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Altered nitric oxide production mediates matrix-specific PAK2 and NF-?B activation by flow.

ABSTRACT: Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-?B activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a protein kinase A-dependent pathway; however, the mechanisms underlying this regulation were unknown. We show that basement membrane proteins limit membrane recruitment of PAK2, the dominant isoform in endothelial cells, by blocking its interaction with the adaptor protein Nck. This uncoupling response requires protein kinase A-dependent nitric oxide production and subsequent PAK2 phosphorylation on Ser-20 in the Nck-binding domain. Of importance, shear stress does not stimulate nitric oxide production in endothelial cells on fibronectin, resulting in enhanced PAK activation, NF-?B phosphorylation, ICAM-1 expression, and monocyte adhesion. These data demonstrate that differential flow-induced nitric oxide production regulates matrix-specific PAK signaling and describe a novel mechanism of nitric oxide-dependent NF-?B inhibition.

SUBMITTER: Yurdagul A

PROVIDER: S-EPMC3564533 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Altered nitric oxide production mediates matrix-specific PAK2 and NF-κB activation by flow.

Yurdagul Arif A Chen Jie J Funk Steven Daniel SD Albert Patrick P Kevil Christopher G CG Orr A Wayne AW

Molecular biology of the cell 20121121 3

Shear stress generated by distinct blood flow patterns modulates endothelial cell phenotype to spatially restrict atherosclerotic plaque development. Signaling through p21-activated kinase (PAK) mediates several of the deleterious effects of shear stress, including enhanced NF-κB activation and proinflammatory gene expression. Whereas shear stress activates PAK in endothelial cells on a fibronectin matrix, basement membrane proteins limit shear-induced PAK activation and inflammation through a p ...[more]

PMID: 23171552

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Project description:Recently, it has been reported that a ?-receptor antagonist could reduce inflammation-induced edema. Lymphatic vessels play an essential role in removing excess interstitial fluid. We tested the hypothesis that activation of ?-receptors would reduce or weaken collecting lymphatic contractions. We used isolated, cannulated rat mesenteric collecting lymphatic vessels to study contractions in response to the ?-receptor agonist afobazole in the absence and presence of different ?-receptor antagonists. We used RT-PCR and Western blot analysis to investigate whether these vessels express the ?1-receptor and immunofluorescence confocal microscopy to examine localization of the ?1-receptor in the collecting lymphatic wall. Using N-nitro-l-arginine methyl ester (l-NAME) pretreatment before afobazole in isolated lymphatics, we tested the role of nitric oxide (NO) signaling. Finally, we used 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence as an indicator to test whether afobazole increases NO release in cultured lymphatic endothelial cells. Our results show that afobazole (50-150 µM) elevated end-systolic diameter and generally reduced pump efficiency and that this response could be partially blocked by the ?1-receptor antagonists BD 1047 and BD 1063 but not by the ?2-receptor antagonist SM-21. ?1-Receptor mRNA and protein were detected in lysates from isolated rat mesenteric collecting lymphatics. Confocal images with anti-?1-receptor antibody labeling suggested localization in the lymphatic endothelium. Blockade of NO synthases with l-NAME inhibited the effects of afobazole. Finally, afobazole elicited increases in NO production from cultured lymphatic endothelial cells. Our findings suggest that the ?1-receptor limits collecting lymphatic pumping through a NO-dependent mechanism.NEW & NOTEWORTHY Relatively little is known about the mechanisms that govern contractions of lymphatic vessels. ?1-Receptor activation has been shown to reduce the fractional pump flow of isolated rat mesenteric collecting lymphatics. The ?1-receptor was localized mainly in the endothelium, and blockade of nitric oxide synthase inhibited the effects of afobazole.

Dataset Information

Altered nitric oxide production mediates matrix-specific PAK2 and NF-?B activation by flow.

Publications

Altered nitric oxide production mediates matrix-specific PAK2 and NF-κB activation by flow.

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