Valproic acid suppresses Nrf2/Keap1 dependent antioxidant protection through induction of endoplasmic reticulum stress and Keap1 promoter DNA demethylation in human lens epithelial cells

Highlights

• Valproic acid (VPA) activates ER stress mediated UPR in human lens epithelial cells.

• VPA induces Ca²⁺ release from ER to cytoplasm in human lens epithelial cells.

• VPA suppresses Nrf2 dependent antioxidant protection in human lens epithelial cells.

• VPA demethylates Keap1 promoter by modifying DNA demethylation enzyme expressions.

Abstract

Recent epidemiological studies confirm the prevalence of cataract in epileptic patients. Similarly, the drugs used to treat epilepsy also show the connection with increased cataract formation. In this present study, we investigated the suppression of Nrf2/Keap1 dependent antioxidant protection through induction of endoplasmic (ER) stress and Keap1 promoter DNA demethylation in human lens epithelial cells (HLECs) treated with valproic acid (VPA), an antiepileptic drug. 20 mM VPA induces ER stress and activates the unfolded protein response (UPR) within 4 h by activating the ER stress sensor proteins, such as PERK, IRE1α, and ATF6 in HLECs. Consequently, the integrated ER stress signals, such as eIF2α, ATF4, BiP, and CHOP are altered accordingly to induce ER-Ca²⁺ release, reactive oxygen species (ROS) overproduction, and cell death in HLECs treated with VPA. VPA also suppresses the Nrf2, catalase, and glutathione reductase expressions with significant increases in Keap1 protein. Bisulphite genomic DNA sequencing reveals the promoter DNA demethylation in the Keap1 promoter, which results in the overexpression of Keap1 mRNA and protein in HLECs treated with 20 mM VPA. VPA also alters the expression profiles of passive DNA demethylation pathway enzymes such Dnmt1, Dnmt3a, Dnmt3b, and active DNA demethylation pathway enzyme, TET1 leading to DNA demethylation in the Keap1 promoter of HLECs. Overexpressed Keap1 decreases the Nrf2 level, thereby abolishing the Nrf2 dependent antioxidant protection. This might be responsible for lenticular proteins oxidation and cataract formation.

Introduction

Epilepsy is a most common neurological disorder in the United States. Various epidemiological studies indicate the associations between cataract prevalence and epilepsy is equivalent to association of cataract with aging, diabetes, arterial hypertension and smoking (Hanhart et al., 2010, Isaac et al., 1991, Kato et al., 1990, Kinoshita et al., 2004, Kuwabara et al., 2009, Mathers et al., 1987). The onset of epilepsy is due to over-synchronous firing of brain neurons (Kerr et al., 2013) and these firing patterns are modulated by a major excitatory neurotransmitter, glutamate, through the family of glutamate receptors (Kim et al., 2004).

The interaction of glutamate with postsynaptic membrane glutamate receptors and its overstimulation increases intracellular Ca²⁺ by direct opening of postsynaptic ion channels and secondarily affecting calcium homeostatic mechanisms (Chen et al., 2000). Since, glutamate receptors, especially GluR2 directly open the postsynaptic ion channels; they are eventually considered as a drug target of epilepsy (Rogawski, 2011, Stephen and Brodie, 2011). However, drugs used to treat epilepsy also showed associations with cataract formation (Isaac et al., 1991, Kinoshita et al., 2004, Mathers et al., 1987), because of the existence and overexpression of GluR2, RNA editing, and membrane localization in the lens, which results in Ca²⁺ influx in the lens leading to cataract formation (Barbon et al., 2006, Farooq et al., 2012, Sawada et al., 2009).

There are several drugs used for epilepsy treatment, such as carbamazepine, lamotrigine, phenobarbital, phenytoin and valproic acid (VPA), with alone or multidrug therapy. VPA is widely used anticonvulsant agent for suppressing epileptic seizures, depression and migraine headaches and also a mood stabilizer for the treatment of manic depression or bipolar disorder (Singh et al., 2012, Singh et al., 2005, Tunnicliff, 1999). Carbamazepine, phenobarbital and phenytoin are reported to induce cataract in small numbers of epileptic patients (Bar et al., 1983, Hanhart et al., 2010, Mathers et al., 1987). However, in utero exposure of VPA induced the formation of bilateral congenital cataracts in a child out of 37 children (Glover et al., 2002), and these congenital cataracts have little to do with age-related cataracts (ARCs). VPA is never prescribed during pregnancy in the United States and Canada, because of its teratogenic effects known as fetal valproate syndrome, which includes multiple birth defects, dysmorphic facies, developmental delay, learning difficulties and behavioral problems (Wlodarczyk et al., 2012).

VPA is known to induce rapid dose-dependent hyperacetylation of the histones H3 and H4, and can cause growth arrest and induce differentiation of transformed cells in culture (Gurvich et al., 2004). VPA also down-regulates numerous structural maintenance of chromatin proteins, DNA methyltransferases (Dnmts), and heterochromatins leading to chromatin decondensation (Marchion et al., 2005). Since the inhibitors of histone deacetylation cause epigenetic DNA modifications (Dong et al., 2010, Milutinovic et al., 2007, Vire et al., 2006), VPA also induces DNA demethylation in the non-replicating cells by a dose dependent manner (Detich et al., 2003, Dong et al., 2008). In addition, VPA stimulates intracellular reactive oxygen species (ROS) (Defoort et al., 2006, Na et al., 2003) leading to the activation of redox-sensitive transcription factors that interact with the antioxidant response element-driven genes (Achachi et al., 2005, Kawai and Arinze, 2006, Sands et al., 2000).

We found the cataractogenic stressors such as hypoxia with low glucose (Elanchezhian et al., 2012b) or high glucose (Ikesugi et al., 2006), homocysteine (Elanchezhian et al., 2012a), and galactose (Ikesugi et al., 2006), induce endoplasmic reticulum (ER) stress mediated activation of unfolded protein response (UPR) and overproduction of ROS and cell death in lens epithelial cells (LECs). We also reported the suppression of nuclear factor erythroid-2-related factor 2 (Nrf2; also known as NFE2L2) dependent antioxidant protection due to ROS overproduction in LECs (Elanchezhian et al., 2012a, Elanchezhian et al., 2012b). Recently, we found the promoter DNA demethylation of Kelch like ECH-associated protein 1 (Keap1), which is a negative regulator of Nrf2, in human lens epithelial cells (HLECs) as well as in diabetic cataractous lenses (Palsamy et al., 2012). These sequential events might be responsible for ARC formation in humans. Here, we hypothesized that VPA also induces suppression of Nrf2/Keap1 dependent stress protection through induction of ER stress and Keap1 promoter DNA demethylation in HLECs. To experiment this hypothesis, we have studied the UPR activation, Ca²⁺ release from ER, ROS production, and suppression of Nrf2/Keap1 dependent stress protection in HLECs. In addition, we also analyzed the promoter DNA demethylation status of Keap1 gene along with expression profiles of DNA methylation and demethylation pathway enzymes. Overall, this work reveals the VPA mediated ER stress activation, Ca²⁺ release from ER, ROS overproduction, and loss of Keap1 promoter methylation as a result of altered DNA demethylation pathway enzymes that leads to the failure of Nrf2 dependent antioxidant system in HLECs.