The role of STAT1/IRF-1 on synergistic ROS production and loss of mitochondrial transmembrane potential during hepatic cell death induced by LPS/d-GalN.

Previously, we demonstrated that signal transducer and activator of transcription factor 1 (STAT1) plays an essential role in liver injury induced by lipopolysaccharide (LPS)/D-galactosamine (D-GalN); however, the underlying mechanism involved remains unclear. Here, we showed that LPS/D-GalN administration induced secretion of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma), which mediated apoptosis synergistically. Moreover, LPS/D-GalN-induced apoptosis was associated with increased inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production, as well as elevated reactive oxygen species (ROS) production, which were all strongly inhibited by treatment with the antioxidant N-acetyl-L-cysteine (NAC) and an iNOS/NO inhibitor, L-NMMA. Although STAT1 activation and expression did not change significantly in TNF-alpha/IFN-gamma-cotreated cells compared with cells treated with IFN-gamma alone, the absence of STAT1 or interferon regulatory factor 1 (IRF-1) in genetic knockout mice strongly abrogated the observed effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, loss of mitochondrial transmembrane potential (DeltaPsim), and apoptosis compared with STAT1(+/+) and IRF-1(+/+) mice. Additionally, the synergistic effects of TNF-alpha/IFN-gamma on iNOS/NO induction, ROS production, and apoptosis were significantly inhibited by overexpression of dominant negative STAT1 in contrast to overexpression of wild-type STAT1. In STAT1-deficient mice, nuclear factor kappaB (NF-kappaB) activation by TNF-alpha/IFN-gamma was attenuated and strongly inhibited by both NAC and L-NMMA. Moreover, the proteasome inhibitor, MG132, inhibited NF-kappaB activation and strongly inhibited iNOS/NO induction, ROS production, and loss of DeltaPsim induced by TNF-alpha/IFN-gamma, thereby inhibiting apoptosis. Interestingly, it appears peroxynitrite, which is produced by TNF-alpha/IFN-gamma, may interfere with STAT1 phosphorylation by inducing STAT1 nitration. Collectively, these findings demonstrate that TNF-alpha/IFN-gamma synergistically potentiates iNOS/NO induction, ROS production, and loss of DeltaPsim via STAT1 overexpression, playing an important role in promoting apoptosis and liver injury induced by LPS/D-GalN.

Lee HJ ，Oh YK ，Rhee M ，Lim JY ，Hwang JY ，Park YS ，Kwon Y ，Choi KH ，Jo I ，Park SI ，Gao B ，Kim WH ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Requirement for STAT1 in LPS-induced gene expression in macrophages.

Ohmori Y ，Hamilton TA 《JOURNAL OF LEUKOCYTE BIOLOGY》

Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma- and bacterial lipopolysaccharide-stimulated macrophages.

(5R)-5-Hydroxytriptolide (LLDT-8) is a novel analog of triptolide that has antiarthritic, hepatoprotective, and antiallogenic transplantation-rejective effects. In the present study, we report that LLDT-8 inhibited nitric oxide (NO) production and inducible nitric-oxide synthase (iNOS) expression in macrophages. LLDT-8 significantly attenuated NO production, in a dose-dependent manner, in primary peritoneal macrophages and a macrophage cell line of Raw 264.7 cells following stimulation with interferon (IFN)-gamma, lipopolysaccharide (LPS), and IFN-gamma plus LPS. It also reduced the production of tumor necrosis factor-alpha from LPS-stimulated Raw 264.7 cells. To further elucidate the mechanism responsible for the inhibition of NO, we examined the effect of LLDT-8 on IFN-gamma and LPS-induced iNOS expression. Indeed, LLDT-8 prevented NO generation by inhibiting iNOS expression at mRNA level and protein level, rather than by interfering its enzymatic activity. In IFN-gamma-stimulated Raw 264.7 cells, LLDT-8 suppressed the gene transcription of signal transducer and activator of transcription 1alpha and interferon regulatory factor (IRF)-1, but it displayed no apparent effect on IFN-gamma receptor level on cell surface. After LPS challenge, LLDT-8 further abrogated the expression of LPS receptor complex, including CD14, Toll-like receptor 4, and myeloid differentiation protein-2; decreased the LPS-induced phosphorylation of stress-activated protein kinase/c-Jun NH(2)-terminal kinase, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase (MAPK); retarded the degradation of IkappaBalpha; and ameliorated the DNA binding activity of nuclear factor-kappaB (NF-kappaB) to nuclear proteins that accounts for transcriptional regulation of iNOS. Taken together, these results suggest that LLDT-8 reduces NO production and iNOS expression by inhibiting IFN-gamma-triggered IRF-1 expression and LPS-triggered MAPK phosphorylation and NF-kappaB activation.

Zhou R ，Zheng SX ，Tang W ，He PL ，Li XY ，Yang YF ，Li YC ，Geng JG ，Zuo JP ... -  《JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS》

Requirement of tumor necrosis factor alpha and nuclear factor-kappaB in the induction by IFN-gamma of inducible nitric oxide synthase in macrophages.

IFN-gamma induces NO production, inducible NO synthase (iNOS) protein, and promoter expression in mouse macrophage cells. Mutation of IFN regulatory factor 1 responsive element, gamma-activated site, as well as NF-kappaB elements in the murine iNOS promoter strongly reduced IFN-gamma-induced iNOS transcriptional activity. The role of NF-kappaB activation in iNOS induction by IFN-gamma was corroborated by overexpression of the NF-kappaB inhibitory protein IkappaBalpha, which inhibited iNOS promoter activity induced by IFN-gamma. In addition, IFN-gamma treatment induced p65 binding to the iNOS promoter by chromatin immunoprecipitation assay and NF-kappaB binding to DNA by EMSA, although with a delayed kinetics, suggesting an indirect autocrine role for another cytokine produced in response to IFN-gamma. It is interesting that we found that IFN-gamma induced TNF-alpha secretion, and the induction of iNOS expression by IFN-gamma was abolished in primary peritoneal macrophages from TNF-alpha-deficient (TNF-alpha-/-) mice or in RAW 264.7 cells treated with anti-TNF-alpha neutralizing antibodies. Moreover, exogenous addition of recombinant mouse TNF-alpha restored iNOS expression induced by IFN-gamma in TNF-alpha-/- mice. It is intriguing that NF-kappaB binding to DNA in response to IFN-gamma treatment was absent in TNF-alpha-/- mice. Taken together, our data suggest that the TNF-alpha produced in response to IFN-gamma is required for iNOS induction by activating NF-kappaB transcription factor.

Vila-del Sol V ，Díaz-Muñoz MD ，Fresno M 《JOURNAL OF LEUKOCYTE BIOLOGY》

NF-kappaB stimulates inducible nitric oxide synthase to protect mouse hepatocytes from TNF-alpha- and Fas-mediated apoptosis.

Hepatocyte apoptosis is induced by tumor necrosis factor alpha (TNF-alpha) and Fas ligand. Although nuclear factor-kappaB (NF-kappaB) activation protects hepatocytes from TNF-alpha-mediated apoptosis, the NF-kappaB responsive genes that protect hepatocytes are unknown. Our aim was to study the role of NF-kappaB activation and inducible nitric oxide synthases (iNOSs) in TNF-alpha- and Fas-mediated apoptosis in hepatocytes. Primary cultures of hepatocytes from wild-type and iNOS knockout mice were treated with TNF-alpha, the Fas agonistic antibody Jo2, a nitric oxide (NO) donor (S-nitroso-N-acetylpenicillamine), an NO inhibitor (N(G)-methyl-L-arginine acetate), and/or adenovirus-expressing NF-kappaB inhibitors. The IkappaB superrepressor and a dominant-negative form of IkappaB kinase beta (IKKbeta) inhibited NF-kappaB binding activity by TNF-alpha or Jo2 and sensitized hepatocytes to TNF-alpha- and Jo2-mediated apoptosis. TNF-alpha and Jo2 induced iNOS messenger RNA and protein levels through the induction of NF-kappaB. S-nitroso-N-acetylpenicillamine inhibited Bid cleavage, the mitochondrial permeability transition, cytochrome c release, and caspase-8 and -3 activity, and reduced TNF-alpha- and Fas-mediated death in hepatocytes expressing IkappaB superrepressor. N(G)-methyl-L-arginine acetate partially sensitized hepatocytes to TNF-alpha- and Fas-mediated cell killing. TNF-alpha alone or Jo2 alone induced moderate cell death in hepatocytes from iNOS(-)/(-) mice. NO protects hepatocytes from TNF-alpha- and Fas-mediated apoptosis. Endogenous iNOS, which is activated by NF-kappaB via IKKbeta, provides partial protection from apoptosis.

Hatano E ，Bennett BL ，Manning AM ，Qian T ，Lemasters JJ ，Brenner DA ... -  《GASTROENTEROLOGY》