Abstract
Excess nitric oxide (NO) induces apoptosis in some cell types including macrophages; however, the cascade of NO-mediated apoptosis is not fully understood. We investigated the initial steps of NO-mediated apoptosis in mouse macrophage-like RAW 264.7 cells. When cells were treated with bacterial lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma), NO-mediated apoptosis occurred. Under these conditions, p53 accumulation was not observed, indicating that DNA damage is not the main trigger of NO-mediated apoptosis. On the other hand, mRNA and protein for CHOP, a transcription factor known to be induced by endoplasmic reticulum (ER) stress, were induced. The CHOP induction by LPS/IFN-gamma treatment preceded cytochrome c release from mitochondria. In addition, p90ATF6, an ER membrane-bound transcription factor involved in ER stress response, was cleaved to its active soluble form p50ATF6, which was transported to nucleus and bound to the ER stress response element of the CHOP gene. In the luciferase reporter assay, both the CHOP-binding element of the Rous sarcoma virus long terminal repeat and ER stress response element of the CHOP gene were activated by LPS/IFN-gamma treatment. When RAW 264.7 cells or COS-7 cells were transfected with expression plasmids for CHOP, p90ATF6, or p50ATF6, cell death was observed. In addition, apoptosis induced by p50ATF6 was prevented by a CHOP dominant negative form as well as by an ATF6 dominant negative form, and LPS/IFN-gamma-induced apoptosis was prevented by the CHOP dominant negative form. Peritoneal macrophages from CHOP knockout mice showed resistance to NO-induced apoptosis. These results indicate that the ER stress pathway involving ATF6 and CHOP plays a key role in NO-mediated apoptosis in macrophages.
Highlights
Nitric oxide (NO) is a multifunctional biomolecule involved in a variety of physiological and pathological processes [1]
P90ATF6, an endoplasmic reticulum (ER) membranebound transcription factor involved in ER stress response, was cleaved to its active soluble form p50ATF6, which was transported to nucleus and bound to the ER stress response element of the CHOP gene
Peritoneal macrophages from CHOP knockout mice showed resistance to nitric oxide (NO)-induced apoptosis. These results indicate that the ER stress pathway involving ATF6 and CHOP plays a key role in NO-mediated apoptosis in macrophages
Summary
Plasmids—A luciferase reporter assay plasmid pGL3-promoter vector that harbors the SV40 promoter was obtained from Promega Corp. (Madison, WI). A partial mouse CHOP cDNA clone was isolated by reverse transcription-PCR using total RNA from immunostimulated RAW 264.7 cells. A full-length mouse CHOP cDNA clone was isolated by reverse transcription-PCR, using total RNA from immunostimulated RAW 264.7 cells. The mammalian expression plasmid for CHOP dominant negative form (pOPRSVI-L133A/L140ACHOP) [10] was obtained from Shizuo Akira (Osaka University, Japan). Transfection of RAW 264.7 cells and COS-7 cells with plasmids was carried out using TransIT-LT1 polyamine (PanVera Corp., Madison, WI) according to the protocol provided by the manufacturer. Hybridization was performed using digoxigenin-labeled antisense RNA as a probe for mouse CHOP or rat GAPDH. The sequence of double-stranded oligonucleotide used for the ERSE of the CHOP gene is as follows: 5Ј-CCTACCAATCAGAAAGTGGCACGC-3Ј, 3Ј-GGATGGTTAGTCTTTCACCGTGCG-5Ј This probe was 3Ј end-labeled with digoxigenin-11-ddUTP and terminal transferase using a DIG gel shift kit (Roche Molecular Biochemicals). Cells were cultured with or without LPS plus IFN-␥ or thapsigargin for 10 h prior to harvesting. 34 h after transfection, luciferase activity of cell extracts was measured using Luciferase Assay Systems (Promega Corp.) and normalized for -galactosidase activity
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