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 A₂ 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-Δ12,14-prostaglandin D₂-activated peroxisome proliferator-activated receptor-γ (PPARγ) receptors. This note was confirmed by transfection with PPARγ 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 PPARγ were associated with the proximal antioxidant response element (ARE)-binding site on HO-1 promoter, suggesting that Nrf2/PPARγ 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 PPARγ 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.