Our results demonstrate convergence between AMPK and Nrf2 pathways and this intersection is essential for anti-inflammatory effect of BBR in LPS-stimulated macrophages and endotoxin-shocked mice. Uncovering this intersection is significant for understanding the relationship between energy homeostasis and antioxidative responses and may be beneficial for developing new therapeutic strategies against inflammatory diseases. Antioxid. Redox Signal. 20, 574-588.

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of antioxidative proteins, including heme oxygenase-1 (HO-1). Nrf2 is sequestered in the cytoplasm by Keap1 under unstimulated conditions but translocates into the nucleus and transactivates the antioxidant responsive element (ARE) upon exposure to oxidative insults. It has recently been demonstrated that in vitro phosphorylation of Nrf2 on Ser40 by protein kinase C (PKC) facilitates the dissociation of Nrf2 from the Keap1 complex (Huang HC, Nguyen T, and Pickett CB. J Biol Chem 277: 42769–42774, 2002). The present study was designed to examine whether PKC is involved in oxidative stress-mediated nuclear translocation of Nrf2 in vivo and, if so, which PKC isoforms are involved. Induction of HO-1 gene expression by phorone, a glutathione depletor, and 4-hydroxy-2,3-nonenal (4-HNE), an end product of lipid peroxidation, was suppressed by a specific PKC inhibitor, Ro-31-8220, at concentrations that inhibit all isoforms in WI-38 cells. The induction of HO-1 was not affected by prolonged exposure of the cells to 12- O-tetradecanoylphorbol-13 acetate (TPA), suggesting that TPA-insensitive atypical PKC (aPKC) isoforms are involved. An immunocomplex kinase assay revealed that phorone and 4-HNE increased aPKCι activity. In COS-7 cells, 4-HNE induced nuclear translocation of the Nrf2-green fluorescent protein (GFP) fusion protein, but not the Nrf2(S40A)-GFP mutant. In the absence of oxidative insults, the Nrf2(S40E)-GFP mutant was distributed in the nucleus. The Nrf2-GFP accumulation in the nucleus was induced by coexpression of aPKCι, but not by a kinase inactive mutant aPKCι(K274W). The activity of an ARE-driven reporter was increased by coexpression of aPKCι, and this effect was eliminated by Ro-31-8220 in HepG2 cells. The reporter activity induced by 4-HNE was inhibited by coexpression of aPKCι(K274W). These results suggest that phosphorylation of Nrf2 Ser40 by aPKC(s) is involved in the nuclear translocation and ARE transactivation of Nrf2 by oxidative stress.

We used lipopolysaccharide (LPS) to activate microglia that play an important role in the brain immune system. LPS injected into the rat hippocampus CA1 region activated microglial cells resulting in an increased production of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha in the hippocampus during the initial stage of treatment. Immunostaining for IL-1beta was increased at 6 hr after LPS injection. IL-1beta-immunopositive cells were co-localized with immunostaining for CD11b. Subacute treatment with LPS by the same route for 5 days caused long-term activation of microglia and induced learning and memory deficits in animals when examined with a step-through passive avoidance test, but histochemical analysis showed that neuronal cell death was not observed under these experimental conditions. The increased expression of the heme oxygenase-1 (HO-1) gene, an oxidative stress maker, was observed. However, the genetic expression of brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, decreased during the course of LPS treatment. We found decreases in [3H]MK801 binding in the hippocampus CA1 region by LPS-treatment for 5 days. The data shows that glutamatergic transmission was attenuated in the LPS-treated rats. These results suggest that long-term activation of microglia induced by LPS results in a decrease of glutamatergic transmission that leads to learning and memory deficits without neuronal cell death. The physiologic significance of these findings is discussed.

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