1. ¹Department of Community, Occupational and Family Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
2. ²Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
3. ³Institute of Biomedical and clinical sciences, Peninsula Medical School, Devon, UK
4. ⁴Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

Correspondence: H-M Shen, Department of Community, Occupational and Family Medicine, Yong Loo Lin School of Medicine, National University of Singapore, MD3, 16 Medical Drive, Singapore 117597, Singapore. Tel: +65 6516 4998; Fax: +65 6779 1489; E-mail: cofshm@nus.edu.sg

Received 15 May 2007; Revised 17 September 2007; Accepted 25 September 2007; Published online 16 November 2007.

Abstract

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.