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Establishment of stable reporter expression for in vivo imaging of nuclear factor-?B activation in mouse liver.

ABSTRACT: The nuclear factor-?B (NF-?B) signaling pathway plays a critical role in a multitude of cellular processes. Activation of the NF-?B transcription factor family is essential for the initiation of inflammation, immunity, cell proliferation and apoptosis through a list of responsive genes. In hepatic tissue, activation of the NF-?B pathway has been implicated in a number of pathological conditions. Here we described a mouse model for noninvasive quantification of NF-?B activation in the hepatic tissues. Mice were subjected to hydrodynamic delivery with a mixture of pattB-NF-?B-Fluc reporter and ?C31o integrase vector. Hepatic expression of ?C31o integrase mediated chromosomal integration of the pattB-NF-?B-Fluc reporter, resulting in stable luciferase expression at 300 days post transfection. We applied noninvasive imaging and were able to detect NF-?B activation under acute liver injury and hepatitis conditions. During hepatectomy-induced liver regeneration, NF-?B activation was detected locally in the tissues at the surgery site. Treatment with Sorafenib suppressed NF-?B activation, accompanied with perturbation of liver regeneration. In conclusion, we established a method for stable transfection of the hepatic tissues and applied the transfected mice to longitudinal monitoring of NF-?B activity under pathological conditions. Further exploration of this methodology for establishment of other disease models and for evaluation of novel pharmaceuticals is likely to be fruitful.

SUBMITTER: Yan S

PROVIDER: S-EPMC3841335 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Establishment of stable reporter expression for in vivo imaging of nuclear factor-κB activation in mouse liver.

Yan Shaoduo S Fu Qiuxia Q Zhou Yong Y Zhang Ning N Zhou Qianqian Q Wang Xiaoying X Yuan Zhennan Z Wang Xiaohui X Du Juan J Zhang Jingang J Zhan Linsheng L

Theranostics 20131015 11

The nuclear factor-κB (NF-κB) signaling pathway plays a critical role in a multitude of cellular processes. Activation of the NF-κB transcription factor family is essential for the initiation of inflammation, immunity, cell proliferation and apoptosis through a list of responsive genes. In hepatic tissue, activation of the NF-κB pathway has been implicated in a number of pathological conditions. Here we described a mouse model for noninvasive quantification of NF-κB activation in the hepatic tis ...[more]

PMID: 24312154

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Project description:ObjectiveThromboxane A2 receptor (TPr) has been reported to trigger vascular inflammation. Nuclear factor κ B (NF-κB) is a known transcription factor. The aims of the present study were to determine the contributions of NF-κB activation to TPr-triggered vascular inflammation and elucidate the mechanism(s) underlying TPr activation of NF-κB.Approach and resultsThe effects of TPr activators, [1S-[1 alpha,2 alpha(Z),3beta(1E,3S*), 4 alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) and U46619, on NF-κB activation, phosphorylation of rhoA/rho-associated kinases and liver kinase B1, cell adhesion and migration, proliferation, and endothelium-dependent vasorelaxation were assayed in cultured human umbilical vein endothelial cells, human monocytes, or isolated mouse aortas. Exposure of human umbilical vein endothelial cells to TPr agonists I-BOP and U46619 induced dose-dependent and time-dependent phosphorylation of inhibitor of κB α in parallel with aberrant expression of inflammatory markers cyclooxygenase-2, inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Inhibition of NF-κB by pharmacological or genetic means abolished TPr-triggered expression of inflammatory markers. Consistently, exposure of human umbilical vein endothelial cells to either I-BOP or U46619 significantly increased phosphorylation of inhibitor of κB α, I kappaB kinase, rhoA, rho-associated kinases, and liver kinase B1. Pretreatment of human umbilical vein endothelial cells with the TPr antagonist SQ29548 or rho-associated kinases inhibitor Y27632 or silencing of the LKB1 blocked TPr-enhanced phosphorylation of inhibitor of κB α and its upstream kinase, I kappaB kinase. Finally, exposure of isolated mouse aortas to either U46619 or I-BOP enhanced NF-κB activation and vascular inflammation in parallel with reduced endothelium-dependent relaxation in intact vessels.ConclusionsTPr stimulation instigates aberrant inflammation and endothelial dysfunction via rho-associated kinases/liver kinase B1/I kappaB kinase-dependent NF-κB activation in vascular endothelial cells.

Dataset Information

Establishment of stable reporter expression for in vivo imaging of nuclear factor-?B activation in mouse liver.

Publications

Establishment of stable reporter expression for in vivo imaging of nuclear factor-κB activation in mouse liver.

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