Transactivation Of iNOS Promoter Research Articles

Cinnamomi Cortex and Scutellariae Radix in the Japanese herbal medicine Kampo saireito inhibit expression of iNOS through different mechanisms in hepatocytes

ABSTRACTAimThe Japanese herbal medicine, saireito, prevents the induction of inducible nitric oxide synthase (iNOS) expression and NO production, which is an indicator of liver protection. Saireito is composed of 12 kinds of crude drug. This study aimed to examine whether these crude drugs influence the induction of iNOS gene expression and if so, the mechanisms involved.MethodsHepatocytes were isolated from rats by collagenase perfusion and cultured. Each crude drug was added to primary cultured cells stimulated by interleukin (IL)‐1β. Induction of iNOS/NO production and its signaling pathway were analyzed.ResultsIL‐1β induced iNOS expression and NO production. Eleven of the saireito crude drugs inhibited NO production. Among them, Cinnamomi Cortex and Scutellariae Radix had higher effects than the others and inhibited the expression of iNOS and tumor necrosis factor (TNF)‐α. Transfection experiments revealed that both crude drugs reduced the activities of iNOS promoter transactivation (mRNA synthesis) and its mRNA stability. However, Cinnamomi Cortex and Scutellariae Radix showed that different mechanisms were involved in their inhibitory effects on two signaling pathways of iNOS induction, IκBα degradation/nuclear factor (NF)‐κB activation and Akt activation/type I IL‐1 receptor (IL‐1RI) upregulation.ConclusionCinnamomi Cortex and Scutellariae Radix can prevent the induction of iNOS gene expression, in part through NF‐κB activation and IL‐1RI upregulation with different mechanisms, indicating that saireito containing both crude drugs may be a potential therapeutic treatment for injured livers.

PyroGlu-Leu inhibits the induction of inducible nitric oxide synthase in interleukin-1β-stimulated primary cultured rat hepatocytes

Pyroglutamyl leucine (pyroGlu-Leu), which is a peptide isolated from wheat gluten hydrolysate, has been reported to be a hepatoprotective compound in acute liver failure. In inflamed liver, proinflammatory cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)-α stimulate the induction of inducible nitric oxide synthase (iNOS). Excess production of nitric oxide (NO) by iNOS is an inflammatory biomarker in liver injury. We examined proinflammatory cytokine-stimulated hepatocytes as a simple “in vitro inflammation model” to determine liver protective effects of pyroGlu-Leu and its mechanisms of action. We hypothesized that pyroGlu-Leu inhibits the induction of iNOS gene expression, resulting in the attenuation of hepatic inflammation. Hepatocytes were isolated from rats by collagenase perfusion and cultured. Primary cultured cells were treated with IL-1β in the presence or absence of pyroGlu-Leu. The induction of iNOS and its signaling pathway were analyzed. IL-1β stimulated the enhancement of NO production in hepatocytes and this effect was inhibited by pyroGlu-Leu. pyroGlu-Leu decreased the expression of iNOS protein and its mRNA. Transfection experiments with iNOS-luciferase constructs revealed that pyroGlu-Leu inhibited both of iNOS promoter transactivation and its mRNA stabilization. pyroGlu-Leu also decreased the expression of an iNOS gene antisense transcript, which is involved in iNOS mRNA stability. However, pyroGlu-Leu had no effects on IκB degradation and NF-κB activation. Results demonstrate that pyroGlu-Leu inhibited the induction of iNOS gene expression at transcriptional and post-transcriptional steps through IκB/NF-κB-independent pathway, leading to the prevention of NO production. pyroGlu-Leu may have therapeutic potential for liver injury through the suppression of iNOS.

Peroxidation of n-3 Polyunsaturated Fatty Acids Inhibits the Induction of iNOS Gene Expression in Proinflammatory Cytokine-Stimulated Hepatocytes

Eicosapentaenoic acid and docosahexaenoic acid (EPA/DHA), n-3 polyunsaturated fatty acids (PUFAs), have a variety of biological activities including anti-inflammatory and anticancer effects. We hypothesized that their peroxidized products contributed in part to anti-inflammatory effects. In the liver, the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) has been implicated as one of the factors in hepatic inflammation and injury. We examined whether the peroxidation of EPA/DHA influences the induction of iNOS and NO production in proinflammatory cytokine-stimulated cultured hepatocytes, which is in vitro liver inflammation model. Peroxidized EPA/DHA inhibited the induction of iNOS and NO production in parallel with the increased levels of their peroxidation, whereas unoxidized EPA/DHA had no effects at all. Peroxidized EPA/DHA reduced the activation of transcription factor, NF-κB, and the expression of the iNOS antisense transcript, which are involved in iNOS promoter transactivation (mRNA synthesis) and its mRNA stabilization, respectively. These findings demonstrated that peroxidized products of EPA/DHA suppressed the induction of iNOS gene expression through both of the transcriptional and posttranscriptional steps, leading to the prevention of hepatic inflammation.

EDARAVONE INHIBITS THE INDUCTION OF INOS GENE EXPRESSION AT TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL STEPS IN MURINE MACROPHAGES

Edaravone, a free radical scavenger, plays crucial roles in the prevention of injuries to the brain, heart, and liver. Our in vivo study indicated that edaravone prevented endotoxin-induced liver injury through inhibition of NO production in addition to reductions in oxidative products and proinflammatory cytokine induction. Studies were performed to determine whether edaravone directly influences the induction of iNOS in murine RAW264 macrophages as a substitute for Kupffer cells (resident macrophages) in the liver. RAW264 cells were treated with LPS (1 microg/mL) in the presence or absence of edaravone. NO production, iNOS induction, and its related signaling were analyzed. Edaravone (0.5 - 5 mM) decreased the production of NO stimulated by LPS in time- and dose-dependent manners, and these concentrations of edaravone had no cytotoxic effects. Edaravone decreased the levels of iNOS protein and mRNA. Transfection experiments with iNOS promoter-luciferase constructs revealed that edaravone inhibited the activities of both iNOS promoter transactivation and iNOS mRNA stabilization. However, edaravone did not have any effects on I kappaB alpha degradation or nuclear factor-kappaB activation. In contrast, edaravone markedly suppressed the LPS-stimulated expression of iNOS antisense-transcript, which stabilizes iNOS mRNA by interacting with its 3'-untranslated region and RNA-binding proteins. Edaravone may inhibit the induction of iNOS gene expression at the steps of its promoter transactivation in a nuclear factor-kappaB-independent manner and mRNA stabilization in RAW264 cells.

Na +/H + exchanger inhibitor, FR183998, has protective effect in lethal acute liver failure and prevents iNOS induction in rats

Selective inhibition of Na(+)/H(+) exchanger (NHE) improves organ dysfunctions including heart ischemia-reperfusion injury. In vivo and in vitro studies were designed to investigate whether NHE inhibitor has a protective effect in lethal acute liver failure, and if so, what are the mechanisms involved. NHE inhibitor (FR183998) was administered to rats treated with d-galactosamine/lipopolysaccharide (GalN/LPS), or incubated with cultured hepatocytes stimulated by pro-inflammatory cytokine, interleukin (IL)-1beta. FR183998 reduced the increases of pro-inflammatory cytokines such as TNF-alpha, interferon-gamma and CINC-1, but enhanced the anti-inflammatory cytokine, IL-10, leading to the prevention of liver injury and increased survival rate in GalN/LPS-treated animals. FR183998 prevented the activation of transcription factor NF-kappaB induced by GalN/LPS. In vivo and in vitro experiments revealed that FR183998 reduced inducible nitric oxide synthase (iNOS) induction and NO production. Further FR183998 decreased levels of iNOS antisense-transcript in GalN/LPS-treated liver and IL-1beta-treated hepatocytes. FR183998 may reduce a variety of inflammatory mediators such as cytokines and NO in part through the inhibition of NF-kappaB activation, resulting in the prevention of fulminant liver failure, and may inhibit iNOS gene expression at steps of iNOS promoter transactivation and its mRNA stabilization through NF-kappaB and iNOS antisense-transcript, respectively.

Rebamipide, anti-gastric ulcer drug, up-regulates the induction of iNOS in proinflammatory cytokine-stimulated hepatocytes

Nitric oxide (NO) generated from inducible NO synthase (iNOS) during hepatic injury has been reported to contribute to cytoprotection or cellular damage. Rebamipide, anti-gastric ulcer drug, has protective effects in a variety of tissue and organ injury. However, it remains unknown whether rebamipide is involved in the regulation of iNOS gene expression under pathological conditions. We examined whether rebamipide influences the induction of iNOS in hepatocytes exposed to pro-inflammatory cytokine. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of rebamipide. Pretreatment of cells with rebamipide resulted in up-regulation of iNOS induction by IL-1β, followed by increased NO production. Rebamipide enhanced the degradation of IκBα and the activation of NF-κB. Further, rebamipide super-induced the up-regulation of type I IL-1 receptor (IL-1RI), which is essential for iNOS induction in addition to the IκB/NF-κB pathway. Transfection experiments revealed that rebamipide increased the transactivation of iNOS promoter and the stability of iNOS mRNA. In the latter, rebamipide increased the antisense-transcript corresponding to the 3′-UTR of iNOS mRNA, which stabilizes iNOS mRNA by interacting with the 3′-UTR and RNA-binding proteins. These findings demonstrate that rebamipide up-regulates iNOS by iNOS promoter activation through NF-κB, and by its mRNA stabilization presumably through the super-induction of IL-1RI and antisense-transcript. Rebamipide may contribute to a novel potentiated treatment in liver injuries.

Characterization of alternatively spliced isoforms of the type I interleukin-1 receptor on iNOS induction in rat hepatocytes

In animal models of liver injury, proinflammatory cytokines are implicated in inducing iNOS, which is followed by the production of NO in hepatocytes. Previously we have reported that the up-regulation of type I IL-1 receptor (IL-1RI) is required for the transcriptional activation of iNOS gene, in concert with the activation of transcription factor NF-κB. In this study, we found three alternatively spliced isoforms of IL-1RI in primary cultured rat hepatocytes: two (long and short) membrane-bound and one soluble IL-1RI. Interleukin (IL)-1β markedly augmented the mRNA levels of long and short IL-1RI with time, but was less effective for soluble IL-1RI. Two membrane-bound IL-1RI were localized in the intracellular fraction, whereas soluble IL-1RI was released into the culture medium. Cotransfection experiments with iNOS promoter-luciferase constructs revealed that the overexpression of long and short IL-1RI, but not soluble IL-1RI, significantly increased the transactivation of iNOS promoter and the stabilization of its mRNA. In contrast, the addition of conditioned medium containing soluble IL-1RI reduced the induction of iNOS and NO production stimulated by IL-1β. These results further suggest that the enhancement of IL-1RI isoforms may contribute to the regulation of iNOS induction in hepatocytes.

HEPATIC ISCHEMIA/REPERFUSION UPREGULATES THE SUSCEPTIBILITY OF HEPATOCYTES TO CONFER THE INDUCTION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE EXPRESSION

During hepatic ischemia/reperfusion (I/R), proinflammatory cytokines such as tumor necrosis factor alpha and interleukin (IL) 1beta stimulate the induction of inducible nitric oxide synthase (iNOS) in hepatocytes, followed by massive production of nitric oxide. We hypothesized that I/R upregulated the susceptibility of hepatocytes to confer the induction of iNOS gene expression. This study was designed to investigate whether cell susceptibility occurs in response to I/R and to delineate the mechanisms underlying the susceptibility. Hepatocytes were isolated from rats with hepatic I/R or sham, cultured, and treated with IL-1beta. The iNOS induction and its signal including inhibitor kappaB (IkappaB) kinase/nuclear factor kappaB (NF-kappaB) and Akt/type 1 interleukin 1 receptor (IL-1R1) were analyzed. Hepatocytes isolated from rats with I/R markedly increased the production of nitric oxide when stimulated by IL-1beta as compared with sham control. Ischemia/R also increased the levels of iNOS protein and its messenger RNA. Furthermore, I/R enhanced the activation of transcription factor NF-kappaB and the transactivation of iNOS promoter. However, I/R had no effects on the degradation of IkappaB and the nuclear translocation of p65 subunit of NF-kappaB. In contrast, I/R increased the phosphorylation of Akt and the upregulation of IL-1R1 induction, which is essential signal for the transcriptional activation of iNOS in addition to IkappaB kinase/NF-kappaB. These results demonstrate that I/R may augment hepatocyte susceptibility for the induction of iNOS gene expression through the enhancement of IL-1R1.

Hepatitis C virus core protein transactivates the inducible nitric oxide synthase promoter via NF-κB activation

Intrahepatic levels of the inducible nitric oxide synthase (iNOS) are increased in chronic hepatitis C patients. As iNOS gene promoter contains Nuclear Factor (NF)-κB binding sites and hepatitis C virus (HCV) core protein activates NF-κB, the aim of this work was to study if HCV core protein transactivates iNOS promoter through NF-κB activation. iNOS mRNA and protein were determined by RT-PCR and western blot in HepG2 cells. The effect of HCV core protein on iNOS promoter was assayed by cotransfecting HepG2 cells with the core protein expression plasmid pHCV-Co and p1iNOS-CAT or p2iNOS-CAT plasmids. Formation of NF-κB–DNA complexes was determined by electrophoretic mobility shift assay. Transfection of HepG2 cells with pHCV-Co plasmid results in an increase in iNOS mRNA and protein levels. Cotransfection with pHCV-Co and p1iNOS-CAT or p2iNOS-CAT plasmids results in a transactivation of iNOS promoter, the presence of the proximal NF-κB binding site in the promoter being sufficient for the transactivation. Furthermore, the HCV core protein increases the formation of complexes between NF-κB and its binding sequence in the iNOS promoter. The expression of the NF-κB inhibitor IKB reverts the effect of the HCV core protein on the iNOS promoter. In conclusion, HCV core protein transactivates iNOS gene promoter through NF-κB activation.

Hepatocyte Nuclear Factor-4α Mediates Redox Sensitivity of Inducible Nitric-oxide Synthase Gene Transcription

The underlying redox-sensitive mechanisms that regulate hepatocyte expression of inducible nitric-oxide synthase (iNOS) and its antioxidant functions are largely unknown. We have demonstrated previously that oxidative stress induced by benzenetriol-mediated superoxide production increases interleukin-1beta-induced iNOS protein synthesis, steady state iNOS mRNA expression, NO production, iNOS gene transcription, and trans-activation of the iNOS promoter in primary cultures of rat hepatocytes. In this study, we extend these studies by establishing the sequence specificity and binding of nuclear protein to the previously described 15-base cis-regulatory element of the rat hepatocyte iNOS promoter, isolating and identifying the cis-regulatory element transcription factor as hepatocyte nuclear factor-4alpha (HNF-4alpha), and confirming the functional role of HNF-4alpha in mediating redox-sensitive iNOS promoter trans-activation. In addition, we demonstrate that binding of HNF-4alpha to the transcriptional coactivator, PC4, in the presence of oxidative stress and interleukin-1beta stimulation is essential for increased iNOS promoter activity in this setting. Our results indicate that HNF-4alpha is the transcription factor that mediates redox regulation of hepatocyte iNOS gene transcription.