Limits...
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.

Show MeSH

Related in: MedlinePlus

Nnat expression in HAECs activates NF-κB–dependent gene expression in time- and concentration-dependent manner. A: Western blots of Nnat, VCAM-1, ICAM-1, and IκB-α protein from endothelial cells infected with Ad-Empty, Ad-GFP, and increasing number of Ad-Nnat adenovirus per cell (MOI). B: Real-time PCR measurement of IL-6 mRNA expression in endothelial cells infected with Ad-Empty, Ad-GFP, or Ad-Nnat at different MOI. (n = 3). C: Endothelial cells were collected at various times after infection with Ad-Nnat and Western blots were probed with antibodies to Nnat, NAK, IκB-α, phospho-p65, p65, and β-actin. D: Time course of IL-6 mRNA expression after Ad-Nnat infection of endothelial cells (n = 3). IL-6 mRNA expression levels were normalized to GAPDH. ***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
getmorefigures.php?uid=PMC2551689&req=5

f2: Nnat expression in HAECs activates NF-κB–dependent gene expression in time- and concentration-dependent manner. A: Western blots of Nnat, VCAM-1, ICAM-1, and IκB-α protein from endothelial cells infected with Ad-Empty, Ad-GFP, and increasing number of Ad-Nnat adenovirus per cell (MOI). B: Real-time PCR measurement of IL-6 mRNA expression in endothelial cells infected with Ad-Empty, Ad-GFP, or Ad-Nnat at different MOI. (n = 3). C: Endothelial cells were collected at various times after infection with Ad-Nnat and Western blots were probed with antibodies to Nnat, NAK, IκB-α, phospho-p65, p65, and β-actin. D: Time course of IL-6 mRNA expression after Ad-Nnat infection of endothelial cells (n = 3). IL-6 mRNA expression levels were normalized to GAPDH. ***P < 0.001, **P < 0.01, *P < 0.05. Data are a representative of three to four experiments.

Mentions: HAECs were infected with an Ad-Nnat to determine the downstream effects of Nnat expression. Western blotting revealed high levels of the Nnat protein in infected cells (Fig. 2A and C; see below). Because the function of Nnat in endothelium was unknown, we compared gene expression in endothelial cells infected with Ad-Nnat to that of an empty adenovirus control (Ad-Empty) using an endothelial cell biology RT2 Profiler PCR array. Of 84 endothelial-specific genes, 27 were differentially regulated in Ad-Nnat–infected cells. Twenty-one genes were increased, and six were decreased (supplementary Table S4). Among the increased genes, four major groups were apparent: 1) cytokines (IL-1β, IL-6, IL-7, IL-11, and TNF-α) and chemokines (CX3CL1 and CCL2); 2) adhesion molecules (ICAM-1, VCAM-1, and SELE); 3) growth factors (vascular endothelial growth factor, endothelial cell growth factor-1, and placental growth factor); and 4) extracellular matrix proteases/inhibitors (matrix metallopeptidase-1; serpin peptidase inhibitor, clade E, member-1; and plasminogen activator urokinase). The changes in TNF-α, IL-1β, IL-6, MCP-1 (CCL2), ICAM-1, VCAM-1, and SELE expression in Ad-Nnat–infected cells were verified by real-time PCR (Table 3). These results suggest that Nnat expression activates endothelial cells.


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)

Nnat expression in HAECs activates NF-κB–dependent gene expression in time- and concentration-dependent manner. A: Western blots of Nnat, VCAM-1, ICAM-1, and IκB-α protein from endothelial cells infected with Ad-Empty, Ad-GFP, and increasing number of Ad-Nnat adenovirus per cell (MOI). B: Real-time PCR measurement of IL-6 mRNA expression in endothelial cells infected with Ad-Empty, Ad-GFP, or Ad-Nnat at different MOI. (n = 3). C: Endothelial cells were collected at various times after infection with Ad-Nnat and Western blots were probed with antibodies to Nnat, NAK, IκB-α, phospho-p65, p65, and β-actin. D: Time course of IL-6 mRNA expression after Ad-Nnat infection of endothelial cells (n = 3). IL-6 mRNA expression levels were normalized to GAPDH. ***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

f2: Nnat expression in HAECs activates NF-κB–dependent gene expression in time- and concentration-dependent manner. A: Western blots of Nnat, VCAM-1, ICAM-1, and IκB-α protein from endothelial cells infected with Ad-Empty, Ad-GFP, and increasing number of Ad-Nnat adenovirus per cell (MOI). B: Real-time PCR measurement of IL-6 mRNA expression in endothelial cells infected with Ad-Empty, Ad-GFP, or Ad-Nnat at different MOI. (n = 3). C: Endothelial cells were collected at various times after infection with Ad-Nnat and Western blots were probed with antibodies to Nnat, NAK, IκB-α, phospho-p65, p65, and β-actin. D: Time course of IL-6 mRNA expression after Ad-Nnat infection of endothelial cells (n = 3). IL-6 mRNA expression levels were normalized to GAPDH. ***P < 0.001, **P < 0.01, *P < 0.05. Data are a representative of three to four experiments.
Mentions: HAECs were infected with an Ad-Nnat to determine the downstream effects of Nnat expression. Western blotting revealed high levels of the Nnat protein in infected cells (Fig. 2A and C; see below). Because the function of Nnat in endothelium was unknown, we compared gene expression in endothelial cells infected with Ad-Nnat to that of an empty adenovirus control (Ad-Empty) using an endothelial cell biology RT2 Profiler PCR array. Of 84 endothelial-specific genes, 27 were differentially regulated in Ad-Nnat–infected cells. Twenty-one genes were increased, and six were decreased (supplementary Table S4). Among the increased genes, four major groups were apparent: 1) cytokines (IL-1β, IL-6, IL-7, IL-11, and TNF-α) and chemokines (CX3CL1 and CCL2); 2) adhesion molecules (ICAM-1, VCAM-1, and SELE); 3) growth factors (vascular endothelial growth factor, endothelial cell growth factor-1, and placental growth factor); and 4) extracellular matrix proteases/inhibitors (matrix metallopeptidase-1; serpin peptidase inhibitor, clade E, member-1; and plasminogen activator urokinase). The changes in TNF-α, IL-1β, IL-6, MCP-1 (CCL2), ICAM-1, VCAM-1, and SELE expression in Ad-Nnat–infected cells were verified by real-time PCR (Table 3). These results suggest that Nnat expression activates endothelial cells.

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