Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA.

Heyninck K ，Sabbe L ，Chirumamilla CS ，Szarc Vel Szic K ，Vander Veken P ，Lemmens KJA ，Lahtela-Kakkonen M ，Naulaerts S ，Op de Beeck K ，Laukens K ，Van Camp G ，Weseler AR ，Bast A ，Haenen GRMM ，Haegeman G ，Vanden Berghe W ... -  《-》

Functional dissection of Nrf2-dependent phase II genes in vascular inflammation and endotoxic injury using Keap1 siRNA.

Keap1 is a cytoplasmic repressor of the transcription factor Nrf2, and its degradation induces Nrf2 activation, leading to upregulation of antioxidant phase II genes. We investigated the roles of phase II genes in vascular inflammation and septic injury using Keap1 siRNA and elucidated its underlying mechanism. Selective knockdown of Keap1 with siRNA promoted Nrf2-dependent expression of phase II genes in endothelial cells, such as heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), and peroxiredoxin-1 (Prx1), resulting in the elevation of cellular glutathione levels and suppression of tumor necrosis factor (TNF)-α-induced intracellular H(2)O(2) accumulation. Keap1 knockdown inhibited TNF-α-induced expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by suppressing NF-κB activation via inhibition of its upstream modulators, Akt, NIK, and IKK, resulting in the elevation of monocyte adhesion to endothelial cells. Importantly, these events were reversed by HO-1 and GCL inhibitors and Prx1-specific siRNA. Keap1 knockdown also inhibited endotoxin-induced expression of inducible nitric oxide synthase (iNOS) and TNF-α by upregulating HO-1, GCL, and Prx1 expression in macrophages. Moreover, in vivo Keap1 knockdown increased the expression of phase II genes and suppressed the expression of ICAM-1, VCAM-1, iNOS, and TNF-α in an endotoxemic mouse model, resulting in significant protection against liver and lung injuries and lethality. Our results indicate that Keap1 knockdown prevents NF-κB-mediated vascular inflammation and endotoxic shock by suppressing NF-κB-mediated inflammatory gene expression via upregulation of Nrf2-mediated antioxidant genes. Thus, siRNA targeting Keap1 may provide a new therapeutic approach for inflammation-associated vascular diseases and sepsis.

Kim JH ，Choi YK ，Lee KS ，Cho DH ，Baek YY ，Lee DK ，Ha KS ，Choe J ，Won MH ，Jeoung D ，Lee H ，Kwon YG ，Kim YM ... -  《-》

Piceatannol induces heme oxygenase-1 expression in human mammary epithelial cells through activation of ARE-driven Nrf2 signaling.

Piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene), derived from the seeds of Euphorbia lagascae, has been reported to have anti-proliferative, anti-inflammatory, and antioxidant properties. However, the mechanisms underlying its chemoprotective effects remain largely unresolved. In the present study, we found that piceatannol treatment (30 microM) significantly upregulated the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) and its mRNA transcript at 6h and 3h, respectively in human breast epithelial (MCF10A) cells. A redox-sensitive transcription factor NF-E2-related factor (Nrf2) plays a pivotal role in induced expression of many cytoprotective enzymes including HO-1. Piceatannol induced translocation of Nrf2 into the nucleus and its transcriptional activities when treated to the MCF10A cells for 6h. Upregulation of HO-1 expression by piceatannol through direct binding of Nrf2 to antioxidant response element (ARE) was verified by the chromatin immunoprecipitation (ChIP) assay. siRNA knock down of Nrf2 gene abolished piceatannol-induced HO-1 expression. In addition, piceatannol-induced activation of Nrf2 and/or HO-1 expression was abrogated by the pharmacological inhibitor (LY294002) as well as the kinase-dead form of Akt. In an attempt to elucidate the molecular mechanisms underlying cytoprotective or chemoprotective activity exerted by piceatannol, we examined its effect on the signaling pathways responsible for induction of HO-1 expression. We hypothesize that an electrophilic quinone formed as a consequence of oxidation of piceatannol bearing the catechol moiety may bind directly to Kelch-like ECH-associated protein 1 (Keap1), an inhibitory protein that sequesters Nrf2 in the cytoplasm. This will diminish the affinity of Keap1 for Nrf2. The thiol reducing agents, dithiothreitol (100 microM) or beta-mercaptoethanol (1.4 microM), attenuated piceatannol-induced Nrf2 activation and HO-1 expression. It is hence likely that piceatannol modifies specific cysteine residues of Keap1, which allows Nrf2 to translocate into the nucleus and bind to ARE, leading to enhancement of the expression of HO-1. The characteristic catechol moiety of piceatannol appears to be critical for induction of Nrf2 activation and subsequent upregulation of HO-1.

Lee HH ，Park SA ，Almazari I ，Kim EH ，Na HK ，Surh YJ ... -  《-》

Withaferin A induces Nrf2-dependent protection against liver injury: Role of Keap1-independent mechanisms.

Small molecules of plant origin offer presumptively safe opportunities to prevent carcinogenesis, mutagenesis and other forms of toxicity in humans. However, the mechanisms of action of such plant-based agents remain largely unknown. In recent years the stress responsive transcription factor Nrf2 has been validated as a target for disease chemoprevention. Withania somnifera (WS) is a herb used in Ayurveda (an ancient form of medicine in South Asia). In the recent past, withanolides isolated from WS, such as Withaferin A (WA) have been demonstrated to be preventive and therapeutic against multiple diseases in experimental models. The goals of this study are to evaluate withanolides such as WA as well as Withania somnifera root extract as inducers of Nrf2 signaling, to probe the underlying signaling mechanism of WA and to determine whether prevention of acetaminophen (APAP)-induced hepatic toxicity in mice by WA occurs in an Nrf2-dependent manner. We observed that WA profoundly protects wild-type mice but not Nrf2-disrupted mice against APAP hepatotoxicity. WA is a potent inducer of Nrf2-dependent cytoprotective enzyme expression both in vivo and in vitro. Unexpectedly, WA induces Nrf2 signaling at least in part, in a Keap1-independent, Pten/Pi3k/Akt-dependent manner in comparison to prototypical Nrf2 inducers, sulforaphane and CDDO-Im. The identification of WA as an Nrf2 inducer that can signal through a non-canonical, Keap1-independent pathway provides an opportunity to evaluate the role of other regulatory partners of Nrf2 in the dietary and pharmacological induction of Nrf2-mediated cytoprotection.

Palliyaguru DL ，Chartoumpekis DV ，Wakabayashi N ，Skoko JJ ，Yagishita Y ，Singh SV ，Kensler TW ... -  《-》

Curcumin induces stabilization of Nrf2 protein through Keap1 cysteine modification.

The present study was aimed to investigate the effects of curcumin, a representative chemopreventive phytochemical with pronounced antioxidant and anti-inflammatory properties, on activation of Nrf2 and expression of its target protein heme oxygenase-1 (HO-1) in mouse skin in vivo and in cultured murine epidermal cells. Treatment of mouse epidermal JB6 cells with curcumin resulted in the induction of HO-1 expression, and this was abrogated in cells transiently transfected with Nrf2 siRNA. While curcumin treatment increased protein expression of Nrf2, it did not alter the steady-state level of the Nrf2 mRNA transcript. Treatment of cells with curcumin stabilized Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of this transcription factor. Tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,β-unsaturated carbonyl group, failed to induce HO-1 expression as well as nuclear translocation of Nrf2 and its binding to the antioxidant/electrophile response elements. Cells transfected with a mutant Keap1 protein in which cysteine 151 (Cys151) is replaced by serine exhibited marked reduction in curcumin-induced Nrf2 transactivation. Mass spectrometric analysis revealed that curcumin binds to Keap1 Cys151, supporting that this amino acid is a critical target for curcumin modification of Keap1, which facilitates the liberation of Nrf2. Thus, it is likely that the α,β-unsaturated carbonyl moiety of curcumin is essential for its binding to Keap1 and stabilization of Nrf2 by hampering ubiquitination and proteasomal degradation.

Shin JW ，Chun KS ，Kim DH ，Kim SJ ，Kim SH ，Cho NC ，Na HK ，Surh YJ ... -  《-》