, GRP94 and calreticulin as molecular chaperones and calcium binding proteins, suggest that these results might have functional relevance to the therapeutic action of valproate.

Introduction

Bipolar disorder (BD) is a well-characterized mood disorder with clinical features and genetics that strongly indicate a biological basis for the illness. While 60% of BD patients respond well to long-term pharmacotherapy with mood stabilizing drugs, the precise defect of the disorder has yet to be elucidated. It has been proposed that adaptational changes in gene expression are critical to the prophylactic effects of these drugs (Post, 1992).

Sodium valproate (VPA) is a branched chain fatty acid (2-propylpentanoic acid), that was originally prescribed as an anticonvulsant, but has more recently become a first line treatment for bipolar disorder (Pope et al., 1991). In addition to its role in the treatment of epilepsy, VPA has been shown to effectively treat mania when administered acutely as well as helping to prevent relapses when given chronically (Bowden et al., 1994). Like other mood stabilizing drugs, the precise mechanism of action of VPA has yet to be determined.

Our lab has used differential display polymerase chain reaction (PCR) to study the regulation of gene expression by the mood stabilizers, VPA and lithium (Wang and Young, 1996, Wang et al., 1999). One gene shown to be regulated by VPA was identified as the molecular chaperone, 78-kilodalton glucose-regulated protein (GRP78). GRP78, along with GRP94 and calreticulin, comprise the resident endoplasmic reticulum (ER) stress proteins (Gething, 1997). These proteins are all constitutively expressed and act as molecular chaperones capable of binding Ca2+ (Nigam et al., 1994). In addition to its ER lumenal functions, calreticulin has also been shown to have additional cellular functions, including cell adhesion and gene expression (reviewed by Burns et al., 1994). ER stress proteins have been shown to be up-regulated in response to various cellular insults (Gething, 1997). In turn, up-regulation of ER stress proteins has been shown to prevent ER Ca2+ depletion and inhibit unfolded protein aggregation, thus protecting the cell from damage or death (Gething and Sambrook, 1992, Liu et al., 1997, Yu et al., 1999).

As GRP78 was identified as a VPA regulated gene (Wang et al., 1999), an interesting question arises concerning the effects of VPA on the expression of closely related ER stress proteins, GRP94 and calreticulin. This study investigates further the effects of VPA on the regulation of GRP78 in addition to that of GRP94 and calreticulin. The identification of cellular targets that are regulated by mood stabilizers will allow for the future development of novel drugs that target specific abnormalities of mood disorders and have superior efficacy.

Section snippets

Cell culture and treatment

Rat C6 glioma cells (American Type Tissue Collection) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% l-glutamine at 37°C in a 5.0% CO2 atmosphere. Drug treatment was carried out by supplementing the culture media with sodium valproate (ICN Biomedicals Inc). Cell viability was greater than 98% as confirmed by Tryptan Blue exclusion and was not different across drug treatments.

Northern and slot blot hybridization

Isolation of total RNA from rat C6

Results

To determine whether chronic treatment with VPA could increase the expression of grp78, grp94 and calreticulin, rat C6 glioma cells were treated for seven days at 1 mM concentration. This cell line was used since both mood stabilizing drugs and cellular insults can increase ER stress proteins in these cells (Brostrom et al., 1991, Chen et al., 1996, Chen et al., 1997, Chen et al., 1999a, Wang et al., 1999). Following seven days of treatment, total RNA was isolated and expression of grp78, grp94

Discussion

The goal of this study was to examine the effect of VPA on the expression of the ER stress proteins. Previously, our lab used differential display PCR on rat cerebral cortex to identify genes regulated by VPA treatment and found a resulting increase in GRP78 expression (Wang et al., 1999). This finding illustrated the importance of studying the effects of VPA on ER stress proteins, and here we report these effects for GRP78, GRP94 and calreticulin. A dose-dependent relationship between grp78,

Conclusion

Valproate, a commonly prescribed anticonvulsant and mood stabilizing drug, is capable of up-regulating the expression of GRP78, GRP94 and calreticulin, collectively referred to as the ER stress proteins. The roles of these ER stress proteins as molecular chaperones as well as calcium binding proteins, suggest that these results might have functional relevance to the therapeutic action of VPA. The identification of genes that are regulated by anticonvulsant and mood stabilizing drugs may allow

Acknowledgements

This work was supported by a Stanley Foundation Research Grant (L.T.Y.). J.F.W. is a Canadian Psychiatric Research Foundation fellow. L.T.Y. is a career scientist of the Ontario Ministry of Health.

References (32)

Cited by (64)

  • Proteotoxicity and endoplasmic reticulum stress-mediated cell death

    2022, Mechanisms of Cell Death and Opportunities for Therapeutic Development
  • A cell-based, multiparametric sensor approach characterises drug-induced cytotoxicity in human liver HepG2 cells

    2013, Toxicology in Vitro
    Citation Excerpt :

    The reduced cell respiration was compensated by an increase in metabolic activity for a similar time range. The observed reduction of intracellular concentration of ATP led to strong respiratory and metabolic stress and caused an initiation of energy-independent necrotic-like pathways, which was detected in a previous study (Bown et al., 2000). Consequently, metabolic activity levelled off to 82% over the entire experiment.

  • Endoplasmic reticulum: The unfolded protein response is tangled in neurodegeneration

    2012, International Journal of Biochemistry and Cell Biology
    Citation Excerpt :

    Several studies have addressed the UPR as a target for drug therapy to restore protein homeostasis and reduce neurodegeneration. Valproate, a drug widely prescribed in the treatment of bipolar disorder and epilepsy, has been shown to increase the levels of BiP and other ER chaperones and protect against ER stress (Bown et al., 2000; Wang et al., 1999). In addition, valproate has been shown to inhibit GSK-3β activity (Kim et al., 2005).

  • Histone deacetylase inhibition activates transcription factor Nrf2 and protects against cerebral ischemic damage

    2012, Free Radical Biology and Medicine
    Citation Excerpt :

    HDAC inhibitors not only regulate gene expression directly via promoter hyperacetylation but also selectively modulate transcription factors via hyperacetylation that is independent of histones [37]. For example, HDAC inhibitors acetylate the neuroprotective transcription factor Sp-1 [38], as well as glucose-regulated protein 78 [39]. Additionally, these drugs inhibit the excitotoxicity-induced nuclear accumulation of glyceraldehyde-3-phosphate dehydrogenase [40], which has been shown to have a proapoptotic role in neurons.

View all citing articles on Scopus
View full text