Arachidonic Acid Induces ARE/Nrf2-Dependent Heme Oxygenase-1 Transcription in Rat Brain Astrocytes

Arachidonic acid (AA) is a major product of phospholipid hydrolysis catalyzed by phospholipase A(2) during neurodegenerative diseases. AA exerts as a second messenger to regulate various signaling components which may be involved in different pathophysiological processes. Astrocytes are the main types of CNS resident cells which maintain and support the physiological function of brain. AA has been shown to induce ROS generation through activation of NADPH oxidases (Noxs) which may play a key role in the expression of heme oxygenase-1 (HO-1). Therefore, this study was designed to investigate the mechanisms underlying AA-induced HO-1 expression in rat brain astrocytes (RBA-1). We found that AA induced HO-1 protein and mRNA expression and promoter activity in RBA-1, which was mediated through the synthesis of 15-deoxy-Delta 12,14-prostaglandin D-2-activated peroxisome proliferator-activated receptor-gamma (PPAR gamma) receptors. This note was confirmed by transfection with PPAR gamma small interfering RNAs (siRNA) which attenuated the AA-mediated responses. AA-induced HO-1 expression was mediated through Nox/ROS generation, which was inhibited by Nox inhibitors (diphenyleneiodonium and apocynin) and ROS scavengers (N-acetyl cysteine). Moreover, AA-induced HO-1 expression was mediated through phosphorylation of Src, Pyk2, platelet-derived growth factor, PI3K/Akt, and ERK1/2 which were inhibited by the pharmacological inhibitors including PP1, PF431396, AG1296, LY294002, and U0126 or by transfection with respective siRNAs. AA-enhanced Nrf2 expression and HO-1 promoter activity was inhibited by transfection with Nrf2 siRNA or by these pharmacological inhibitors. Furthermore, chromatin immunoprecipitation assay confirmed that Nrf2 and PPAR gamma were associated with the proximal antioxidant response element (ARE)-binding site on HO-1 promoter, suggesting that Nrf2/PPAR gamma are key transcription factors modulating HO-1 expression. AA-induced ARE promoter activity was also reduced by these pharmacological inhibitors. These findings suggested that AA increases formation of Nrf2 and PPAR gamma complex and binding with ARE1 binding site through Src, Pyk2, PI3K/Akt, and ERK1/2, which further induced HO-1 expression in RBA-1 cells.