Dominant-negative Jun N-terminal protein kinase (JNK-1) inhibits metabolic oxidative stress during glucose deprivation in a human breast carcinoma cell line

Abstract

Signal transduction pathways involved in glucose deprivation-induced oxidative stress were investigated in human breast carcinoma cells (MCF-7/ADR). In MCF-7/ADR, glucose deprivation-induced prolonged activation of c-Jun N-terminal kinase (JNK1) as well as cytotoxicity and the accumulation of oxidized glutathione. Glucose deprivation also caused significant increases in total glutathione, cysteine, γ-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of γ-glutamylcysteine synthetase, suggesting that the synthesis of glutathione increased as an adaptive response. Expression of a catalytically inactive dominant negative JNK1 in MCF-7/ADR inhibited glucose deprivation-induced cell death and the accumulation of oxidized glutathione as well as altered the duration of JNK activation from persistent (>2 h) to transient (30 min). In addition, stimulation of glutathione synthesis during glucose deprivation was not observed in cells expressing the highest levels of dominant negative protein. Finally, a linear dose response suppression of oxidized glutathione accumulation was noted for clones expressing increasing levels of dominant negative JNK1 during glucose deprivation. These results show that expression of a dominant negative JNK1 protein was capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR. These findings support the hypothesis that JNK signaling pathways may control the expression of proteins contributing to cell death mediated by metabolic oxidative stress during glucose deprivation. Finally, these results support the concept that JNK signaling-induced shifts in oxidative metabolism may provide a general mechanism for understanding the diverse biological effects seen during the activation of JNK signaling cascades.

Introduction

We have previously observed that glucose deprivation induces cell death [1], activates c-Jun N-terminal kinase 1 (JNK1) [2], and increases intracellular pro-oxidant production and oxidized glutathione content [3] in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). During glucose deprivation the thiol antioxidant, N-acetylcysteine, suppresses JNK1 activation and glucose deprivation-induced cytotoxicity as well as suppressing increased pro-oxidant production and the accumulation of oxidized glutathione [3], [4]. These results have led us to investigate the involvement of JNK1 signaling pathways in the process of glucose deprivation-induced cytotoxicity and oxidative stress in MCF-7/ADR cells.

Several studies have demonstrated that JNK, also called stress-activated protein kinases (SAPK), signal transduction pathways can be activated by a diverse array of cellular stimuli. JNKs are activated in response to mitogenic signals, including growth factors [5], T cell activation and proliferation signaling [6], [7], [8], oncogenic Ras [9], and CD40 ligation [10], [11]. JNKs are also involved in cellular responses to the engagement of several classes of cell surface receptors, including cytokine receptors [12], [13], [14], and G protein-coupled receptors [15]. In addition, JNKs participate in cellular responses to environmental stresses such as heat shock [16], [17], oxidative stress [15], [18], [19], UV light [9], [17], [20], [21], osmotic shock [22], and γ-radiation [23], [24]. JNKs are also activated by various chemical compounds, including protein synthesis inhibitors [25], ceramide [26], [27], DNA-damaging agents [17], [28], [29], and chemopreventive drugs [30], [31]. Following activation, JNK phosphorylates several transcription factors including activating transcription factor-2 (ATF-2) [32], Elk-1 [33], Sap-1a [34], and c-Jun [9], which are thought to be involved in regulating numerous genes implicated in metabolism, cell proliferation, transformation, differentiation, and DNA repair [35], [36], [37], [38], [39], [40], [41], [42].

One interesting aspect of the cellular responses following JNK activation is the fate of the cells. It is thought that JNK is involved in both cell growth and cell death pathways [43], [44], [45]. However, the factor(s), which determine the various outcomes of JNK signaling, are still unknown. Recent studies have shown that the duration of JNK activation appears to be one factor, which may determine the fate of the cells. Bost et al. [45] observed that epidermal growth factor induced a rapid and prolonged activation of the JNK pathway that correlated with growth stimulation. In contrast, Chen et al. [21], [24], [30] reported that a prolonged activation of JNK was associated with the initiation of cell death. In the current study, we demonstrate that expression of catalytically inactive dominant negative JNK1 protein is capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR multidrug resistant human breast adenocarcinoma cells. These results suggest that JNK signaling pathways may not only be activated during oxidative stress, but may also control the expression of proteins that contribute to oxidative stress. These results also support the notion that shifts in the balance of intracellular oxidation/reduction reactions (cellular redox status) dependent upon the metabolic state of the cell at the time of JNK activation, may contribute to the diversity of biological effects seen following activation of JNKs.