Activation of the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2) is one of the major cellular defense lines against oxidative and xenobiotic stress, but also influences genes involved in lipid and glucose metabolism. It is unresolved whether the cytoprotective and metabolic responses mediated by Nrf2 are connected or separable events in non-malignant cells. In this study we show that activation of Nrf2, either by the small molecule sulforaphane or knockout of the Nrf2 inhibitor Keap1, leads to increased cellular glucose uptake and increased glucose addiction in fibroblasts. Upon Nrf2 activation glucose is preferentially metabolized through the pentose phosphate pathway with increased production of NADPH. Interference with the supply of glucose or the pentose phosphate pathway and NADPH generation not only hampers Nrf2-mediated detoxification of reactive oxygen species on the enzyme level but also Nrf2-initiated expression of antioxidant defense proteins, such as glutathione reductase and heme-oxygenase1. We conclude that the Nrf2-dependent protection against oxidative stress relies on an intact pentose phosphate pathway and that there is crosstalk between metabolism and detoxification already at the level of gene expression in mammalian cells.

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BACKGROUND AND PURPOSEThe transcription factor NF-κB orchestrates many pro-inflammatory signals and its inhibition is considered a promising strategy to combat inflammation. Here we report the characterization of the natural product plumericin as a highly potent inhibitor of the NF-κB pathway with a novel chemical scaffold, which was isolated via a bioactivity-guided approach, from extracts of Himatanthus sucuuba, an Amazonian plant traditionally used to treat inflammation-related disorders.EXPERIMENTAL APPROACHA NF-κB luciferase reporter gene assay was used to identify NF-κB pathway inhibitors from H. sucuuba extracts. Monitoring of TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin by flow cytometry was used to confirm NF-κB inhibition in endothelial cells, and thioglycollate-induced peritonitis in mice to confirm effects in vivo. Western blotting and transfection experiments were used to investigate the mechanism of action of plumericin.KEY RESULTSPlumericin inhibited NF-κB-mediated transactivation of a luciferase reporter gene (IC50 1 μM), abolished TNF-α-induced expression of the adhesion molecules VCAM-1, ICAM-1 and E-selectin in endothelial cells and suppressed thioglycollate-induced peritonitis in mice. Plumericin exerted its NF-κB pathway inhibitory effect by blocking IκB phosphorylation and degradation. Plumericin also inhibited NF-κB activation induced by transfection with the constitutively active catalytic subunit of the IκB kinase (IKK-β), suggesting IKK involvement in the inhibitory action of this natural product.CONCLUSION AND IMPLICATIONSPlumericin is a potent inhibitor of NF-κB pathways with a new chemical scaffold. It could be further explored as a novel anti-inflammatory lead compound.

The aim of our study was to examine in detail the impact of NF-E2-related factor (Nrf2) activation on endothelial cell function with focus on redox homeostasis and the endothelial nitric oxide synthase (eNOS) system. We administered 2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide (CDDO-IM), a known activator of Nrf2, to primary human umbilical vein endothelial cells. Activation of Nrf2 by CDDO-IM increased the amount of bioavailable nitric oxide (NO), a major contributor to vascular homeostasis, in naive and stressed cells. Concomitantly, intracellular reactive oxygen species were dose-and timedependently reduced. In apparent contrast to elevated NO levels, eNOS protein expression was transiently decreased in an Nrf2-dependent manner. Employing pharmacological inhibitors as well as a small interfering RNA approach, we identified de novo protein synthesis of heme oxygenase 1 (HO-1) and its enzymatic activity as cause for the observed reduction of eNOS. We hypothesize that under redox stress, when the availability of tetrahydrobiopterin, a pivotal stoichiometric cofactor for eNOS, is limited, activation of Nrf2 leads (a) to transient reduction of eNOS protein levels and (b) to an antioxidant defense in human umbilical vein endothelial cells. Both activities ensure that a stoichiometric ratio of eNOS and tetrahydrobiopterin is sustained and that the risk of eNOS uncoupling is reduced. Our study is the first to provide a causal link between Nrf2 activation and eNOS expression and NO levels, respectively.

Objective-Our goal was to examine the influence of indirubin-3Ј-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. Methods and Results-I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G 0 /G 1 phase of the cell cycle as assessed by 5-bromo-2Ј-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38 MAPK was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-␥-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via Key Words: indirubin Ⅲ vascular smooth muscle cell Ⅲ proliferation Ⅲ neointima Ⅲ STAT3 R estenosis is the major factor hampering the beneficial effect of angioplasty and stenting. Vascular smooth muscle cell (VSMC) proliferation, next to local vascular inflammation, is a critical factor in neointima formation and vascular lumen loss during restenosis. Thus, one current strategy to maintain proper vascular function after angioplasty is to inhibit VSMC proliferation by targeting cell cycle regulation, eg, by drug-eluting stents. 2,3 Two products, the rapamycin-eluting Cypher stent and the paclitaxel-eluting Taxus stent, were approved by the US Food and Drug Administration in 2003 and 2004, respectively. Although successfully introduced into the market, there are now concerns about an increased risk of late stent thrombosis. 4 Thus, compounds inhibiting neointima formation with mechanisms differing from those of rapamycin and paclitaxel 5 may have the potential to be efficient with fewer side effects.Indirubin, a red isomer of indigo, is the active ingredient of the traditional Chinese medicinal formulation Danggui Longhui Wan, used against chronic myelocytic leukemia. 6 Enzyme-based in vitro studies have indicated that indirubin and its derivatives are potent inhibitors of the cyclindependent kinases (CDKs) 1, 2, 4, and 5. 6 -9 Furthermore, different indirubin derivatives showed antitumor activity in several human cancer cells. 6,10 -14 Moreover, for indirubin-3Ј-monoxime (I3MO), an in vitro antiinflammatory and anticancer activity had been described on the basis of the inhibition of tumor necrosis factor-induced nuclear factor B activation and the subsequent suppression of antiapoptotic and proproliferative gene expression. 14 The aim of this study was to investigate the potential use of I3MO in the prevention of restenosis. We therefore analyzed a possible effect on platelet-derived growth factor (PDGF)- MethodsAn expanded Methods section can be found in the Supplemental Data, available online at http://atvb.ahajournals.org. Cell CultureVSMCs were isolated from male Sprague-Dawley rat thoracic aortas by enzyma...