Carbon Monoxide Produced by Heme Oxygenase-1 in Response to Nitrosative Stress Induces Expression of Glutamate-Cysteine Ligase in PC12 Cells via Activation of Phosphatidylinositol 3-Kinase and Nrf2 Signaling

Induction of heme oxygenase-1 (HO-1) expression has been associated with adaptive cytoprotection against a wide array of toxic insults, but the underlying molecular mechanisms remain largely unresolved. In this study, we investigated the potential role of carbon monoxide (CO), one of the by-products of the HO-1 reaction, in the adaptive survival response to peroxynitrite-induced PC12 cell death. Upon treatment of rat pheochromocytoma (PC12) cells with the peroxynitrite generator 3-morpholinosydnonimine hydrochloride (SIN-1), the cellular GSH level decreased initially, but was gradually restored to the basal level. This was accompanied by increased expression of the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in GSH biosynthesis. The SIN-1-induced GCLC up-regulation was preceded by induction of HO-1 and subsequent CO production. Inhibition of HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression by small interfering RNA abrogated the up-regulation of GCLC expression and the subsequent GSH restoration induced by SIN-1. In contrast, additional exposure to the CO-releasing molecule (CO-RM) restored the GSH level previously reduced by inhibition of CO production using zinc protoporphyrin IX. Furthermore, CO-RM treatment up-regulated GCLC expression through activation of Nrf2. The CO-RM-induced activation of Nrf2 was under the control of the phosphatidylinositol 3-kinase/Akt signaling pathway. In conclusion, CO produced by HO-1 rescues PC12 cells from nitrosative stress through induction of GCLC, which is mediated by activation of phosphatidylinositol 3-kinase/Akt and subsequently Nrf2 signaling.