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Neuronatin: a new inflammation gene expressed on the aortic endothelium of diabetic mice.

Mzhavia N, Yu S, Ikeda S, Chu TT, Goldberg I, Dansky HM - Diabetes (2008)

Bottom Line: Nnat expression stimulated p38, Jun NH(2)-terminal kinase, extracellular signal-related kinase, and AKT kinase phosphorylation.Phosphatidylinositol 3-kinase and p38 inhibitors prevented Nnat-mediated activation of NF-kappaB-induced gene expression.The effects of Nnat on inflammatory pathways in vitro and in vivo suggest a pathophysiological role of this new gene in diabetic vascular diseases.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Columbia University, New York, New York, USA. nm2170@columbia.edu

ABSTRACT

Objective: Identification of arterial genes and pathways altered in obesity and diabetes.

Research design and methods: Aortic gene expression profiles of obese and diabetic db/db, high-fat diet-fed C57BL/6J, and control mice were obtained using mouse Affymetrix arrays. Neuronatin (Nnat) was selected for further analysis. To determine the function of Nnat, a recombinant adenovirus (Ad-Nnat) was used to overexpress the Nnat gene in primary endothelial cells and in the mouse aorta in vivo.

Results: Nnat, a gene of unknown vascular function, was upregulated in the aortas of db/db and high-fat diet-fed mice. Nnat gene expression was increased in db/db mouse aorta endothelial cells. Nnat protein was localized to aortic endothelium and was selectively increased in the endothelium of db/db mice. Infection of primary human aortic endothelial cells (HAECs) with Ad-Nnat increased expression of a panel of nuclear factor-kappaB (NF-kappaB)-regulated genes, including inflammatory cytokines, chemokines, and cell adhesion molecules. Infection of mouse carotid arteries in vivo with the Ad-Nnat increased expression of vascular cell adhesion molecule 1 protein. Nnat activation of NF-kappaB and inflammatory gene expression in HAECs was mediated through pathways distinct from tumor necrosis factor-alpha. Nnat expression stimulated p38, Jun NH(2)-terminal kinase, extracellular signal-related kinase, and AKT kinase phosphorylation. Phosphatidylinositol 3-kinase and p38 inhibitors prevented Nnat-mediated activation of NF-kappaB-induced gene expression.

Conclusions: Nnat expression is increased in endothelial cells of obese and diabetic mouse blood vessels. The effects of Nnat on inflammatory pathways in vitro and in vivo suggest a pathophysiological role of this new gene in diabetic vascular diseases.

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TNF-α augments Nnat-induced activation of NF-κB. A: Western blot for IκB-α protein in HAECs infected with Ad-Nnat in the presence or absence of exogenous TNF-α (top). Quantitative densitometry of Western blot (bottom) for IκB-α protein expression was normalized to β-actin (n = 3). B: IκB-α and endothelial cell adhesion molecules VCAM-1 and ICAM-1 protein. C: ICAM-1 mRNA levels in endothelial cells infected with Ad-GFP or Ad-Nnat in the presence or absence of TNF-α neutralizing antibody. D: Effect of TNF-α blocking antibody on Nnat induced increase in cytokine IL-6 mRNA expression. Endothelial cells were infected with Ad-GFP or Ad-Nnat and treated with TNF-α or TNF-α neutralizing antibody. IL-6 and ICAM-1 expression levels were normalized to GAPDH. E: Western blot analysis of p38, SAPK/JNK1/2, and ERK1/2 MAPK phosphorylation in HAECs infected with Ad-Nnat or Ad-Empty in the presence or absence of exogenous TNF-α. F: Western blot analysis of Akt phosphorylation: P-AKT (S473), AKT, and β-actin. ***P < 0.001; **P < 0.01; *P < 0.05. Data are a representative of three to four experiments.
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f3: TNF-α augments Nnat-induced activation of NF-κB. A: Western blot for IκB-α protein in HAECs infected with Ad-Nnat in the presence or absence of exogenous TNF-α (top). Quantitative densitometry of Western blot (bottom) for IκB-α protein expression was normalized to β-actin (n = 3). B: IκB-α and endothelial cell adhesion molecules VCAM-1 and ICAM-1 protein. C: ICAM-1 mRNA levels in endothelial cells infected with Ad-GFP or Ad-Nnat in the presence or absence of TNF-α neutralizing antibody. D: Effect of TNF-α blocking antibody on Nnat induced increase in cytokine IL-6 mRNA expression. Endothelial cells were infected with Ad-GFP or Ad-Nnat and treated with TNF-α or TNF-α neutralizing antibody. IL-6 and ICAM-1 expression levels were normalized to GAPDH. E: Western blot analysis of p38, SAPK/JNK1/2, and ERK1/2 MAPK phosphorylation in HAECs infected with Ad-Nnat or Ad-Empty in the presence or absence of exogenous TNF-α. F: Western blot analysis of Akt phosphorylation: P-AKT (S473), AKT, and β-actin. ***P < 0.001; **P < 0.01; *P < 0.05. Data are a representative of three to four experiments.

Mentions: Additional experiments were performed to identify the signal transduction pathways by which Nnat activated NF-κB in endothelial cells. Because Nnat induced TNF-α mRNA expression (Table 3), we entertained the possibility that Nnat might induce inflammatory gene expression through stimulation of TNF-α release by the cell, leading to ligation of TNF-α cell surface receptors. Cells were first infected with Ad-Nnat to determine whether Nnat expression would result in activation of NF-κB in the presence of exogenous TNF-α. Treatment of Ad-Empty–infected HAECs with TNF-α decreased IκB-α protein level (Fig. 3A). Treatment of Nnat-expressing HAECs with TNF-α further decreased IκB-α protein.


Neuronatin: a new inflammation gene expressed on the aortic endothelium of diabetic mice.

Mzhavia N, Yu S, Ikeda S, Chu TT, Goldberg I, Dansky HM - Diabetes (2008)

TNF-α augments Nnat-induced activation of NF-κB. A: Western blot for IκB-α protein in HAECs infected with Ad-Nnat in the presence or absence of exogenous TNF-α (top). Quantitative densitometry of Western blot (bottom) for IκB-α protein expression was normalized to β-actin (n = 3). B: IκB-α and endothelial cell adhesion molecules VCAM-1 and ICAM-1 protein. C: ICAM-1 mRNA levels in endothelial cells infected with Ad-GFP or Ad-Nnat in the presence or absence of TNF-α neutralizing antibody. D: Effect of TNF-α blocking antibody on Nnat induced increase in cytokine IL-6 mRNA expression. Endothelial cells were infected with Ad-GFP or Ad-Nnat and treated with TNF-α or TNF-α neutralizing antibody. IL-6 and ICAM-1 expression levels were normalized to GAPDH. E: Western blot analysis of p38, SAPK/JNK1/2, and ERK1/2 MAPK phosphorylation in HAECs infected with Ad-Nnat or Ad-Empty in the presence or absence of exogenous TNF-α. F: Western blot analysis of Akt phosphorylation: P-AKT (S473), AKT, and β-actin. ***P < 0.001; **P < 0.01; *P < 0.05. Data are a representative of three to four experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2551689&req=5

f3: TNF-α augments Nnat-induced activation of NF-κB. A: Western blot for IκB-α protein in HAECs infected with Ad-Nnat in the presence or absence of exogenous TNF-α (top). Quantitative densitometry of Western blot (bottom) for IκB-α protein expression was normalized to β-actin (n = 3). B: IκB-α and endothelial cell adhesion molecules VCAM-1 and ICAM-1 protein. C: ICAM-1 mRNA levels in endothelial cells infected with Ad-GFP or Ad-Nnat in the presence or absence of TNF-α neutralizing antibody. D: Effect of TNF-α blocking antibody on Nnat induced increase in cytokine IL-6 mRNA expression. Endothelial cells were infected with Ad-GFP or Ad-Nnat and treated with TNF-α or TNF-α neutralizing antibody. IL-6 and ICAM-1 expression levels were normalized to GAPDH. E: Western blot analysis of p38, SAPK/JNK1/2, and ERK1/2 MAPK phosphorylation in HAECs infected with Ad-Nnat or Ad-Empty in the presence or absence of exogenous TNF-α. F: Western blot analysis of Akt phosphorylation: P-AKT (S473), AKT, and β-actin. ***P < 0.001; **P < 0.01; *P < 0.05. Data are a representative of three to four experiments.
Mentions: Additional experiments were performed to identify the signal transduction pathways by which Nnat activated NF-κB in endothelial cells. Because Nnat induced TNF-α mRNA expression (Table 3), we entertained the possibility that Nnat might induce inflammatory gene expression through stimulation of TNF-α release by the cell, leading to ligation of TNF-α cell surface receptors. Cells were first infected with Ad-Nnat to determine whether Nnat expression would result in activation of NF-κB in the presence of exogenous TNF-α. Treatment of Ad-Empty–infected HAECs with TNF-α decreased IκB-α protein level (Fig. 3A). Treatment of Nnat-expressing HAECs with TNF-α further decreased IκB-α protein.

Bottom Line: Nnat expression stimulated p38, Jun NH(2)-terminal kinase, extracellular signal-related kinase, and AKT kinase phosphorylation.Phosphatidylinositol 3-kinase and p38 inhibitors prevented Nnat-mediated activation of NF-kappaB-induced gene expression.The effects of Nnat on inflammatory pathways in vitro and in vivo suggest a pathophysiological role of this new gene in diabetic vascular diseases.

View Article: PubMed Central - PubMed

Affiliation: Division of Cardiology, Columbia University, New York, New York, USA. nm2170@columbia.edu

ABSTRACT

Objective: Identification of arterial genes and pathways altered in obesity and diabetes.

Research design and methods: Aortic gene expression profiles of obese and diabetic db/db, high-fat diet-fed C57BL/6J, and control mice were obtained using mouse Affymetrix arrays. Neuronatin (Nnat) was selected for further analysis. To determine the function of Nnat, a recombinant adenovirus (Ad-Nnat) was used to overexpress the Nnat gene in primary endothelial cells and in the mouse aorta in vivo.

Results: Nnat, a gene of unknown vascular function, was upregulated in the aortas of db/db and high-fat diet-fed mice. Nnat gene expression was increased in db/db mouse aorta endothelial cells. Nnat protein was localized to aortic endothelium and was selectively increased in the endothelium of db/db mice. Infection of primary human aortic endothelial cells (HAECs) with Ad-Nnat increased expression of a panel of nuclear factor-kappaB (NF-kappaB)-regulated genes, including inflammatory cytokines, chemokines, and cell adhesion molecules. Infection of mouse carotid arteries in vivo with the Ad-Nnat increased expression of vascular cell adhesion molecule 1 protein. Nnat activation of NF-kappaB and inflammatory gene expression in HAECs was mediated through pathways distinct from tumor necrosis factor-alpha. Nnat expression stimulated p38, Jun NH(2)-terminal kinase, extracellular signal-related kinase, and AKT kinase phosphorylation. Phosphatidylinositol 3-kinase and p38 inhibitors prevented Nnat-mediated activation of NF-kappaB-induced gene expression.

Conclusions: Nnat expression is increased in endothelial cells of obese and diabetic mouse blood vessels. The effects of Nnat on inflammatory pathways in vitro and in vivo suggest a pathophysiological role of this new gene in diabetic vascular diseases.

Show MeSH
Related in: MedlinePlus