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

Effect of kinase inhibitors on Nnat-induced NF-κB activation. A: Western blot analysis of Iκ B-α, Phospho-p65 (P-p65), VCAM-1, β-actin P-p38, P-JNK1/2, P-ERK1/2, and P-AKT protein levels in the presence of kinase inhibitors. IL-6 (B) and SELE (C) mRNA expression in HAECs infected with Ad-Nnat. Effect of kinase inhibitors on Nnat-induced increase in IL-6 and SELE expression levels, normalized to GAPDH. I, no inhibitor; U0, U0126; SB, SB202190; SP, SP600125; LY, LY294002; W, Wortmannin; and IK, IKK-VII. ***P < 0.001, **P < 0.01. Data are representative of three to four experiments. D: Schematic describing Nnat-induced signal transduction in endothelial cells. Inhibitors of PI 3-kinase and p38 completed blocked Nnat-induced NF-κB activation, JNK1/2 inhibition led to a partial block, and inhibition of ERK1/2 increased NF-κB activation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2551689&req=5

f4: Effect of kinase inhibitors on Nnat-induced NF-κB activation. A: Western blot analysis of Iκ B-α, Phospho-p65 (P-p65), VCAM-1, β-actin P-p38, P-JNK1/2, P-ERK1/2, and P-AKT protein levels in the presence of kinase inhibitors. IL-6 (B) and SELE (C) mRNA expression in HAECs infected with Ad-Nnat. Effect of kinase inhibitors on Nnat-induced increase in IL-6 and SELE expression levels, normalized to GAPDH. I, no inhibitor; U0, U0126; SB, SB202190; SP, SP600125; LY, LY294002; W, Wortmannin; and IK, IKK-VII. ***P < 0.001, **P < 0.01. Data are representative of three to four experiments. D: Schematic describing Nnat-induced signal transduction in endothelial cells. Inhibitors of PI 3-kinase and p38 completed blocked Nnat-induced NF-κB activation, JNK1/2 inhibition led to a partial block, and inhibition of ERK1/2 increased NF-κB activation.

Mentions: To test whether Nnat-induced kinase phosphorylation was involved in activation of NF-κB, we treated Ad-Nnat–infected endothelial cells with p38 (SB202190), JNK1,2,3 (SP600125), ERK1/2 kinase (U0126), PI 3-kinase (LY294002 and wortmannin), or IκB kinase (IKK) (IKK-VII) inhibitors (Fig. 4). SB202190, LY294002, wortmannin, and IKKVII blocked the Nnat-induced IκB-α degradation and increase in VCAM-1 protein. U0126 had the opposite effect; it further increased VCAM-1 expression. SP600125 deceased VCAM-1 protein expression without affecting IκB-α protein levels. LY294002 and wortmannin partially decreased phosphorylation of p65 subunit. SB202190, SP600125, and IKK-VII had no effect. Both LY294002 and SB202190 blocked (∼95%) the increase in IL-6 and SELE gene expression in Ad-Nnat–infected endothelial cells (Fig. 4B and C). U0126 had the opposite effect and increased IL-6 and SELE expression. SP600125 inhibitor only partially (∼40%) blocked the Nnat-mediated increase in IL-6 but not SELE expression. U0126 inhibited ERK phosphorylation and increased p-JNK and p-AKT. SB202190 decreased AKT and increased ERK phosphorylation. SP600125 decreased JNK and p38 phosphorylation without having an effect on others. This inhibitor is able to inhibit kinases upstream of p38 and JNK. We could speculate that partial inhibition of IL-6 but not SELE expression by this compound is a result of blocking kinase upstream from p38 but not JNK itself. LY294002 and wortmannin blocked Nnat-induced phosphorylation of NAK, p38, and JNK, suggesting that these kinases are downstream from PI 3-kinase. IKK-VII treatment blocked IκB-α degradation and did not effect p65 phosphorylation. On the other hand, IKK-VII inhibited phosphorylation of all three MAPKs. As summarized in Fig. 4D, Nnat effects on NF-κB appear to be mediated via PI 3-kinase and p38 and inhibited by ERK1/2.


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)

Effect of kinase inhibitors on Nnat-induced NF-κB activation. A: Western blot analysis of Iκ B-α, Phospho-p65 (P-p65), VCAM-1, β-actin P-p38, P-JNK1/2, P-ERK1/2, and P-AKT protein levels in the presence of kinase inhibitors. IL-6 (B) and SELE (C) mRNA expression in HAECs infected with Ad-Nnat. Effect of kinase inhibitors on Nnat-induced increase in IL-6 and SELE expression levels, normalized to GAPDH. I, no inhibitor; U0, U0126; SB, SB202190; SP, SP600125; LY, LY294002; W, Wortmannin; and IK, IKK-VII. ***P < 0.001, **P < 0.01. Data are representative of three to four experiments. D: Schematic describing Nnat-induced signal transduction in endothelial cells. Inhibitors of PI 3-kinase and p38 completed blocked Nnat-induced NF-κB activation, JNK1/2 inhibition led to a partial block, and inhibition of ERK1/2 increased NF-κB activation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Effect of kinase inhibitors on Nnat-induced NF-κB activation. A: Western blot analysis of Iκ B-α, Phospho-p65 (P-p65), VCAM-1, β-actin P-p38, P-JNK1/2, P-ERK1/2, and P-AKT protein levels in the presence of kinase inhibitors. IL-6 (B) and SELE (C) mRNA expression in HAECs infected with Ad-Nnat. Effect of kinase inhibitors on Nnat-induced increase in IL-6 and SELE expression levels, normalized to GAPDH. I, no inhibitor; U0, U0126; SB, SB202190; SP, SP600125; LY, LY294002; W, Wortmannin; and IK, IKK-VII. ***P < 0.001, **P < 0.01. Data are representative of three to four experiments. D: Schematic describing Nnat-induced signal transduction in endothelial cells. Inhibitors of PI 3-kinase and p38 completed blocked Nnat-induced NF-κB activation, JNK1/2 inhibition led to a partial block, and inhibition of ERK1/2 increased NF-κB activation.
Mentions: To test whether Nnat-induced kinase phosphorylation was involved in activation of NF-κB, we treated Ad-Nnat–infected endothelial cells with p38 (SB202190), JNK1,2,3 (SP600125), ERK1/2 kinase (U0126), PI 3-kinase (LY294002 and wortmannin), or IκB kinase (IKK) (IKK-VII) inhibitors (Fig. 4). SB202190, LY294002, wortmannin, and IKKVII blocked the Nnat-induced IκB-α degradation and increase in VCAM-1 protein. U0126 had the opposite effect; it further increased VCAM-1 expression. SP600125 deceased VCAM-1 protein expression without affecting IκB-α protein levels. LY294002 and wortmannin partially decreased phosphorylation of p65 subunit. SB202190, SP600125, and IKK-VII had no effect. Both LY294002 and SB202190 blocked (∼95%) the increase in IL-6 and SELE gene expression in Ad-Nnat–infected endothelial cells (Fig. 4B and C). U0126 had the opposite effect and increased IL-6 and SELE expression. SP600125 inhibitor only partially (∼40%) blocked the Nnat-mediated increase in IL-6 but not SELE expression. U0126 inhibited ERK phosphorylation and increased p-JNK and p-AKT. SB202190 decreased AKT and increased ERK phosphorylation. SP600125 decreased JNK and p38 phosphorylation without having an effect on others. This inhibitor is able to inhibit kinases upstream of p38 and JNK. We could speculate that partial inhibition of IL-6 but not SELE expression by this compound is a result of blocking kinase upstream from p38 but not JNK itself. LY294002 and wortmannin blocked Nnat-induced phosphorylation of NAK, p38, and JNK, suggesting that these kinases are downstream from PI 3-kinase. IKK-VII treatment blocked IκB-α degradation and did not effect p65 phosphorylation. On the other hand, IKK-VII inhibited phosphorylation of all three MAPKs. As summarized in Fig. 4D, Nnat effects on NF-κB appear to be mediated via PI 3-kinase and p38 and inhibited by ERK1/2.

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