Summary We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine‐phospholipase C (PC‐PLC) and phosphatidylinositol‐phospholipase C (PI‐PLC) to induce protein kinase C activation, which in turn initiates nuclear factor‐κB (NF‐κB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3‐kinase (PI3K)/Akt, and p38 mitogen‐activated protein kinase (MAPK) in LTA‐induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA‐induced iNOS expression and NO release in concentration‐dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time‐dependent activations of Akt and p38 MAPK. The LTA‐induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA‐induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA‐induced formation of an NF‐κB‐specific DNA–protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in κB‐luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF‐κB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.

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In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1) in denbinobin-induced apoptosis in human lung adenocarcinoma (A549) cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN), two antioxidants (N-acetyl-L-cysteine (NAC) and glutathione (GSH)), a c-Jun N-terminal kinase (JNK) inhibitor (SP600125), and an activator protein-1 (AP-1) inhibitor (curcumin). Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS) production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobininduced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

This study investigates the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release caused by Staphylococcus aureus lipoteichoic acid (LTA) in RAW 264.7 macrophages. A phosphatidylcholine-phospholipase C (PC-PLC) inhibitor (D-609) and a phosphatidylinositol-phospholipase C (PI-PLC) inhibitor (U-73122) attenuated LTA-induced iNOS expression and NO release. Two PKC inhibitors (Go 6976 and Ro 31-8220), an NF-kappaB inhibitor (pyrrolidine dithiocarbamate; PDTC), and long-term (24 h) 12-phorbol-13-myristate acetate (PMA) treatment each also inhibited LTA-induced iNOS expression and NO release. Treatment of cells with LTA caused an increase in PKC activity; this stimulatory effect was inhibited by D-609, U-73122, or Ro 31-8220. Stimulation of cells with LTA caused IkappaB-alpha phosphorylation and IkappaB-alpha degradation in the cytosol, and translocation of p65 and p50 NF-kappaB from the cytosol to the nucleus. Treatment of cells with LTA caused NF-kappaB activation by detecting the formation of NF-kappaB-specific DNA-protein complexes in the nucleus; this effect was inhibited by Go 6976, Ro 31-8220, long-term PMA treatment, PDTC, L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), and calpain inhibitor I. These results suggest that LTA might activate PC-PLC and PI-PLC to induce PKC activation, which in turn initiates NF-kappaB activation, and finally induces iNOS expression and NO release in RAW 264.7 macrophages.

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