Activation of NFκB and MnSOD gene expression by free radical scavengers in human microvascular endothelial cells

Abstract

The effect of nonprotein thiol (NPT) free radical scavengers WR-1065 (SH) and WR-33278 (SS), the active thiol and disulfide metabolites of amifostine, N-acetylcysteine (NAC; both L- and D- isomers), mesna, captopril, and dithiothreitol (DTT) on NFκB activation in human microvascular endothelial cells (HMEC) was investigated and contrasted to TNFα. The use of each of these NPTs at millimolar concentrations independent of oxidative damage-inducing agents resulted in a marked activation of NFκB, with the maximum effect observed between 30 min and 1 h after treatment. Only the SH and SS forms of amifostine, however, were effective in activating NFκB when administered at micromolar levels. Using a supershift assay, SH and SS equally affected the p50–p65 heterodimer, but not homodimers or heterodimers containing p52 or c-Rel subunits of NFκB. Neither catalase nor pyruvate when added to the culture medium to minimize hydrogen peroxide production had an effect on NFκB activation by SH. Thus, while oxidative damage is known to activate NFκB, the intracellular redox environment may also be affected by the addition of free radical scavenging agents such as NPT, and these in turn are capable of activating the redox sensitive transcription factor NFκB. There does not appear to be a significant role, if any, for the production of H₂O₂ as an intermediate step in the activation of NFκB by either the SH or the SS form of amifostine. Rather, the underlying mechanism of action, especially for the SS form, may be related to the close structural and functional similarities of these agents to polyamines, which have been reported to be capable of activating NFκB. In contrast to TNFα, exposure of cells to either 40 μM or 4 mM of SH for 30 min did not induce intercellular adhesion molecule-1 (ICAM-1) gene expression, but did increase manganese superoxide dismutase (MnSOD) gene expression. MnSOD expression rose by 2-fold and remained elevated from 4 to 22 h following SH exposure.

Introduction

Nonprotein thiols (NPT) such as N-acetylcysteine (NAC), amifostine, mesna, oltipraz, and captopril are currently used in a wide range of clinical applications ranging from protection of tissues against oxidative damage [1], [2], [3], to reduction of mucus formation [4], to control of hypertension [5]. Common among these therapeutic NPT is their ability to scavenge free radicals and participate in reductive/oxidative reactions that can affect redox-sensitive cellular processes. These include the activation of certain transcription factors, expression levels of genes, and activities of certain proteins [6], [7], [8], [9]. Examples include reports on the activation of nuclear transcription factor κB (NFκB) and enhancement of manganese superoxide dismutase (MnSOD) gene expression in cells exposed to NAC [10] and oltipraz [11]. Recently the prodrug amifostine was reported to inhibit, when evaluated under in vitro conditions, the process of apoptosis in hematopoietic progenitor cells and this was attributed to its ability to also activate the transcription factor NFκB in the absence of oxidative stress-inducing agents [12].

NFκB is an inducible transcription factor that plays an essential role in the expression of a number of gene families that include cytokines and their receptors, cell adhesion molecules, growth factors, and MnSOD [13]. It consists of a family of proteins that bind as homodimers and heterodimers to κB-enhancing elements in the promotor regions of selected genes. These include c-Rel, p52, p50, and p65. In most cells, NFκB is found in an inactive form in the cytoplasm bound to the inhibitory protein IκB. In response to activating signals, IκB is phosphorylated and subsequently degraded by the ubiquitin-proteasome complex, allowing NFκB to translocate to the nucleus and activate gene expression. However, it has been reported that NFκB can also be activated in the absence of IκB degradation through an iron-mediated mechanism in alveolar epithelial cells [14]. It is also known that NFκB activation and binding to DNA can be affected by reducing agents that are capable of altering the redox state of the cysteine 62 residue of its p50 subunit [15].

The most effective inducers of NFκB activation are primarily oxidative stress-related agents. These include the multifunctional cytokine tumor necrosis factor α (TNFα) [16], phorbol esters [7], ionizing radiation [17], hydrogen peroxide, and anti-neoplastic drugs [18]. Activation of NFκB by these oxidative damage-inducing agents is also linked to a number of disease states which include enhanced human immunodeficiency virus (HIV) gene expression [19], atherogenesis [20], diabetic vasculopathy [21], and cancer [9]. It is, therefore, difficult to reconcile the ability of NAC, oltipraz, and amifostine to activate NFκB with their well-characterized cytoprotective effects [22], [23], [24], [25], [26].

DNA-binding motifs for NFκB binding are found in the promotor regions of a large number of genes involved in inflammatory processes [27], apoptosis [28], [29], and intracellular redox-related processes [10]. Two important genes induced following NFκB activation that are representative of this diversity are MnSOD [30], [31] and intercellular adhesion molecule-1 (ICAM-1) [10], [32]. MnSOD is an inducible antioxidant enzyme that protects against oxidative damage caused by reactive oxygen species generated during normal aerobic metabolism and by oxidative stress-inducing agents. It catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. Overexpression of MnSOD has been correlated with suppression of tumorigenicity and tumor metastasis [31]. The mechanism by which MnSOD suppresses malignancy is not clear, but it has been proposed that changes in the cellular redox environment due to the accumulation of hydrogen peroxide or other hydroperoxides lead to growth inhibition of tumor cells [30]. ICAM-1, on the other hand, is an inducible glycoprotein that is expressed during inflammatory reactions. Its upregulation on the surface of endothelial cells mediates stable leukocyte adhesion to the vascular endothelium that precedes leukocyte extravasation into sites of infection or injury. Because NPTs are usually associated with antioxidative, chemopreventive, and cytoprotective effects, and not inflammatory processes, it is of interest to determine whether their ability to activate NFκB is also accompanied by an enhanced expression of these two genes that are representative of such diverse processes. The present study was undertaken to assess NFκB activation by clinically used NPTs, including the thiol and disulfide metabolites of the prodrug amifostine, using a human microvascular endothelial cell (HMEC) system and standard gel shift assays.