Vol. 62, Issue 1, 90-101, July 2002

Aurintricarboxylic Acid Protects against Cell Death Caused by Lipopolysaccharide in Macrophages by Decreasing Inducible Nitric-Oxide Synthase Induction via IB Kinase, Extracellular Signal-Regulated Kinase, and p38 Mitogen-Activated Protein Kinase Inhibition

Chin-Ju Tsi, Yee Chao, Ching-Wen Chen, and Wan Wan Lin

Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan (C.-J.T., C.-W.C., W.W.L.); and Cancer Center, Veterans General Hospital, Taipei, Taiwan (Y.C.)

To elucidate the mechanisms involved in cell protection by aurintricarboxylic acid (ATA), an endonuclease inhibitor, high nitric oxide (NO)-induced macrophage apoptosis was studied. In RAW 264.7 macrophages, a high level of NO production accompanied by cell apoptosis was apparent with lipopolysaccharide (LPS) treatment. Direct NO donor sodium nitroprusside (SNP) also dramatically induced cell death, with an EC₅₀ of 1 mM. Coincubation of ATA (1-500 µM) in LPS-stimulated RAW 264.7 cells resulted in a striking reduction of NO production and cell apoptosis, whereas only a partial cell protection was achieved in response to SNP. This suggests that abrogation of inducible nitric-oxide synthase (iNOS)-dependent NO production might contribute to ATA protection of LPS-treated cells. Immunoblotting and reverse transcription-polymerase chain reaction analysis revealed that ATA down-regulated iNOS protein through transcriptional inhibition of iNOS gene expression but was unrelated to iNOS protein stability. ATA not only inhibited nuclear factor-B (NF-B) activation through impairment of the targeting and degradation of IBs but also reduced LPS-induced activator protein-1 (AP-1) activation. These actions of ATA were not caused by the influence on LPS binding to macrophage membrane. Kinase assays indicated that ATA inhibited IB kinase (IKK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK) activity both in vivo and in vitro, suggesting a direct interaction between ATA and these signaling molecules. Taken together, these results provide novel action targets of ATA and indicate that ATA protection of macrophages from LPS-mediated cell death is primarily the result of its inhibition of NO production, which closely relates to the inactivation of NF-B and AP-1 and inhibition of IKK, ERK and p38 MAPK.