Peroxynitrite induces GADD34, 45, and 153 VIA p38 MAPK in human neuroblastoma SH-SY5Y cells

Abstract

Peroxynitrite, one of the most reactive radicals, is produced from superoxide anion and nitric oxide. A peroxynitrite generator, 3-morpholinosydonimine (SIN-1), was found to induce the expression of three different growth arrest and DNA damage-inducible (GADD) mRNA, GADD34, GADD45, and GADD153, at the early phase during cell death in human neuroblastoma SH-SY5Y cells. In addition, peroxynitrite activated p38 MAPK just before induction of three GADD mRNA. A specific inhibitor of p38 MAPK, SB202190, markedly suppressed peroxynitrite-induced expression of three GADD mRNA in SH-SY5Y cells. The expression of three GADD genes and also p38 MAPK phosphorylation were suppressed by treatment with radical scavengers, superoxide dismutase plus catalase and glutathione. Glutathione depletion by L-buthionine-S, R-sulfoximine (BSO), increased the vulnerability of the cells to peroxynitrite. These findings indicate that peroxynitrite-mediated oxidative stress activated p38 MAPK to induce three GADD genes.

Introduction

Growth arrest and DNA damage-inducible (GADD) genes are stress response genes and classified as GADD34, GADD45, and GADD153. GADD34, GADD45, and GADD153 are reported to be induced by stressors such as UV irradiation, chemical carcinogens, and starvation, respectively [1], [2]. GADD34 is the human/hamster homologue of mouse MyD116 cDNA, which was isolated as a primary response transcription induced during myeloid differentiation of M1 cells [3]. Treatment of M1 cells with methylmethane sulfonate (MMS), UV irradiation, and interleukin-6 leads to a rapid increase in MyD116 mRNA [3], [4]. GADD45 is a p53 responsive gene encoding a protein that interacts with p21/WAF1/CIP1 and exists predominantly in nuclei [5]. GADD153 is a member of the CCAAT enhancer-binding protein family of transcription factors and becomes heterodimer by binding with other CCAAT enhancer-binding protein family proteins [6]. Overexpression of each GADD gene causes growth inhibition and/or apoptosis and combined overexpression of these three GADD genes lead to synergistic or cooperative effects on antiproliferative effects [7]. However, the precise roles of GADD on apoptotic pathways remain to be determined.

Oxidative stress is one of the most important stimuli to mediate growth arrest and cell death. A number of apoptotic mediators such as TNF, chemotherapeutic drugs, and amyloid β-peptides are reported to induce apoptosis through increase in reactive oxygen species [8], [9]. Peroxynitrite, a reaction product of nitric oxide and superoxide anion, is one of the most reactive radicals and damage cellular functions. It is suggested to be involved in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease; a reaction product of peroxynitrite with tyrosine, 3-nitrotyrosine, was found to increase in the brains of the patients with these neurodegenerative disorders [10], [11], [12].

Recently, p38 MAPK was shown to play an important role in the apoptotic signal transduction by various extracellular stimuli. p38 MAPK is a distant member of the MAPK family [13], [14]. p38 MAPK is activated followed by sequential activation of MAPK kinase kinase-MAPK kinase, although the physiological relationship of these MAPK kinase kinases remains to be determined [15], [16], [17]. Several factors are reported to be regulated as the downstream targets of p38 MAPK [18], [19]. Recently, it was reported that hyperosmolality stimulus increased GADD45 and GADD153 protein levels in murine kidney cells via activation of p38 MAPK pathway [20]. However, involvement of GADD34 in p38-dependent apoptotic signal transduction has never been reported.

In this study, we investigated the effect of peroxynitrite on expression of GADD34, GADD45, and GADD153 genes using human neuroblastoma, SH-SY5Y cells. The signal transduction was studied in relation to intracellular redox status and p38 MAPK activation.