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