Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation

Xue Wang,Yong Wang,Hong Pyo Kim,Kiichi Nakahira,Stefan W Ryter,Augustine M.K Choi

doi:10.1074/jbc.m607610200

Abstract

Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47(phox) subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved in both intrinsic and extrinsic apoptotic signaling. Carbon monoxide inhibited the activation of Bid and the expression and mitochondrial translocation of Bax, whereas promoted Bcl-X(L)/Bax interaction and increased Bad phosphorylation. We also show that carbon monoxide promoted an interaction of heme oxygenase-1 with Bax. These results define novel mechanisms underlying the antiapoptotic effects of carbon monoxide during hyperoxic stress.

Highlights

Vated oxygen tension in animal models causes acute and chronic lung injury that resembles acute respiratory distress syndrome
Larly, necrosis, but not apoptosis, was observed in type II cells hyperoxia induced the time-dependent cleavage of Bid, isolated from rats exposed to hyperoxia [10, 38]
A549 cells exposed to hyperoxia, vation and translocation from cytosol to mitochondria

Summary

EXPERIMENTAL PROCEDURES

Chemicals and Reagents—Antibodies anti-Bax, anti-Bid, anti-caspase-8, anti-caspase-9, anti-cytochrome c, anti-Fas, and protein A-agarose were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). For mitochondrial iso- immunoprecipitation, 1 ␮g of antibody (anti-Fas, anti-6A7, lation, at various times after exposure to hypoxia, MLEC were anti-Bax, or anti-phosphoserine) was added to 500 ␮g of total harvested in 0.05% of digitonin in extraction buffer containing protein in 500 ␮l, rotated for 2 h at 4 °C, and incubated. Overexpression of HO-1 by infection with ho-1 containing adenovirus inhibited hyperoxia-induced cell death, as assessed by cell viability and LDH release assays The presence of CO (250 ppm) inhibited hyperoxia-dependent cytochrome c release from the mitochondria and its corresponding accumulation in the cytosol (Fig. 2C). CO inhibbuffered saline with 0.2% Tween 20, the horseradish peroxi- ited the time-dependent activation of caspase-3 during dase-conjugated secondary antibody was applied, and the blot hyperoxic treatment of MLEC (Fig. 2E). The presence of CO during the hyperoxia dent’s t test, and a value of p Ͻ 0.05 was considered significant. treatment decreased the DISC formation and delayed the cleav-

RESULTS

Findings

DISCUSSION