Dong-Keon Lee

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.

Ginseng Berry Extract Prevents Atherogenesis via Anti-Inflammatory Action by Upregulating Phase II Gene Expression

Ginseng berry possesses higher ginsenoside content than its root, which has been traditionally used in herbal medicine for many human diseases, including atherosclerosis. We here examined the antiatherogenic effects of the Korean ginseng berry extract (KGBE) and investigated its underlying mechanism of action in vitro and in vivo. Administration of KGBE decreased atherosclerotic lesions, which was inversely correlated with the expression levels of phase II genes to include heme oxygenase-1 (HO-1) and glutamine-cysteine ligase (GCL). Furthermore, KGBE administration suppressed NF-κB-mediated expression of atherogenic inflammatory genes (TNF-α, IL-1β, iNOS, COX-2, ICAM-1, and VCAM-1), without altering serum cholesterol levels, in ApoE−/− mice fed a high fat-diet. Treatment with KGBE increased phase II gene expression and suppressed lipopolysaccharide-induced reactive oxygen species production, NF-κB activation, and inflammatory gene expression in primary macrophages. Importantly, these cellular events were blocked by selective inhibitors of HO-1 and GCL. In addition, these inhibitors reversed the suppressive effect of KGBE on TNF-α-mediated induction of ICAM-1 and VCAM-1, resulting in decreased interaction between endothelial cells and monocytes. These results suggest that KGBE ameliorates atherosclerosis by inhibiting NF-κB-mediated expression of atherogenic genes via upregulation of phase II enzymes and thus has therapeutic or preventive potential for atherosclerosis.

Changes and expressions of Redd1 in neurons and glial cells in the gerbil hippocampus proper following transient global cerebral ischemia.

Redd1 (known as RTP801/Dig2/DDIT4) is a stress-induced protein, and it is known to be regulated in response to some stresses including hypoxia and oxidative stress. In the present study, we investigated the time-dependent changes in Redd1 immunoreactivity and its protein levels in the gerbil hippocampus proper (CA1-3 regions) after 5 min of transient global cerebral ischemia using immunohistochemistry and Western blot analysis. Redd1 immunoreactivity was apparently changed in the pyramidal neurons of the ischemic CA1 region, not in the pyramidal neurons of the ischemic CA2/3 region. Redd1 immunoreactivity in the CA1 pyramidal neurons was significantly increased at 6 h post-ischemia, decreased until 1 day post-ischemia, increased again at 2 days post-ischemia and weakly observed at 5 days post-ischemia. Especially, at 5 days after ischemic damage, Redd1 immunoreactivity was newly expressed in astrocytes and GABAergic interneurons in the CA1 region. Redd1 protein levels in the ischemic CA1 region were changed like the pattern of the Redd1 immunoreactivity. These results indicate that Redd1 immunoreactivity and protein levels are increased in the ischemic CA1 region at an early time after ischemic damage and that the increased Redd1 expression may be closely related to the delayed neuronal death of the CA1 pyramidal neurons following 5 min of transient global cerebral ischemia.

Specific activation of insulin-like growth factor-1 receptor by ginsenoside Rg5 promotes angiogenesis and vasorelaxation.

Background: The mechanism by which ginsenoside Rg5 regulates vascular function remains unclear. Results: Rg5 increases angiogenesis and vasorelxation by activating multiple signal transduction pathways downstream of insulin-like growth fastor-1 receptor (IGF-1R). Conclusion: Rg5 promotes endothelial cell function through activation of IGF-1R. Significance: These findings reveal a mechanism for the positive regulation of vascular function by Rg5-mediated IGF-1R activation. Ginsenoside Rg5 is a compound newly synthesized during the steaming process of ginseng; however, its biological activity has not been elucidated with regard to endothelial function. We found that Rg5 stimulated in vitro angiogenesis of human endothelial cells, consistent with increased neovascularization and blood perfusion in a mouse hind limb ischemia model. Rg5 also evoked vasorelaxation in aortic rings isolated from wild type and high cholesterol-fed ApoE−/− mice but not from endothelial nitric-oxide synthase (eNOS) knock-out mice. Angiogenic activity of Rg5 was highly associated with a specific increase in insulin-like growth factor-1 receptor (IGF-1R) phosphorylation and subsequent activation of multiple angiogenic signals, including ERK, FAK, Akt/eNOS/NO, and Gi-mediated phospholipase C/Ca2+/eNOS dimerization pathways. The vasodilative activity of Rg5 was mediated by the eNOS/NO/cGMP axis. IGF-1R knockdown suppressed Rg5-induced angiogenesis and vasorelaxation by inhibiting key angiogenic signaling and NO/cGMP pathways. In silico docking analysis showed that Rg5 bound with high affinity to IGF-1R at the same binding site of IGF. Rg5 blocked binding of IGF-1 to its receptor with an IC50 of ∼90 nmol/liter. However, Rg5 did not induce vascular inflammation and permeability. These data suggest that Rg5 plays a novel role as an IGF-1R agonist, promoting therapeutic angiogenesis and improving hypertension without adverse effects in the vasculature.

REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation

Regulated in development and DNA damage responses 1 (REDD‐1), an inhibitor of mammalian target of rapamycin (mTOR), is induced by various cell stressors, including LPS, a major player in the pathogenesis of endotoxemic shock. However, the pathologic role of REDD‐1 in endotoxemia is largely unknown. We found that LPS increased REDD‐1 expression, nuclear transcription factor‐kB(NF‐kB) activation, and inflammation and that these responses were suppressed by REDD‐1 knockdown and in REDD‐1+/− macrophages. REDD‐1 overexpression stimulated NF‐κB‐dependent inflammation without additional LPS stimulation. REDD‐1‐induced NF‐κBactivation was independent of 2 classic IKK‐dependent NF‐κB pathways and the mTOR signaling pathway; however, REDD‐1, particularly its C‐terminal region (178‐229), interacted with and sequestered IκBα, to elicit atypical NF‐κB activation during the delayed and persistent phases of inflammation after stimulation. Moreover, REDD‐1 knockdown mitigated vascular inflammation and permeability in endotoxemic mice, resulting in decreases in immune cell infiltration, systemic inflammation, caspase‐3 activation, apoptosis, and consequent mortality. We further confirmed the inflammatory and cytotoxic effects of REDD‐1 in endotoxemic REDD‐1+/− mice. Our data support the likelihood that REDD‐1 exacerbates endotoxemic inflammation via atypical NF‐κB activation by sequestering IκBα.—Lee, D.‐K., Kim, J.‐H., Kim, J., Choi, S., Park, M., Park, W., Kim, S., Lee, K.‐S., Kim, T., Jung, J., Choi, Y.K., Ha, K.‐S., Won, M.‐H., Billiar, T.R., Kwon, Y.‐G., Kim, Y.‐M. REDD‐1 aggravatesendotoxin‐induced inflammation via atypical NF‐κB activation. FASEB J. 32, 4585‐4599 (2018). www.fasebj.org