Suppression of Interleukin-1β-induced Nitric-oxide Synthase Promoter/Enhancer Activity by Transforming Growth Factor-β1 in Vascular Smooth Muscle Cells

Perrella, Mark A.; Patterson, Cam; Tan, Larissa; Yet, Shaw Fang; Hsieh, Chung Ming; Yoshizumi, Masao; Lee, Mu En

doi:10.1074/jbc.271.23.13776

Cited by 77 publications

(86 citation statements)

References 31 publications

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“…27 Similarly, when the iNOS promoter was transfected into cultured RASMC, a key region was located at Ϫ234 bp from the 5Ј-region. 28 Data have also demonstrated that LPS activation of the human iNOS promoter exhibits cell-type specificity. When the 1.1-kb human iNOS promoter was transfected into macrophages and SMC, the induction in response to LPS was observed only in macrophages, not in vascular SMC, including the A7r5 cell line, freshly cultured RASMC, and human saphenous-vein SMC.…”

Section: Discussionmentioning

confidence: 97%

Nitric Oxide Differentially Regulates Induction of Type II Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells Versus Macrophages

Zhang

Snead

Catravas

2001

ATVB

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Abstract-We studied effects of nitric oxide (NO) released by different NO donors on induction of inducible NO synthase (iNOS) in rat aortic smooth muscle cells (RASMC) and rat macrophage cell line NR8383. iNOS protein expression induced by a CM (interleukin-1␤ 250 U/mL, interferon-␥ 150 U/mL, and tumor necrosis factor-␣ 150 U/mL) was not affected by the NO donor SNAP (0.2 to 1 mmol/L) in RASMC at 24 hours of incubation but was dose-dependently decreased by SNAP in macrophages (maximal 60% inhibition). A fully functional Ϫ3.2-kb rat iNOS promoter was transfected into RASMC and macrophages. The CM-induced promoter activity in transfected macrophages was inhibited by SNAP (maximal 67% inhibition), but this inhibitory effect by SNAP was not observed in transfected RASMC. Electrophoretic mobility-shift assays demonstrated that nuclear factor-B (NF-B) binding patterns were different in 2 cell types and that the ratio of p50:p65 subunits was significantly lower in macrophages than in RASMC. Furthermore, NF-B activity was not affected by SNAP in RASMC but was reduced by SNAP in macrophages. Another putative NO donor, NOR3 (1 mmol/L), completely inhibited iNOS induction by CM in RASMC, but this was accompanied by severe cytotoxicity, which resulted in cell death. Similar concentrations of SNAP did not exhibit cytotoxicity in RASMC, whereas macrophages demonstrated 88% viability compared with cells without SNAP. NO synthase inhibitor N g -monomethyl-L-arginine significantly inhibited CM-induced nitrite production in both cell types and stimulated iNOS protein expression in macrophages but did not affect iNOS expression in RASMC. These data strongly suggest that NO may affect transcriptional regulation of iNOS differently in RASMC

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Section: Discussionmentioning

confidence: 97%

Nitric Oxide Differentially Regulates Induction of Type II Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells Versus Macrophages

Zhang

Snead

Catravas

2001

ATVB

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“…After overnight incubation, LPS (2 g/ml), IL-1␤ (10 ng/ml), and IFN-␥ (100 units/ml) were added to NIH/3T3 cells. Fibroblasts, similar to smooth muscle cells (15), are not as sensitive to LPS stimulation alone as are macrophages and therefore require the addition of proinflammatory cytokines for NOS2 induction. After 24 h of treatment, RNA was harvested for Northern blot analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmid Constructs and DNA Probes-The mouse NOS2 luciferase reporter plasmids, iNOS(Ϫ1485/ϩ31), iNOS(Ϫ815/ϩ31), iNOS(Ϫ518/ ϩ31), and iNOS(Ϫ234/ϩ31) in the pGL2-Basic vector (Promega) were described previously (14,15). Ets-1, Ets-2, NERF2, and Elk-3 (Erp)-cloned into pCI expression vector (Promega), and iNOS(Ϫ234/ ϩ31)ETSm with a mutation in the Ets binding site located at bp Ϫ190 to Ϫ180, were generous gifts from Dr. Peter Oettgen, Boston, MA.…”

Section: Methodsmentioning

confidence: 99%

“…cytokines or LPS in vascular smooth muscle cells and macrophages, respectively (14,15,17). Since endogenous Elk-3 expression inversely correlates with NOS2 message, and overexpression of Elk-3 represses the induction of NOS2 by an inflammatory stimulus, we wanted to determine whether TGF-␤1 (10 ng/ml) would increase Elk-3 mRNA that had been down-regulated by LPS (10 ng/ml).…”

Section: Elk-3 Inhibition Of Nos2mentioning

confidence: 99%

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Elk-3 Is a Transcriptional Repressor of Nitric-oxide Synthase 2

Chen

Layne

Chung

et al. 2003

Journal of Biological Chemistry

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The inducible isoform of nitric-oxide synthase (NOS2), a key enzyme catalyzing the dramatic increase in nitric oxide by lipopolysaccharide (LPS), plays an important role in the pathophysiology of endotoxemia and sepsis. Recent evidence suggests that Ets transcription factors may contribute to NOS2 induction by inflammatory stimuli. In this study, we investigated the role of Ets transcription factors in the regulation of NOS2 by LPS and transforming growth factor (TGF)-␤1. Transient transfection assays in macrophages showed that Ets-2 produced an increase in NOS2 promoter activity, whereas the induction by Ets-1 was modest and NERF2 had no effect. Elk-3 (Net/Erp/Sap-2a) markedly repressed NOS2 promoter activity in a dose-dependent fashion, and overexpression of Elk-3 blunted the induction of endogenous NOS2 message. Mutation of the Net inhibitory domain of Elk-3, but not the C-terminal-binding protein interaction domain, partially alleviated this repressive effect. We also found that deletion of the Ets domain of Elk-3 completely abolished its repressive effect on the NOS2 promoter. LPS administration to macrophages led to a dose-dependent decrease in endogenous Elk-3 mRNA levels, and this decrease in Elk-3 preceded the induction of NOS2 mRNA. In a mouse model of endotoxemia, the expression of Elk-3 in kidney, lung, and heart was significantly down-regulated after systemic administration of LPS, and this down-regulation also preceded NOS2 induction. Moreover, TGF-␤1 significantly increased endogenous Elk-3 mRNA levels that had been down-regulated by LPS in macrophages. This increase in Elk-3 correlated with a TGF-␤1-induced down-regulation of NOS2. Taken together, our data suggest that Elk-3 is a strong repressor of NOS2 promoter activity and mRNA levels and that endogenous expression of Elk-3 inversely correlates with NOS2. Thus, Elk-3 may serve as an important mediator of NOS2 gene expression.Sepsis is a systemic inflammatory response to severe infection that frequently progresses to refractory hypotension, multiple organ system failure, and death (1). In a subset of patients, the release of bacterial cell wall-derived lipopolysaccharide (LPS 1 or endotoxin) initiates a cascade of inflammatory events (2). This response to infection is mediated, in part, by macrophage activation and the release of proinflammatory cytokines (3). Among the mediators induced by these signal cascades, nitric oxide (NO) acts as a key regulator of hypotension and end organ damage (4, 5). The production of nitric oxide from L-arginine is catalyzed by NO synthases (NOS) (6). Of the three isoforms of NOS identified in mammalian cells, the expression of NOS2 is inducible by many proinflammatory stimuli, such as interleukin-1␤, tumor necrosis factor-␣, interferon-␥, and bacterial LPS, depending on the target cell. Thus, the study of NOS2 gene regulation is of particular importance in our understanding and management of sepsis and other inflammatory disorders. Ets proteins are a family of transcription factors that share a unique and highly...

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“…The generation of the pGL3 MMP-12 promoter construct (Ϫ1046/ϩ39) and of the pGL2 iNOS reporter construct (Ϫ1485/ϩ31) have been described previously (19,45).…”

Section: Methodsmentioning

confidence: 99%

Transforming Growth Factor-β1 Inhibition of Macrophage Activation Is Mediated via Smad3

Werner¹,

Jain²,

Feinberg³

et al. 2000

Journal of Biological Chemistry

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155

110

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Suppression of Interleukin-1β-induced Nitric-oxide Synthase Promoter/Enhancer Activity by Transforming Growth Factor-β1 in Vascular Smooth Muscle Cells

Cited by 77 publications

References 31 publications

Nitric Oxide Differentially Regulates Induction of Type II Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells Versus Macrophages

Nitric Oxide Differentially Regulates Induction of Type II Nitric Oxide Synthase in Rat Vascular Smooth Muscle Cells Versus Macrophages

Elk-3 Is a Transcriptional Repressor of Nitric-oxide Synthase 2

Transforming Growth Factor-β1 Inhibition of Macrophage Activation Is Mediated via Smad3

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