scholarly journals A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter.

1995 ◽  
Vol 182 (6) ◽  
pp. 1683-1693 ◽  
Author(s):  
G Melillo ◽  
T Musso ◽  
A Sica ◽  
L S Taylor ◽  
G W Cox ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Picolinic Acid ◽  
Transient Transfection ◽  
Ifn Gamma ◽  
Functional Studies ◽  
Inos Promoter ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Picolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-gamma-treated murine macrophages. We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation of iNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN-gamma and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid- but not LPS-induced CAT activity by more than 70%. Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-gamma or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRE. Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-gamma activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression. These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

scholarly journals Regulation of Inducible Nitric Oxide Synthase Expression by Viral A238L-Mediated Inhibition of p65/RelA Acetylation and p300 Transactivation

2006 ◽  
Vol 80 (21) ◽  
pp. 10487-10496 ◽  
Author(s):  
Aitor G. Granja ◽  
Prado Sabina ◽  
María L. Salas ◽  
Manuel Fresno ◽  
Yolanda Revilla
Keyword(s):  
Gene Expression ◽  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Promoter Activity ◽  
Host Cells ◽  
Inos Promoter ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Uncontrolled generation of nitric oxide (NO) by inducible nitric-oxide synthase (iNOS) can cause damage to host cells and inflammation, two undesirable events for virus spreading. African swine fever virus (ASFV) infection regulates iNOS-induced gene expression through the synthesis of the A238L virus protein. We here explored the role of A238L, an NF-κB and NFAT inhibitor, in the regulation of iNOS transcription in macrophages. NO production and iNOS mRNA and protein levels as well as iNOS promoter activity after lipopolysaccharide (LPS)-gamma interferon (IFN-γ) treatment were down-regulated both during ASFV infection and in Raw 264.7 cells stably expressing the viral protein. Overexpression of p300, but not of a histone acetyltransferase (HAT) defective mutant, reverted the A238L-mediated inhibition of both basal and LPS-IFN-γ-induced iNOS promoter activity. Following stimulation with LPS-IFN-γ, p65 and p300 interaction was abolished in Raw-A238L cells. Expression of A238L also inhibited p65/relA and p300 binding to the distal NF-κB sequence of the iNOS promoter together with p65 acetylation. Finally, A238L abrogated p300 transactivation mediated by a GAL4-p300 construction. These results provide evidence for an unique viral mechanism involved in transcriptional regulation of iNOS gene expression.

scholarly journals Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major.

1994 ◽  
Vol 180 (3) ◽  
pp. 783-793 ◽  
Author(s):  
S Stenger ◽  
H Thüring ◽  
M Röllinghoff ◽  
C Bogdan
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Leishmania Major ◽  
Tissue Expression ◽  
Ifn Gamma ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Previous studies with inhibitors of inducible nitric oxide synthase (iNOS) suggested that high-output production of nitric oxide (NO) is an important antimicrobial effector pathway in vitro and in vivo. Here, we investigated the tissue expression of iNOS in mice after infection with Leishmania major. Immunohistochemical staining with an iNOS-specific antiserum revealed that in the cutaneous lesion and draining lymph nodes (LN) of clinically resistant mice (C57BL/6), iNOS protein is found earlier during infection and in significantly higher amounts than in the nonhealing BALB/c strain. Similar differences were seen on the mRNA level as quantitated by competitive polymerase chain reaction. Anti-CD4 treatment of BALB/c mice not only induced resistance to disease, but also restored the expression of iNOS in the tissue. In situ, few or no parasites were found in those regions of the skin lesion and the draining LN which were highly positive for iNOS. By double labeling experiments, macrophages were identified as iNOS expressing cells in vivo. In the lesions of BALB/c mice, cells staining positively for transforming growth factor beta (TGF-beta), a potent inhibitor of iNOS in vitro, were strikingly more prominent than in C57BL/6, whereas no such difference was found for interleukin 4 or interferon gamma (IFN-gamma). In vitro, production of NO was approximately threefold higher in C57BL/6 than in BALB/c macrophages after stimulation with IFN-gamma. We conclude that the pronounced expression of iNOS in resistant mice is an important mechanism for the elimination of Leishmania in vivo. The relative lack of iNOS in susceptible mice might be a consequence of macrophage deactivation by TGF-beta and reduced responsiveness to IFN-gamma.

Expression profile of a human inducible nitric oxide synthase promoter reporter in transgenic mice during endotoxemia

2005 ◽  
Vol 288 (1) ◽  
pp. F214-F220 ◽  
Author(s):  
Zhiyuan Yu ◽  
Xuefeng Xia ◽  
Bruce C. Kone
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Transgenic Mice ◽  
Inducible Nitric Oxide Synthase ◽  
Transgene Expression ◽  
Inos Gene ◽  
Inos Promoter ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Inducible nitric oxide synthase (iNOS) is involved in many physiological and pathophysiological processes, including septic shock and acute kidney failure. Little is known about transcriptional regulation of the human iNOS gene in vivo under basal conditions or in sepsis. Accordingly, we developed transgenic mice carrying an insertional human iNOS promoter-reporter gene construct. In these mice, the proximal 8.3 kb of the human iNOS 5′-flanking region controls expression of the reporter gene of enhanced green fluorescent protein (EGFP). Patterns of human iNOS promoter/EGFP transgene expression in tissues were examined by fluorescence microscopy and immunoblotting. Endogenous murine iNOS was basally undetectable in kidney, intestine, spleen, heart, lung, liver, stomach, or brain. In contrast, EGFP from the transgene was basally expressed in kidney, brain, and spleen, but not the other tissues of the transgenic mice. Bacterial lipopolysaccharide induced endogenous iNOS expression in kidney, intestine, spleen, lung, liver, stomach, and heart, but not brain. In contrast, human iNOS promoter/EGFP transgene expression was induced above basal levels only in intestine, spleen, brain, stomach, and lung. Within kidney, human iNOS promoter/EGFP fluorescence was detected most prominently in proximal tubules of the outer cortex and collecting ducts and colocalized with endogenous mouse iNOS. Within the collecting duct, both endogenous iNOS and the human iNOS promoter/EGFP transgene were expressed in cells lacking aquaporin-2 immunoreactivity, consistent with expression in intercalated cells. Although it remains possible that essential regulatory elements reside in remote locations of the gene, our data concerning this 8.3-kb region provide the first in vivo evidence suggesting differential transcriptional control of the human iNOS gene in these organs and marked differences in transcriptional regulatory regions between the murine and human genes.

scholarly journals Recipient humoral immunity against leukoreduced allogeneic platelets is suppressed by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2959-2966 ◽  
Author(s):  
A Bang ◽  
ER Speck ◽  
VS Blanchette ◽  
J Freedman ◽  
JW Semple
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Selective Inhibitor ◽  
Interleukin 4 ◽  
Foreign Protein ◽  
Ifn Gamma ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Leukoreduced allogeneic platelet transfusions have been previously shown to initially stimulate an in vitro cellular cytotoxicity and subsequently Induce the formation of immunoglobulin G (IgG) antidonor alloantibodies. To further characterize these responses and determine if they are related, recipient BALB/c H-2d mice were treated with aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and transfused weekly with 2 x 10(8) C57BL/6 H2b platelets. In control, non-AMG-treated mice, transfusion significantly (P < .01) increased serum levels of interferon-gamma (IFN-gamma) by day 1 posttransfusion (PT). IFN-gamma returned to pretransfusion levels by day 3 PT, and its production was not affected by AMG treatment. Serum interleukin-4 (IL-4), on the other hand, was undetectable before and during the transfusion protocol. By day 3 PT, recipient spleen cells could mediate in vitro anti-P815 (auto), anti-EL4 (allo), and anti-R1.1 (third-party MHC) cytotoxicity, and these responses were maximal by day 7 PT. Concurrently, a significant reduction in the vitro ability of recipient splenocytes to respond to Concanavalin A (ConA) was observed; this was not seen with lipopolysaccharide (LPS) stimulation. Elevated levels of NO2- were found in the ConA culture supernatants from transfused mice at day 3 PT. Serum antidonor alloantibodies were detected by the fifth platelet transfusion. AMG treatment of recipient mice significantly inhibited the transfusion. Induced cytotoxicity and ConA-stimulated NO2- production, and restored ConA-induced proliferation to normal levels. AMG appeared to selectively inhibit platelet-induced alloantibody production in that it did not affect antibody production induced by transfusions with 10(5) allogeneic leukocytes or by immunization with a foreign protein antigen, human gamma globulin, in adjuvant therapy. These results indicate that an in vivo AMG-sensitive mechanism is essential for recipients to initiate a humoral IgG immune response against allogeneic platelets.

scholarly journals Induction of Inducible Nitric Oxide Synthase-NO· by Lipoarabinomannan ofMycobacterium tuberculosis Is Mediated by MEK1-ERK, MKK7-JNK, and NF-κB Signaling Pathways

2001 ◽  
Vol 69 (4) ◽  
pp. 2001-2010 ◽  
Author(s):  
Edward D. Chan ◽  
Kristin R. Morris ◽  
John T. Belisle ◽  
Preston Hill ◽  
Linda K. Remigio ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Map Kinase ◽  
Signaling Pathways ◽  
Inducible Nitric Oxide Synthase ◽  
Promoter Activity ◽  
Inos Promoter ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Nitric oxide (NO· ) expression by inducible nitric oxide synthase (iNOS) is an important host defense mechanism againstMycobacterium tuberculosis in mononuclear phagocytes. The objective of this investigation was to examine the role of mitogen-activated protein (MAP) kinase (MAPK) and nuclear factor κB (NF-κB) signaling pathways in the regulation of iNOS and NO· by a mycobacterial cell wall lipoglycan known as mannose-capped lipoarabinomannan (ManLAM). Specific pharmacologic inhibition of the extracellular-signal-regulated kinase (ERK) or NF-κB pathway revealed that both these signaling cascades were required in gamma interferon (IFN-γ)-ManLAM-induced iNOS protein and NO2 − expression in mouse macrophages. Transient cotransfection of dominant-negative protein mutants of the c-Jun NH2-terminal kinase (JNK) pathway revealed that the MAP kinase kinase 7 (MKK7)-JNK cascade also mediated IFN-γ–ManLAM induction of iNOS promoter activity whereas MKK4 did not. Overexpression of null mutant IκBα, a potent inhibitor of NF-κB activation, confirmed that the IκBα kinase (IKK)–NF-κB signaling pathway enhanced IFN-γ–ManLAM-induced iNOS promoter activity. By contrast, activated p38 mapk inhibited iNOS induction. These results indicate that combined IFN-γ and ManLAM stimulation induced iNOS and NO· expression and that MEK1-ERK, MKK7-JNK, IKK–NF-κB, and p38 mapk signaling pathways play important regulatory roles.

scholarly journals The altered tumoricidal capacity of macrophages isolated from tumor-bearing mice is related to reduce expression of the inducible nitric oxide synthase gene.

1996 ◽  
Vol 183 (4) ◽  
pp. 1323-1329 ◽  
Author(s):  
M R Dinapoli ◽  
C L Calderon ◽  
D M Lopez
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Tumor Cells ◽  
Inducible Nitric Oxide Synthase ◽  
No Production ◽  
Western Blots ◽  
Ifn Gamma ◽  
Tumor Bearing ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Nitric oxide (NO) is a major effector molecule in the destruction of tumor cells by activated macrophages. However, in many cases, developing neoplasms appear to be capable of impairing steps in the complex process leading to NO production as a means of avoiding immune destruction. After activation with lipopolysaccharide (LPS), peritoneal-elicited macrophages (PEM) from mice bearing mammary tumors display alterations in their ability to lyse tumor cells due to reduced production of NO. In contrast, when these same cells are stimulated with LPS in combination with interferon gamma (IFN-gamma), they are able to produce NO and lyse targets at normal levels. Since tumor-associated macrophages are intimately associated with the cells of the developing tumor, their ability to produce NO and lyse tumor targets is likely to be more relevant to controlling tumor growth. This population of macrophages exhibited a more profound inability to produce NO and lyse targets and, unlike the PEM, was not able to upregulate these functions even when treated with combinations of LPS and IFN-gamma. Northern and Western blots revealed that inducible nitric oxide synthase (iNOS) mRNA and protein levels correlated directly with the ability of each macrophage population to produce NO, and the levels of these macromolecules were altered sufficiently in tumor bearers' macrophages to account for the diminished NO production described. These results indicate that a spatial gradient of suppression of macrophage cytolytic activity and iNOS expression exists in mammary tumor-bearing mice, whereby macrophages from within the tumor exhibit a more pronounced suppression than the more distally located PEM. This suppression may be due to proximity of the macrophages to the developing tumor, macrophage maturational state, or both.

289: Roles of Inducible Nitric Oxide Synthase in Voiding Function and Bladder Pain During Experimental Cystitis

2006 ◽  
Vol 175 (4S) ◽  
pp. 96-96
Author(s):  
Masayoshi Nomura ◽  
Hisae Nishii ◽  
Masato Tsutsui ◽  
Naohiro Fujimoto ◽  
Tetsuro Matsumoto
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Voiding Function ◽  
Bladder Pain ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide
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