Nitric Oxide Synthase Gene Transcription Research Articles

Interaction of recombinant octameric hemoglobin with endothelial cells

Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbβG83C. We investigate the potential side effects of rHbβG83C on endothelial cells. The rHbβG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbβG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb βG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

Inflammatory Cytokines Induce Oxidative Stress And Apoptosis In PC12 Cells

Inflammation causes oxidative stress, which plays a critical role in neurodegenerative diseases such as multiple sclerosis and diabetic neuropathy. We have shown that PC12 cells exposed to metals such as manganese and cobalt (which causes oxidative stress) underwent apoptotic cell death. Treatment with reducing agents such as glutathione reversed the effect, suggesting oxidative stress processes were involved. Caspase activity was induced, indicating that cell death was apoptotic. It is known that inflammation contributes to progression of the disease. We investigated whether inflammatory cytokines contribute to cell death by activating oxidative pathways. PC12 cells treated with interferon gamma showed 60% loss in viability. Inhibitors of caspase‐3, −8 and −9 activity prevented cell death. Inducible nitric oxide synthase gene transcription was upregulated within 6 hours of treatment with IFN gamma as well as TNF/LPS treatment. Increase in the level for nitrite was observed using Griess reagent. and might be selectively exposed to iNOS‐mediated oxidative damage as a consequence of elevated TNFα levels. Our results suggest that oxidative stress induces cell death involving iNOS and caspase induction.

Triptolide, an active component of the Chinese herbal remedy Tripterygium wilfordii Hook F, inhibits production of nitric oxide by decreasing inducible nitric oxide synthase gene transcription.

The ethyl acetate (EA) extract of Tripterygium wilfordii Hook F (TWHF) and its major active component, triptolide, have been reported to be effective in the treatment of rheumatoid arthritis and other autoimmune inflammatory diseases. Nitric oxide (NO) has been recognized as an important mediator of inflammation. This study was therefore undertaken to examine the effects of the EA extract and triptolide on the production of NO and inducible NO synthase (iNOS) gene expression and transcription in vivo and in vitro. Peritoneal macrophages from C57BL/6J mice treated orally with the EA extract of TWHF were assayed for NO production and iNOS messenger RNA (mRNA) expression by reverse transcriptase-polymerase chain reaction. The murine fibroblast cell line NIH3T3 was also assessed for NO production and iNOS mRNA expression, as well as for iNOS promoter activation, Oct-1 nuclear binding capacity, and Oct-1 protein content by transient transfection, electrophoretic mobility shift assay, and immunoblotting, respectively. NO production and iNOS mRNA expression by macrophages from C57BL/6J mice immunized with trinitrophenyl-bovine serum albumin in Freund's complete adjuvant were significantly inhibited by oral administration of the EA extract (52.3% and 59.8% of control, respectively, at one-eighth of the dose that is lethal for 50% of the animals [LD(50)] and 21.0% and 38.1% of control, respectively, at one-fourth the LD(50)). Moreover, the EA extract and triptolide significantly inhibited NO production in vitro in activated peritoneal macrophages, which reflected a decreased level of iNOS mRNA. Finally, triptolide inhibited promoter activity of the iNOS gene and induction of the activity of the regulator of iNOS transcription, Oct-1. The EA extract of TWHF and triptolide inhibit transcription of the iNOS gene. This may contribute to the antiinflammatory effects of this traditional herbal remedy.

Evidence of nuclear localization of neuronal nitric oxide synthase in cultured astrocytes of rats

With immunocytochemistry, we have observed the nuclear localization of neuronal nitric oxide synthase (nNOS) in cultured cerebral cortical astrocytes of rats. During the early six days in the subcultures of these cells, nNOS-immunoreactivity was mainly distributed in the cytoplasm. However, nNOS-immunoreactivity was mainly distributed in the nucleus at day 7, and this nuclear localization lasted about ten hours. Meanwhile, inducible nitric oxide synthase expression was significantly inhibited in these cells. Thereafter, nNOS-immunoreactivity was mainly distributed in the cytoplasm again. By confocal microscopy and western blot analysis, the phenomenon of nNOS nuclear localization was further confirmed; and the activity of nNOS in nuclear protein extracts from astrocytes of day 7-subculture could be detected using electron spin resonance (ESR) technique. These results may represent a new pathway of nitric oxide/nNOS participating in inducible nitric oxide synthase gene transcription regulation.

Effect of Uwhangchungsimwon on expression of nitric oxide synthase and vascular cell adhesion molecule-1 in human endothelial cells.

For molecular biological characterization of the effects of Uwhangchungsimwon (UC) on the expression of nitric oxide synthase (NOS) gene and cell adhesion-regulating gene, vascular cell adhesion molecule-1 (VCAM-1), the human endothelial cell line (ECV304) was treated with the extract of UC and transcription of genes was examined using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. UC showed a transcription-activating effect on the NOS gene and a suppressing effect on the VCAM-1 gene in human endothelial cells, and these effects were found in a dose- and time-dependent manner. Down-regulation of VCAM-1 expression by UC was directly mediated by increased nitric oxide (NO) production, which was associated with increased NOS gene transcription. This study strongly suggests that the clinical effects of UC on stroke might be derived at least in part from its ability to induce NOS expression, which was followed by significant reduction of VCAM-1 expression.

CFLIP protein prevents tumor necrosis factor-alpha-mediated induction of caspase-8-dependent apoptosis in insulin-secreting betaTc-Tet cells.

Type 1 diabetes is characterized by the infiltration of activated leukocytes within the pancreatic islets, leading to beta-cell dysfunction and destruction. The exact role played by interferon-gamma, tumor necrosis factor (TNF)-alpha, and interleukin-1beta in this pathogenic process is still only partially understood. To study cytokine action at the cellular level, we are working with the highly differentiated insulin-secreting cell line, betaTc-Tet. We previously reported that it was susceptible to apoptosis induced by TNF-alpha, in combination with interleukin-1beta and interferon-gamma. Here, we report that cytokine-induced apoptosis was correlated with the activation of caspase-8. We show that in betaTc-Tet cells, overexpression of cFLIP, the cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein, completely abolished cytokine-dependent activation of caspase-8 and protected the cells against apoptosis. Furthermore, cFLIP overexpression increased the basal and interleukin-1beta-mediated transcriptional activity of nuclear factor (NF)-kappaB, whereas it did not change cytokine-induced inducible nitric oxide synthase gene transcription and nitric oxide secretion. The presence of cFLIP prevented the weak TNF-alpha-induced reduction in cellular insulin content and secretion; however, it did not prevent the decrease in glucose-stimulated insulin secretion induced by the combined cytokines, in agreement with our previous data demonstrating that interferon-gamma alone could induce these beta-cell dysfunctions. Together, our data demonstrate that overexpression of cFLIP protects mouse beta-cells against TNF-alpha-induced caspase-8 activation and apoptosis and is correlated with enhanced NF-kappaB transcriptional activity, suggesting that cFLIP may have an impact on the outcome of death receptor-triggered responses by directing the intracellular signals from beta-cell death to beta-cell survival.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B.

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between -5.2 and -6.1 kb in the 5'-flanking hiNOS promoter DNA, which contains multiple nuclear factor kappa beta (NF-kappa B) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-alpha, interleukin-1 beta, and IFN-gamma (TNF-alpha, IL-1 beta, and IFN-gamma) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at -5.8 kb in the hiNOS promoter identifies a bifunctional NF-kappa B/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at -5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-kappa B activates hiNOS promoter-reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-kappa B function in these cells. These results indicate that both Stat 1 and NF-kappa B are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

The 5′2 promoter of the neuronal nitric oxide synthase dual promoter complex mediates inducibility by nerve growth factor

Neuronal nitric oxide synthase (nNOS) is induced by nerve growth factor (NGF) in pheochromocytoma PC12 cells. Previous studies from our laboratory identified two closely linked promoters (designated 5′1 and 5′2) that mediate transcription of the human nNOS gene in the brain [J. Xie, P. Roddy, T.K. Rife, F. Murad, A.P. Young, Two closely linked but separable promoters for human neuronal nitric oxide synthase gene transcription, Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 1242–1246]. In this report, we demonstrate that luciferase fusion genes under transcriptional control by the 5′1 and 5′2 dual promoter complex are inducible by NGF in stably transformed PC12 cells. In sharp contrast, neither epidermal growth factor (EGF) nor fibroblast growth factor 2 (FGF2) are able to significantly enhance the expression of NOS–luciferase fusion genes. Deletion studies indicate that the 5′2 promoter plays a major role in mediating NGF inducibility. The 5′2 promoter contains six potential Ets binding sites as well as four potential AP1 binding sites. Thus, it is possible that activation of Ets and/or AP1 transcription factors by the Ras–Raf–MAP kinase cascade contributes to the NGF-mediated induction of nNOS.

Inhibitors of ADP-Ribosylation Impair Inducible Nitric Oxide Synthase Gene Transcription through Inhibition of NF Kappa B Activation

In murine macrophages, inducible NO synthase II (iNOS or NOS-II) is induced at the transcriptional level by IFN-γ, alone or synergistically with LPS. We investigated the possible role of reactions of ADP-ribosylation in triggering the signaling pathways involved in NOS-II gene expression. Stimulation with IFN-γ and/or LPS of RAW 264.7 macrophages, transiently transfected with the NOS-II promoter, was inhibited by ADP-ribosylation inhibitors, indicating that they interfered with the signal(s) responsible for NOS-II gene transcription. We therefore explored the effect of these inhibitors on the activity of IRF-1 and NFκB transcription factors known to be involved in NOS-II induction by IFN-γ and LPS. No effect was observed on IRF-1 activation. However, NFκB binding to its target sequence diminished and transfection experiments with an NFκB-driven reporter plasmid demonstrating that ADP-ribosylation inhibitors suppressed NFκB-dependent promoter activity. These results provide evidence that a step involving ADP-ribosylation is required to activate NFκB-mediated gene transcription.

Inducible Nitric Oxide Synthase Gene Transcription and Protein Activity in the Rat Heart during Endotoxaemia

Septicaemia leads to an impairment of myocardial contractility in animals and humans. Cytokines released during endotoxaemia are capable of increasing inducible nitric oxide synthase (iNOS) expressionin vitroin myocytes, endothelial cells and macrophages. The aim of this study was to assess whether iNOS gene transcription occurs in the myocytein vivo.Rats were injected with intraperitoneal endotoxin. Myocardial sections obtained 4, 6 and 8 hours after infection were hybridised with oligonucleotides complementary to iNOS cDNA. Myocardial homogenates were used to measure NOS enzyme activity and to detect iNOS mRNA. Uninfected control animals did not demonstrate myocardial iNOS expression. Myocardium from endotoxaemic animals contained iNOS mRNA and high calcium-independent NOS enzyme activity.In situhybridisation did not localise iNOS to myocytes but to cells located between myocytes. Endotoxaemia leads to iNOS gene transcription and calcium-independent NOS enzyme activity in the rat myocardium.In situhybridisation demonstrates that iNOS is not transcribed by the myocytein vivo.

Tetrahydrocannabinol inhibition of macrophage nitric oxide production

Δ 9-Tetrahydrocannabinol (THC) inhibited nitric oxide (NO .) production by mouse peritoneal macrophages activated by bacterial endotoxin lipopolysaccharide (LPS) and interferon-γ (IFN)-γ). Inhibition of NO . production was noted at THC concentrations as low as 0.5 μg/mL, and was nearly total at 7 μg/mL. Inhibition was greatest if THC was added 1–4 hr before induction of nitric oxide synthase (NOS) by LPS and IFN-γ, and declined with time after addition of the inducing agents. This suggested that an early step such as NOS gene transcription or NOS synthesis, rather than NOS activity, was affected by THC. Steady-state levels of mRNA for NOS were not affected by THC. In contrast, protein synthesis was inhibited as indicated by immunoblotting. NOS activity was also decreased in the cytosol of cells pretreated with THC. Addition of excess cofactors did not restore activity. Inhibition of NO . production was greater at low levels of IFN-γ, indicating the ability of the cytokine to overcome inhibition. The effectiveness of various THC analogues, in decreasing order of potency, was Δ 8-THC > Δ 9-THC > cannabidiol ⩾ 11-OH-THC > cannabinol. The presumably inactive stereoisomer, (+)Δ 9-THC, and the endogenous ligand for cannabinoid receptors, anandamide, were weakly inhibitory. Inhibition may be mediated by a process that depends partly on stereoselective receptors and partly on a nonselective process. LPS, IFN-γ, hormone receptor agonists, and forskolin increased macrophage cyclic AMP levels. THC inhibited this increase, indicating functional cannabinoid receptors. Addition of 8-bromocyclic AMP increased NO . 2-fold, and partially restored NO . production that had been inhibited by THC. This occurred only under conditions of limited NOS induction, suggesting that the effect of THC on cyclic AMP was responsible for only a small portion of the inhibition of NO . production.

Cytokine-induced nitric oxide synthase gene transcription is blocked by the heat shock response in human liver cells

Previously we demonstrated that the heat shock response (HSR) inhibits cytokine-stimulated nitric oxide (NO) synthesis and inducible NO synthase (NOS2) expression in hepatocytes. In this study we sought to determine the molecular basis of this inhibition using a human liver cell line. After induction of the HSR by sodium arsenite or hyperthermia, the AKN-1 human liver cell line was treated with cytokines to stimulate NOS2 expression and NO production. Western blot analysis for hsp70 was performed, and NOS2 mRNA and 24-hour NO synthesis were quantitated. Cytokine-induced NOS2 promoter activity of AKN-1 cells transfected with a 7.0 kilobase NOS2 promoter luciferase construct and NO production of AKN-1 cells transduced with the NOS2 gene were measured. Sodium arsenite or hyperthermia induced the synthesis of hsp70 protein in AKN-1 cells, indicating activation of the HSR. Cytokines stimulated high levels of NOS2 mRNA and NO production. However, prior induction of the HSR significantly inhibited NOS2 expression and NO synthesis. Cytokine-stimulated NOS2 promoter activity of transfected AKN-1 cells was decreased by 77%, but the HSR did not affect NOS2 enzyme activity in transduced AKN-1 cells. These findings indicate that the HSR inhibits cytokine-induced NOS2 expression and NO synthesis in AKN-1 cells by preventing NOS2 promoter activation. Effects on NOS2 protein translation or stability were not observed. These data suggest that the HSR, which is expressed in the liver after trauma, shock, or ischemia-reperfusion, blocks NOS2 gene expression at the transcriptional level.

Cytokines activate inducible nitric oxide synthase gene transcription in inner medullary collecting duct cells

The effects of lipopolysaccharide (LPS) and/or inflammatory cytokines on the expression of inducible nitric oxide synthase (iNOS) were studied in mIMCD-3 cells, derived from the murine inner medullary collecting duct. Under basal conditions, the production of nitrite, a stable metabolite of NO, was negligible; however, incubation with tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IF-gamma) for 24 h resulted in a 12-fold increase in nitrite synthesis and the appearance of abundant iNOS mRNA and protein. The induction of nitrite production and iNOS mRNA was time dependent, requiring approximately 8 h for expression of significant levels of nitrite or iNOS mRNA. Coincubation with the transcription inhibitor actinomycin D or the translation inhibitor cycloheximide prevented the cytokine induction of iNOS mRNA and NO production, indicating that synthesis of intermediary proteins stimulated transcription of the iNOS gene. Nuclear run-on transcription demonstrated that the iNOS gene was transcriptionally inactive under basal conditions, but was markedly induced by TNF-alpha and IF-gamma. These results indicate that inflammatory cytokines stimulate NO production in mIMCD-3 cells by activating iNOS gene transcription in a process that requires new protein synthesis.

Two closely linked but separable promoters for human neuronal nitric oxide synthase gene transcription.

In this report we demonstrate that the human cerebellum contains neuronal nitric oxide synthase (nNOS) mRNAs with two distinct 5'-untranslated regions that are encoded through use of closely linked but separate promoters. nNOS cDNA clones were shown to contain different 5' terminal exons spliced to a common exon 2. Genomic cloning and sequence analysis demonstrate that the unique exons are positioned within 300 bp of each other but separated from exon 2 by an intron that is at least 20 kb in length. A CpG island engulfs the downstream 5'-terminal exon. In contrast, most of the upstream exon resides outside of this CpG island. Interestingly, the upstream exon includes a GT dinucleotide repeat. A fusion gene with a 414-bp nNOS genomic fragment that includes a portion of the upstream 5'-terminal exon and its immediate 5'-flanking DNA is expressed in transfected HeLa cells. Also expressed is a fusion gene that contains the luciferase reporter under transcriptional control by a 308-bp genomic fragment that includes the region separating both 5'-terminal exons. These results indicate that expression of these exons is subject to transcriptional control by separate promoters. However, the proximity of these promoters raise the possibility that complex interactions may be involved in regulating nNOS gene expression at these sites.