Abstract TP107: Stats Regulate Transcription Of 12/15-lox In Mouse Cortical Neurons Under Glutamate-induced Oxidative Stress

Abstract

Background and Purpose— Oxidative stress during reperfusion following cerebral ischemic stroke causes neuronal death and various forms of cerebrovascular injury. 12/15-lipoxygenase (12/15-LOX) is a major contributor to these processes; however, the regulation of 12/15-LOX expression in the ischemic brain is still unknown. In this study, we investigated the transcriptional regulation of 12/15-LOX in primary cortical neurons subjected to oxidative stress. Methods— Immature cortical neurons were treated with glutamate. Western blots were performed for analysis of 12/15-LOX protein level and phosphorylation of STAT1 and 6 in response to glutamate stimuli. Primary mouse cerebral cortical neurons were transfected with small interfering RNA probes targeted to STAT1 or 6 and were used for real time RT-PCR for analysis of changes in 12/15-LOX mRNA level, and cell viability assay following glutamate treatment. Results— In preliminary screening of putative transcriptional factors on the promoters of mouse and human 12/15-LOX genes, we detected multiple putative binding motifs for STATs in the promoters of 12/15-LOX. Following glutamate treatment of primary cortical neurons, phosphorylation of STAT1 (Y701) and STAT6 (Y641), as well as 12/15-LOX protein levels, were significantly increased. Importantly, we found significant increases in mRNA levels of 12/15-LOX in mouse cerebral cortices subjected to ischemia reperfusion, which was paralleled in glutamate-treated primary cortical neurons. Gene silencing of STAT1 or 6 by siRNA transfection in primary cortical neurons reduced the levels of both 12/15-LOX mRNA and protein under glutamate-induced oxidative stress. Moreover, inhibition of STAT1 or 6 by siRNA transfection protected primary cortical neurons against glutamate-induced oxidative stress. Conclusions— This study increases our understanding of the transcriptional regulation of the 12/15-LOX gene in cerebral ischemic injury and oxidative stress, which will be important to guard against neuronal cell death. Identifying regulators of 12/15-LOX transcription may suggest new molecular targets for stroke therapy.