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

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that are po tentially involved in zinc homeostasis and free radical scavenging. The exp ression pattern of MT-1 and the binding activity of various MT-1 promoter e lements were investigated after mild focal cerebral ischemia in the rat. Tr ansient focal ischemia was induced by occluding both common carotid arterie s 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 i n the cortex 24 hr after reperfusion. In situ hybridization and immunocytoc hemistry showed a rapid increase in MT-1 and -2 mRNA and MT protein in endo thelial cells of microvessels at 6 hr after reperfusion, followed by an inc reased expression in astrocytes of the infarcted cortex at 24 hr after repe rfusion. The early increase in MT expression preceded an increase in cerebr al edema measured with T2-weighted magnetic resonance imaging. Gel shift as says were performed on nuclear extracts prepared from cortices before and a t 6 and 24 hr after reperfusion. Increased binding activity was found at an antioxidant/electrophilic response element (ARE) sequence in the MT-1 prom oter at 6 hr with a lower and variable binding activity at 24 hr after repe rfusion. Constitutive binding activity was found for Sp1 and a metal respon se element in the MT-1 promoter that did not increase after ischemia and re perfusion. This study suggests a role of ARE-binding proteins in inducing c erebral MT-1 expression and implicates MT-1 as one of the early detoxifying genes in an endogenous defense response to cerebral ischemia and reperfusi on.