Increased Binding Activity at an Antioxidant-Responsive Element in the Metallothionein-1 Promoter and Rapid Induction of Metallothionein-1 and -2 in Response to Cerebral Ischemia and Reperfusion

Abstract

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are potentially involved in zinc homeostasis and free radical scavenging. The expression pattern of MT-1 and the binding activity of various MT-1 promoter elements were investigated after mild focal cerebral ischemia in the rat. Transient focal ischemia was induced by occluding both common carotid arteries and the right middle cerebral artery for 30 min. By the use of real-time quantitative PCR, a 10-fold increase in MT-1 and -2 mRNA levels was found in the cortex 24 hr after reperfusion. In situ hybridization and immunocytochemistry showed a rapid increase in MT-1 and -2 mRNA and MT protein in endothelial cells of microvessels at 6 hr after reperfusion, followed by an increased expression in astrocytes of the infarcted cortex at 24 hr after reperfusion. The early increase in MT expression preceded an increase in cerebral edema measured with T2-weighted magnetic resonance imaging. Gel shift assays were performed on nuclear extracts prepared from cortices before and at 6 and 24 hr after reperfusion. Increased binding activity was found at an antioxidant/electrophilic response element (ARE) sequence in the MT-1 promoter at 6 hr with a lower and variable binding activity at 24 hr after reperfusion. Constitutive binding activity was found for Sp1 and a metal response element in the MT-1 promoter that did not increase after ischemia and reperfusion. This study suggests a role of ARE-binding proteins in inducing cerebral MT-1 expression and implicates MT-1 as one of the early detoxifying genes in an endogenous defense response to cerebral ischemia and reperfusion.Key words: antioxidant response element; cerebral ischemia; edema; electrophoretic mobility shift assay; magnetic resonance imaging; metallothionein; promoter Transient focal cerebral ischemia of 1-2 hr duration induces increased transcription of various genes that either serve to protect cells against the consequences of the insult or activate a cell death pathway (Koistinaho and Hokfelt, 1997). Gene expression is profound in the infarct border zone rather than in the infarct core areas where gene transcription and translation are suppressed and cells die rapidly (Akins et al., 1996). In contrast to focal ischemic insults, global cerebral ischemia elicits a pathology with a clearly delayed time course, and here the gene expression patterns are found in the affected areas before overt pathology (Massa et al., 1996). In the present study we used a model of mild focal cerebral ischemia, in which cell death progresses in a delayed manner (Du et al., 1996;van Lookeren Campagne et al., 1999b). The extended time course of infarct development in this model may resemble the situation in a subset of patients with acute stroke (Pantano et al., 1999) and allows us to study gene expression patterns that potentially fulfill a role early in infarct development. Among the early response genes are metallothionein-1 and -2 (MT-1 and -2). These genes belong to the phase II-responsive detoxifying genes that co...

Electrophile Response Element-mediated Induction of the Cystine/Glutamate Exchange Transporter Gene Expression

“…EpREs have been found in the promoter regions of various genes, e.g. those encoding glutathione S-transferase, NADPH: quinone oxidoreductase, the heavy and light subunits of ␥-GCS, ferritin L subunit, metallothionein-1, and thioredoxin (9,10,(22)(23)(24)(25). The common function of these proteins is for cell defense against xenobiotics and oxidants.…”

Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress

“…MT transcripts are most significantly upregulated in cells neighboring sites following ischemic damage with increased expression lasting for extended periods following trauma. 41 MT1 and hemeoxygenase 1 (HO-1) genes, which were also significantly upregulated, have been shown to be increased following hypoxic preconditioning. 42 Thus, taken together, there is evidence not only for oxidative stress responses within the schizophrenia prefrontal cortex but also support that this stress may be partially or specifically due to hypoxic conditions.…”

A Small-Molecule-Inducible Nrf2-Mediated Antioxidant Response Provides Effective Prophylaxis against Cerebral IschemiaIn Vivo

“…؊/؊ mouse brain Induction of Nrf2-dependent gene expression normally occurs in the ischemic penumbra after stroke, raising the possibility that Nrf2 activation may be an endogenous response to limit stroke damage (van Lookeren Campagne et al, 1999;Campagne et al, 2000;Laxton et al, 2001;Liverman et al, 2004). Notably, data from gel-shift assays indicated that binding to the ARE sequence found in Nrf2 target genes such as metallothionein-1 and metallothionein-2 was upregulated after transient forebrain ischemia in rat (Campagne et al, 2000).…”