Repeated transient sulforaphane stimulation in astrocytes leads to prolonged Nrf2-mediated gene expression and protection from superoxide-induced damage

Abstract

Oxidative stress is a major contributor to slowly developing diseases like Parkinson’s disease, Alzheimer’s disease and cancer and one of the main causes of tissue damage following ischemic insults in the brain. Nrf2 is a transcription factor responsible for much of the inducible cellular defense against oxidative stress. Nrf2 can also be activated by xenobiotics like sulforaphane, a component highly enriched in cruciferous vegetables such as broccoli. Ingestion of broccoli or sulforaphane results in long-term protection against radical damage, although absorbed sulforaphane is cleared from the body within a few hours. Here we have examined whether the prolonged protection induced by sulforaphane is explained by a slow down regulation of the Nrf2 response. Furthermore, to simulate daily ingestion of sulforaphane, we examined the hypothesis that repeated transient sulforaphane stimulation results in an accumulation of Nrf2-mediated gene expression and an increased protection against oxidative damage. The kinetics of sulforaphane-induced Nrf2 response was studied in astrocytes, a cell type known to be highly involved in the defense against oxidative stress in the brain. Sulforaphane stimulation for 4 h induced an Nrf2-dependent increase of Nqo1 and Hmox1 mRNA that remained elevated for 24 h, and the corresponding proteins remained elevated for over 48 h. In addition, peroxide-clearing activity and the levels of glutathione were elevated for more than 20 h after stimulation for 4 h with sulforaphane, resulting in an increased resistance to superoxide-induced cell damage. Repeated sulforaphane stimulation resulted in an accumulation of mRNA and protein levels of Nqo1 and a persistent cell protection against oxidative damage. These findings indicate that brief stimulation of the Nrf2 pathway by sulforaphane results in long-lasting elevation of endogenous antioxidants in astrocytes. The findings also demonstrate that part of this response can be built up by repeated transient stimulation, possibly explaining how intermittent intake of sulforaphane can result in long-term protection from radical-induced disease.

Introduction

Oxidative stress is one of the main causes of tissue damage following ischemic insults in the brain (Kuroda and Siesjo, 1997) and contributes to cell death in slowly developing neurological diseases like Parkinson’s disease and Alzheimer’s disease (Calabrese et al., 2007) as well as in atherosclerosis (Kaneto et al., 2010) and cancer (Benz and Yau, 2008). Oxidative stress occurs when the production of free radicals and other reactive substances exceeds the capacity of the cells natural defense systems. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates the expression of many of the enzymes and free radical scavengers that defend the cell against free radical-induced damage. Thus Nrf2 activation represents a key step in endogenous cellular protection (Copple et al., 2008).

Under normal conditions, most Nrf2 is inactive since it is sequestered in the cytoplasm by its repressor kelch-like ECH-associated protein 1 (Keap1) (Itoh et al., 1999). Keap1 targets Nrf2 for ubiquitinylation and proteasome-mediated degradation (Cullinan et al., 2004). In response to oxidative damage or other reactive chemicals, cysteine residues in Keap1 are modified (Hong et al., 2005, Yamamoto et al., 2008), resulting in a conformational change that releases and activates Nrf2 (Eggler et al., 2005, Kobayashi et al., 2006, Tong et al., 2006). Activated Nrf2 is then transported to the nucleus where it binds to promoters containing the antioxidant response element (ARE) motif (Itoh et al., 1997). Binding of Nrf2 to the ARE upregulates transcription of numerous cytoprotective enzymes that induce glutathione (GSH) synthesis and degrade radicals and aldehydes, resulting in increased protection from a range of toxic substances (Ishii et al., 2000). Among these are the neuroprotective enzymes heme oxygenase-1 (Hmox1) (Alam et al., 1999) and NAD(P)H:quinone oxidoreductase 1 (Nqo1) (Venugopal and Jaiswal, 1996).

Studies using genetically modified mice have further demonstrated the critical role of Nrf2 in the defense against oxidative damage. Nrf2−/− mice develop diseases even from minor exposure to cigarette smoke (Rangasamy et al., 2004), sunlight (Hirota et al., 2005) and other radical inducers, likely because they are unable to activate their radical protection system properly (Chan and Kwong, 2000, Chan and Kan, 1999, Itoh et al., 1997, Ramos-Gomez et al., 2001). Nrf2−/− mice also develop larger infarct volumes following stroke (Shih et al., 2005) and are more prone to develop Parkinson’s disease (Burton et al., 2006). Conversely, mice over-expressing Nrf2 are protected against neurodegeneration caused by Parkinson’s disease or amyotrophic lateral sclerosis (Chen et al., 2009, Vargas et al., 2008) and transplanted astrocytes over-expressing Nrf2 confer neuroprotection following brain injury induced by oxidative stress (Calkins et al., 2005, Jakel et al., 2007).

Xenobiotics, like sulforaphane (SF) from broccoli, are also capable of modifying sulfhydryls in Keap1 resulting in the release and activation of Nrf2 (Dinkova-Kostova et al., 2001, Dinkova-Kostova and Talalay, 2008). Sulforaphane-mediated activation of the Nrf2 pathway protects against stroke in rats (Zhao et al., 2006), 6-hydroxydopamine toxicity in rat organotypic nigrostriatal cocultures (Siebert et al., 2009), kainate-induced cell death in the hippocampus (Rojo et al., 2008) and inhibit tumour development in a number of rodent models (Fahey et al., 2002, Pearson et al., 1983, Talalay et al., 1978, Zhang et al., 1994). Importantly, mice lacking the Nrf2 gene do not acquire cancer protection from broccoli, sulforaphane or other known Nrf2-activating drugs (Iida et al., 2004, Xu et al., 2006). This indicates that the positive effect of broccoli or sulforaphane requires a functional Nrf2 response and that activation of the Nrf2 system is involved in protection from free radical-induced disease.

Astrocytes play an essential role in the cellular antioxidant defense in the brain. They are the main source of GSH and supply the neurons with substrate for glutathione synthesis to improve the neuronal antioxidative reserves (Dringen, 2000, Dringen et al., 1999). Astrocytes remain viable and maintain their metabolic properties longer than neurons in a model of cerebral ischemia (Thoren et al., 2005), allowing astrocytes to maintain their nursing function also during oxygen deprivation. In addition, although Nrf2 is active in neurons, recent results indicate that astrocytes constitute the most important target for Nrf2-stimulating therapy in the brain (Vargas and Johnson, 2009). In response to tert-butylhydroquinone, sulforaphane incubation or over-expression of Nrf2, astrocytes exhibit greater Nrf2 activation than neurons and this astrocytic response protects neurons against oxidative insults (Kraft et al., 2004, Shih et al., 2003). Studies in cultured astrocytes have shown that sulforaphane preconditioning for 48 h upregulates Nqo1 and protects cells against oxidative stress and death after oxygen and glucose deprivation in an Nrf2-dependent manner (Danilov et al., 2009, Kraft et al., 2004). Exactly how Nrf2-activated astrocytes contribute to neuroprotection is still unclear. However, genes regulated by Nrf2 control key steps in e.g. heme metabolism (Alam et al., 2000), reduction of quinones (Itoh et al., 1997) and glutathione synthesis (Shih et al., 2003), mechanisms that are all involved in cell protection.

The pharmacokinetics of sulforaphane in humans indicate that it is cleared from the body within a few hours (Ye et al., 2002) but still offers long-term protection from oxidative stress (van Poppel et al., 1999). Although this suggests that brief stimulation of the Nrf2 pathway is sufficient to cause long-term changes in gene expression, previous studies in astrocytes have only investigated Nrf2-mediated gene expression and protection after constant sulforaphane incubation for 1–2 days (Danilov et al., 2009). At present it is not known how intermittent intake of Nrf2-activating drugs or vegetables can result in long-term protection from radical-induced damage. Therefore, in the present study we have examined the kinetics of two well-known Nrf2-mediated genes, Nqo1 and Hmox1, after exposing the astrocytes briefly (1–4 h), constantly (24 h) or repeatedly (4 h daily, up to 4 days) to sulforaphane. Results show that a brief exposure to sulforaphane was sufficient to induce prolonged Nrf2-mediated Nqo1 and Hmox1 expression and that repeated stimulation results in an accumulation of Nqo1 and sustained protection from oxidative stress. These findings potentially explain how intermittent intake of Nrf2 activators like sulforaphane can induce long-term protection from oxidative-related disease.