Lipid Peroxidation Is Involved in the Activation of NF-κB by Tumor Necrosis Factor but Not Interleukin-1 in the Human Endothelial Cell Line ECV304

Abstract

It has been proposed that reactive oxygen species, and in particular H 2 O 2 , may be involved in the activation of NF-B by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-B activation by interleukin-1 and tumor necrosis factor as well as the ability of H 2 O 2 to activate NF-B in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-B and stimulation of IB␣ degradation by H 2 O 2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H 2 O 2 was sensitive to Nacetyl-L-cysteine, and no increase in H 2 O 2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-B that acts independently of activating agent or cell type. However, we found that tumor necrosis factor-but not interleukin-1-driven NF-B activation and IB␣ degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6,6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-B activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-B. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-B activation by tumor necrosis factor but not interleukin-1.Taken together, the results argue against a role for H 2 O 2 in NF-B activation by cytokines in endothelial cells. Furthermore, tumor necrosis factor and interleukin-1 activate NF-B through different mechanisms in ECV304 cells, with the tumor necrosis factor pathway involving iron-catalyzed lipid peroxidation.The inducible, higher eukaryotic transcription factor NF-B has an important role in the regulation of a number of genes involved in immune and inflammatory responses. It is activated in many cell types by a wide range of stimuli including the proinflammatory cytokines interleukin-1 (IL-1) 1 and tumor necrosis factor (TNF) and the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) (reviewed in Ref. 1).In endothelial cells (ECs), activation of NF-B is central to the regulation of many genes by IL-1 and TNF such as the cell adhesion molecules vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin (2) and tissue factor (3). Recently, NF-B has been identified ...

Overexpression of γ-glutamylcysteine synthetase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappa B and activator protein-1

“…This is consistent with a report indicating NAC or dihydrolipoate blocks NF-kB activation by TNF, lymphotoxin and PMA, but not that induced by calyculin A or okadaic acid, both inhibitors of serine/threonine protein phosphatases (Suzuki et al, 1994). Similarly, lipid peroxidation is involved in the activation of NF-kB by TNF but not by IL-1 (Bowie et al, 1997). Indeed, IL-1 induces NFkB activation independently of the production of ROI (Manna et al, 1998).…”

“…Di erent agents may activate NF-kB by di erent mechanisms (Suzuki et al, 1994;Bowie et al, 1997;Bonizzi et al, 1996). For instance NAC blocks TNF-and PMA-induced NF-kB but not that induced by okadaic acid (Suzuki et al, 1994).…”

“…For instance NAC blocks TNF-and PMA-induced NF-kB but not that induced by okadaic acid (Suzuki et al, 1994). Similarly lipid peroxidation is needed for TNF-but not IL-1 induced NF-kB activation (Bowie et al, 1997). Therefore, we examined the e ect of g-GCS on the activation of NF-kB by PMA, LPS, H 2 O 2 , Figure 4 E ect of g-GCS on the expression of CAT gene linked to NF-kB containing mdr1b promoter.…”

Oxidative stress and regulation of glutathione in lung inflammation

“…However, if oxidant or other environmental stress alters this ratio, this shift in the GSH/GSSG redox buffer influences a variety of cellular signalling processes, such as activation of the transcription factors AP-1 and NF-kB. Oxidative stress including the presence of lipid peroxidation products [50] or depletion of GSH and subsequent increases in cytosolic GSSG in response to oxidative stress causes rapid ubiquitination and phosphorylation and thus subsequent degradation of the inhibitor of NF-kB (IkB), which is a critical step for NF-kB activation [51,52]. Under reducing conditions, such as an increase in intracellular GSH following treatment with N-acetyl-L-cysteine (NAC), the phosphorylation of serine groups on IkB-a following TNF-a treatment is inhibited, leading to the downregulation of NF-kB in endothelial cells ( fig.…”

Vitamin C Inhibits NF-κB Activation by TNF Via the Activation of p38 Mitogen-Activated Protein Kinase

“…The human cell line ECV304 and HUVECs were grown and passaged as described previously (29). All experiments were conducted in complete medium at 37°C.…”