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NFATc1 targets cyclin A in the regulation of vascular smooth muscle cell multiplication during restenosis.

ABSTRACT: Platelet-derived growth factor BB (PDGF-BB) induced cyclin A expression and CDK2 activity in vascular smooth muscle cells (VSMC). Inhibition of nuclear factors of activated T cell (NFAT) activation by cyclosporin A (CsA) and VIVIT suppressed PDGF-BB-induced cyclin A expression and CDK2 activity, resulting in blockade of VSMC in the G(1) phase. In addition, CsA- and VIVIT-mediated inhibition of NFATs and small interfering RNA-targeted down-regulation of cyclin A levels suppressed PDGF-BB-induced VSMC DNA synthesis. PDGF-BB also induced cyclin A mRNA levels in VSMC in an NFAT-dependent manner. Cloning and bioinformatic analysis of rat cyclin A promoter revealed the presence of NFAT-binding elements, and PDGF-BB induced the binding of NFATs to these regulatory sequences in a CsA- and VIVIT-sensitive manner. Chromatin immunoprecipitation analysis showed that NFATc1 binds to the cyclin A promoter in response to PDGF-BB in a VIVIT-sensitive manner. Furthermore, PDGF-BB induced cyclin A promoter-luciferase reporter gene activity in VSMC, and it was inhibited by both CsA and VIVIT. Balloon injury induced cyclin A expression and CDK2 activity in rat carotid arteries, and these responses were also blocked by VIVIT. In addition, VIVIT attenuated balloon injury-induced SMC proliferation, resulting in reduced restenosis. Down-regulation of NFATc1 by its small interfering RNA inhibited PDGF-BB-induced cyclin A expression and DNA synthesis both in rat and human VSMC. Together, these findings demonstrate that the cyclin A-CDK2 complex may be a potential effector of NFATs, specifically NFATc1, in mediating SMC multiplication leading to neointima formation. Therefore, NFATs may be used as target molecules for the development of therapeutic agents against vascular diseases such as restenosis.

SUBMITTER: Karpurapu M

PROVIDER: S-EPMC2546564 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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NFATc1 targets cyclin A in the regulation of vascular smooth muscle cell multiplication during restenosis.

Karpurapu Manjula M Wang Dong D Singh Nikhlesh K NK Li Quanyi Q Rao Gadiparthi N GN

The Journal of biological chemistry 20080729 39

Platelet-derived growth factor BB (PDGF-BB) induced cyclin A expression and CDK2 activity in vascular smooth muscle cells (VSMC). Inhibition of nuclear factors of activated T cell (NFAT) activation by cyclosporin A (CsA) and VIVIT suppressed PDGF-BB-induced cyclin A expression and CDK2 activity, resulting in blockade of VSMC in the G(1) phase. In addition, CsA- and VIVIT-mediated inhibition of NFATs and small interfering RNA-targeted down-regulation of cyclin A levels suppressed PDGF-BB-induced ...[more]

PMID: 18667424

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Project description:Vascular smooth muscle (VSM) plays an important role in the regulation of vascular function. Identifying the mechanisms of VSM contraction has been a major research goal in order to determine the causes of vascular dysfunction and exaggerated vasoconstriction in vascular disease. Major discoveries over several decades have helped to better understand the mechanisms of VSM contraction. Ca2+ has been established as a major regulator of VSM contraction, and its sources, cytosolic levels, homeostatic mechanisms and subcellular distribution have been defined. Biochemical studies have also suggested that stimulation of Gq protein-coupled membrane receptors activates phospholipase C and promotes the hydrolysis of membrane phospholipids into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates initial Ca2+ release from the sarcoplasmic reticulum, and is buttressed by Ca2+ influx through voltage-dependent, receptor-operated, transient receptor potential and store-operated channels. In order to prevent large increases in cytosolic Ca2+ concentration ([Ca2+]c), Ca2+ removal mechanisms promote Ca2+ extrusion via the plasmalemmal Ca2+ pump and Na+/Ca2+ exchanger, and Ca2+ uptake by the sarcoplasmic reticulum and mitochondria, and the coordinated activities of these Ca2+ handling mechanisms help to create subplasmalemmal Ca2+ domains. Threshold increases in [Ca2+]c form a Ca2+-calmodulin complex, which activates myosin light chain (MLC) kinase, and causes MLC phosphorylation, actin-myosin interaction, and VSM contraction. Dissociations in the relationships between [Ca2+]c, MLC phosphorylation, and force have suggested additional Ca2+ sensitization mechanisms. DAG activates protein kinase C (PKC) isoforms, which directly or indirectly via mitogen-activated protein kinase phosphorylate the actin-binding proteins calponin and caldesmon and thereby enhance the myofilaments force sensitivity to Ca2+. PKC-mediated phosphorylation of PKC-potentiated phosphatase inhibitor protein-17 (CPI-17), and RhoA-mediated activation of Rho-kinase (ROCK) inhibit MLC phosphatase and in turn increase MLC phosphorylation and VSM contraction. Abnormalities in the Ca2+ handling mechanisms and PKC and ROCK activity have been associated with vascular dysfunction in multiple vascular disorders. Modulators of [Ca2+]c, PKC and ROCK activity could be useful in mitigating the increased vasoconstriction associated with vascular disease.

Project description:RationaleCoronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes.ObjectiveTo identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs.Methods and resultsWe quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3. The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans.ConclusionsOur data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.

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NFATc1 targets cyclin A in the regulation of vascular smooth muscle cell multiplication during restenosis.

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NFATc1 targets cyclin A in the regulation of vascular smooth muscle cell multiplication during restenosis.

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