Cell signaling of the neuroprotective cytokines

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Introduction : Recently, we reported that Stat3 was activated following cerebral ischemia in mouse [1]. However, little is known about the function of activated Stat3 in the ischemic brain. Here we demonstrate the increased expression of activated Stat3 promotes cell death in clutured astrocytes.Methods : Astrocytes from the forebrains of newborn rats were cultured as described [2] with some modifications. Recombinant adenovirus expressing Stat3 wild type (St3.Wt), Stat3 dominant negative type (St3.DN) and LacZ were constructed. Astocytes were exposed to adenovirus vector expressing Stat3.Wt, Stat3.DN or LacZ (multiplicity of infection 10) for 2h and incubated with a virus free fresh medium up to 72h. At 72h later, astrocytes were homogenized in lysis buffer and immunoblotted with antibody against Stat3, p-Stat3 (Tyr-705) and beta-actin. Cell death was quantified by LDH assay. Caspase-3 like activity was evaluated at 12h, 24 and 48h after the virus inoculation using a caspase-3 fluorometric protease assay kit.Results : Western blots analysis revealed feat Ad.St3.wt remarkably induced p-Stat3(Tyr-705) while Ad.St3.DN and Ad-LacZ did not. These results demonstrated that both Ad.St3.Wt and Ad.St3.DN induced Stat3 protein in cultred astrocytes and only Ad.St3.Wt b could phosphorylate Stat3 at Tyr-705 site and activate Stat3. LDH assay revealed that overexpression of St3.wt promoted cell death while overexpression of St3.DN and LacZ did not. In addition, caspase-3 like activity of the St3.Wt treated group was significantly increased than that of the St3.DN group at 2d after virus inoculation.Conclusion : Our Data have shown that over-expression of Stat3 promotes cell death. Activation of caspase-3 activity may be involved in this cell death mechanism. References : [1] Wen et al.; Neurosci Lett, 303:153-156, (2001), [2] Tanaka J et al.; Glia, 28:85-96 (1999)