Signaling Pathways Activated by the Phytochemical Nordihydroguaiaretic Acid Contribute to a Keap1-independent Regulation of Nrf2 Stability: Role of Glycogen Synthase Kinase-3

Defense against oxidative stress is executed by an antioxidant program that is tightly controlled by the transcription factor Nrf2. The stability of Nrf2 involves the interaction of two degradation domains, designated Neh2 and Neh6, with the E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, respectively. The regulation of Nrf2 through the Neh6 degron remains largely unexplored but requires GSK-3 to form a phosphodegron. In this study, the cancer-chemopreventive agent nordihydroguaiaretic acid (NDGA) increased the level of Nrf2 protein and expression of heme oxygenase-1 (HO-1) in kidney-derived LLC-PK1 and HEK293T cells and in wild-type mouse embryo fibroblasts (MEFs). However, NDGA did not induce HO-1 in Nrf2(-/-) MEFs, indicating that Nrf2 is required for induction. The relevance of the Nrf2/HO-1 axis to antioxidant protection was further demonstrated by the finding that the HO-1 inhibitor stannous-mesoporphyrin abolished protection against hydrogen peroxide conferred by NDGA. NDGA increased Nrf2 and HO-1 protein levels in Keap1(-/-) MEFs, implying that Keap1-independent mechanisms regulate Nrf2 stability. Mutants of the Neh2 or Nrh6 domain and chimeric proteins comprising cyan fluorescent protein fused to Neh2 and green fluorescent protein fused to Neh6 exhibited longer half-lives in the presence of NDGA, demonstrating that NDGA targets both the Neh2 and the Neh6 degrons. In common with other chemopreventive agents, NDGA activated the ERK1/2, p38, JNK, and PI3K pathways. By using selective kinase inhibitors we found that PI3K, JNK, and p38 were responsible for the stabilization of Nrf2 and induction of HO-1 by NDGA. To explain how NDGA might up-regulate Nrf2 in a Keap1-independent manner we explored the participation of GSK-3β because it controls the Neh6 phosphodegron. Importantly, NDGA caused inhibitory phosphorylation of GSK-3β at Ser9 and at Thr390, and this was associated with a substantial reduction in Neh6 phosphorylation. Our study demonstrates that NDGA activates Nrf2 through multiple signaling cascades and identifies GSK-3β as an integrator of these signaling pathways and a gatekeeper of Nrf2 stability at the level of the Neh6 phosphodegron.