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

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 M) 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 factorB (NFB) activation through impairment of the targeting and degradation of I Bs 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 I B 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 NFB and AP-1 and inhibition of IKK, ERK and p38 MAPK. Apoptosis is an essential process of the development and tissue homeostasis of most multicellular organisms, and the deregulation of apoptosis has been implicated in the pathogenesis of many disease states. One of the hallmarks of apoptosis is the orderly cleavage of genomic DNA of nucleosomal or oligonucleosomal lengths. To date, a variety of endonucleases responsible for chromatin degradation have been identified. Although some evidence indicates that endonuclease(s) leading to oligonucleosomal DNA fragmentation is common and an essential event in apoptosis, endonucleasemediated DNA fragmentation may not play a central role in apoptosis for some death inducers. For instance, it was reported that endonucleolytic DNA degradation is neither required nor sufficient for K withdrawal-induced apoptosis of cultured cerebellar granule neurons (Schulz et al., 1998) and heat shock-induced apoptosis of the U937 leukemic cell line (Shrivastava et al., 2000). Aurintricarboxylic acid (ATA), a negatively charged triphenylmethane derivative (473 Da), has been demonstrated to prevent apoptosis in a variety of cell models. It was used as an antiapoptotic drug to counteract ischemic or cytotoxic This work was supported by the grants from the National Science Council of Taiwan (NSC 90-2314-B075-077 and NSC 90-2320-B002-087). ABBREVIATIONS: ATA, aurintricarboxylic acid; MAPK, mitogen-activated protein kinase; STAT, signal transducer and activator of transcription; NFB, nuclear factorB; NO, nitric oxide; LPS, lipopolysaccharide; iNOS, inducible nitric-oxide synthase; IKK, I B kinase; ERK, extracellular signal-regulated kinase; AP-1, activator protein-1; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; EMSA, electrophoretic mobility shift assay; PS, phosphatidylserine; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; TBST, Tris-buffered saline/Tween 20; RT, reverse transcription; PCR, polymerase chain reaction; SNP, sodium nitroprusside; NOS, nitric-oxide synthase; GST, glutathione S-transferase; IFN, interferon. 0026-895X/02/10107-90–101$7.00 MOLECULAR PHARMACOLOGY Vol. 101, No. 7 Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics 1451/989395 Mol Pharmacol 101:90–101, 2002 Printed in U.S.A. 90 at A PE T Jornals on July 7, 2017 m oharm .aspeurnals.org D ow nladed from injury to neurons (Rosenbaum et al., 1998; Vincent and Maiese, 1999; Heiduschka and Thanos, 2000). As such, the use of ATA as a therapeutic agent for conditions such as ischemic stroke and Alzheimer’s disease has been proposed (Rosenbaum et al., 1998). ATA also possesses the ability to inhibit apoptosis of non-neuronal cells, such as hemopoietic cells (Rui et al., 1998; Shrivastava et al., 2000), endothelial cells (Escargueil-Blanc et al., 1997), oligodendrocytes (Vollgraf et al., 1999), and lutein cells (Viergutz et al., 2000). The pharmacological action of ATA (10–100 M) as an inhibitor of apoptosis in serumand growth factor-deprived neuronal cultures was first thought to reside in its inhibition of cellular endonucleases (Martin et al., 1988; Batistatou and Greene, 1991). After these observations, ATA was also demonstrated to act in an endonuclease-independent manner to interact with several cellular targets, which might also contribute to its antiapoptotic effects. These include the topoisomerases (Benchokroun et al., 1995), the interferonand N-methyl-D-aspartate receptors (Zeevald et al., 1993), and different important signaling cascades. For example, it is a potent activator of the mitogen-activated protein kinase (MAPK) cascade in PC-12 (Okada and Koizumi, 1995) and Nb2 lymphoma (Rui et al., 1998) cells. It can activate the erbB4 receptor, a member of the epidermal growth factor receptor family, in SH-SY5Y neuroblastoma cells (Okada and Koizumi, 1997). Its activation on both MAPK and erbB4 activation share characteristics with growth factors that can rescue cells from programmed cell death caused by serum starvation. In addition, ATA affects Nb2 lymphocytes through a selective activation of the Janus tyrosine kinase 2-STAT5 pathway, which promotes cell viability and proliferation (Rui et al., 1998). A recent in vitro study showed that ATA inhibited DNA–NFB binding at 30 M (Sharma et al., 2000). All these findings suggest that ATA does not act exclusively as an endonuclease inhibitor but might exert its antiapoptotic effect via its action on signal transduction pathways that promote cell survival. These endonucleaseindependent mechanisms that contribute to ATA antiapoptotic effects might be cell type-specific. How ATA activates these signaling pathways, as mentioned above, remains unclear. Apoptotic pathways depend totally on the insult and cell types, and the biochemical elucidation on target enzyme and signaling pathways will provide insight into the molecular mechanism responsible for the antiapoptotic action of ATA. As such, it is worthwhile to examine the action of ATA on excess nitric oxide (NO)-induced apoptosis, which is a principal cytotoxic mediator implicated in many inflammatory conditions. There is considerable evidence for the apoptotic effects of NO on macrophages exposed to endotoxin lipopolysaccharide (LPS) (Albina and Reichner, 1998; Hortelano et al., 1999). The use of inhibitors of inducible nitric-oxide synthase (iNOS), overexpression of arginase, and scavengers for NO generated by these activated cells have been demonstrated to block cell injury and death (Misko et al., 1998; Gotoh and Mori, 1999). Besides reduction in NO generation, therapies aimed at inhibiting NO-dependent cell apoptosis may contribute to improving the outcome of sepsis, which remains a clinical conundrum. Thus, in this study, we address the beneficial effect of ATA in macrophages that undergo apoptosis caused by the large amounts of NO produced through iNOS induction by LPS. Unexpectedly, we found that ATA could prevent LPS-induced apoptosis through inhibition of iNOS expression at the transcriptional level. The inhibition of I B kinase (IKK), extracellular signal-regulated kinase (ERK), and p38 MAPK may contribute to the blockade of NFB and AP-1 activation, which are two major transcription factors essential for iNOS gene expression. Experimental Procedures