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.