Involvement of Selective Reactive Oxygen Species Upstream of Proapoptotic Branches of Unfolded Protein Response

Makiko Yokouchi,Nobuhiko Hiramatsu,Kunihiro Hayakawa,Maro Okamura,Shuqi Du,Ayumi Kasai,Yosuke Takano,Akihiro Shitamura,Tsuyoshi Shimada,Jian Yao,Masanori Kitamura

doi:10.1074/jbc.m705951200

Abstract

Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either O(2)(*), H(2)O(2), or ONOO(-) caused apoptosis, whereas ER stress was induced only by O(2)(*) or ONOO(-). Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO(-) was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. O(2)(*) caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by O(2)(*) or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O(2)(*) was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by O(2)(*)-triggered activation of XBP1.

Highlights

Suppression of Endoplasmic reticulum (ER) stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress
To examine whether the generation of reactive oxygen species (ROS) is causative of cadmium-induced apoptosis, cells were pretreated with antioxidant N-acetylcysteine (NAC) or SOD-like antioxidant MnTM-2PyP, stimulated by CdCl2, and subjected to phase-contrast microscopy and Hoechst staining
Cadmium-induced apoptosis of renal tubular cells is dependent on ER stress (1)

Summary

Introduction

Suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. To examine whether the generation of ROS is causative of cadmium-induced apoptosis, cells were pretreated with antioxidant N-acetylcysteine (NAC) or SOD-like antioxidant MnTM-2PyP, stimulated by CdCl2, and subjected to phase-contrast microscopy and Hoechst staining. Attenuation of cadmium-induced ER stress in LL/MnSOD cells was evidenced by blunted induction of CHOP (Fig. 3G).

Results

Conclusion