DMPO blocks signaling, activation , and genomic damage induced by LPS in macrophages

ZILI, ZHAI; SANDRA E. GOMEZ-MEJIBA; DARIO C. RAMIREZ

ORLANDO, FL

Congreso; 17TH ANNUAL MEETING OF THE SFRBM; 2010

SFRBM

DMPO BLOCKS SIGNALING, ACTIVATION, AND GENOMIC DAMAGE INDUCED BY LPS IN MACROPHAGES   Zili Zhai, Sandra E. Gomez-Mejiba & Dario C. Ramirez Experimental Therapeutics Research Laboratory, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104   Exposure of macrophages to lipopolysaccharide (LPS) induces inflammatory, oxidative, and endoplasmic reticulum (ER) stresses, which cause cell damage and apoptosis. In these cellular stresses, reactive oxygen species are important messengers. In our earlier studies, we used the spin trap 5,5,-dimethyl-1-pyrroline N-oxide (DMPO) to detect macromolecular free radicals. We found that DMPO inhibits LPS-induced macrophage activation and death, presumably by trapping free radicals. However, alternative mechanisms of action of DMPO such as influences on cellular stress pathways might also be involved. In this study, we sought to determine whether DMPO affects LPS-induced inflammatory activation, ER-stress, and apoptosis in macrophages. RAW 264.7 cells were treated with LPS (1 ng/ml) and/or DMPO (50 mM) for 24 h. LPS induced cell activation as showed by inducible nitric oxide synthase expression and production of inflammatory mediators such as •NO, TNF-á, and IL-1â. However, DMPO blocked these inflammatory responses. LPS-induced TNF-á and •NO are known to induce ER stress and apoptosis. ER stress was shown by activation of PERK and induction of CHOP, which is known to be involved in apoptosis. Notably, DMPO downregulated LPS-induced ER stress. Furthermore, LPS decreased anti-apoptotic protein 14-3-3 expression, but increased caspase-3 activation. As expected, DMPO restored 14-3-3 expression but, unexpectedly, has a synergistic effect on caspase-3 activation by LPS. Even though upregulating the apoptotic marker caspase-3, DMPO protected cells from LPS-induced DNA damage as showed by inhibiting phosphorylation of histone H2A, acetylation of histone H2B, and phosphorylation of DNA damage sensor p53 and checkpoint Chk1. In order to understand the mechanistic basis of DMPO for the above-mentioned effects, we determined the phosphorylation of Akt, mitogen-activated protein kinases (MAPKs), and IêB-á that are linked to NF-êB activation. DMPO inhibited phosphorylation of Akt, MAPKs (ERK, JNK/SAPK and p38), and IêB-á, which led to inhibition of NF-êB activation. Taken together, DMPO prevented macrophage activation, ER-stress and DNA damage triggered by LPS throughout the blocking of Akt and MAPKs signaling pathways. The analysis of DMPO effects on signaling and gene expression in “inflamed” cells would help us develop new therapeutic applications of this old nitrone spin trap. Supported by NIEHS 5R00ES015415.