Dataset Information

Tungstate-targeting of BK??1 channels tunes ERK phosphorylation and cell proliferation in human vascular smooth muscle.

ABSTRACT: Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca2+-dependent large-conductance BK??1 potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BK??1 channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BK??1 channels potentiates the tungstate-induced ERK1/2 phosphorylation in a Gi/o protein-dependent manner. Tungstate activated BK??1 channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDP?S or pertussis toxin. Moreover, analysis of Gi/o protein activation measuring the FRET among heterologously expressed Gi protein subunits suggested that tungstate-targeting of BK??1 channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and ?1-knockout mice indicated that the presence of the regulatory ?1 subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BK??1 channels promotes activation of PTX-sensitive Gi proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.

SUBMITTER: Fernandez-Marino AI

PROVIDER: S-EPMC4320054 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Tungstate-targeting of BKαβ1 channels tunes ERK phosphorylation and cell proliferation in human vascular smooth muscle.

Fernández-Mariño Ana Isabel AI Cidad Pilar P Zafra Delia D Nocito Laura L Domínguez Jorge J Oliván-Viguera Aida A Köhler Ralf R López-López José R JR Pérez-García María Teresa MT Valverde Miguel Ángel MÁ Guinovart Joan J JJ Fernández-Fernández José M JM

PloS one 20150206 2

Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca2+-dependent large-conductance BKαβ1 potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαβ1 channels in the tungstate-induced ERK phosphorylation and its ...[more]

PMID: 25659150

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Project description:Mitochondria are widely described as being highly dynamic and adaptable organelles, and their movement is thought to be vital for cell function. Yet, in various native cells, including those of heart and smooth muscle, mitochondria are stationary and rigidly structured. The significance of the differences in mitochondrial behavior to the physiological function of cells is unclear and was studied in single myocytes and intact resistance-sized cerebral arteries. We hypothesized that mitochondrial dynamics is controlled by the proliferative status of the cells.High-speed fluorescence imaging of mitochondria in live vascular smooth muscle cells shows that the organelle undergoes significant reorganization as cells become proliferative. In nonproliferative cells, mitochondria are individual (? 2 ?m by 0.5 ?m), stationary, randomly dispersed, fixed structures. However, on entering the proliferative state, mitochondria take on a more diverse architecture and become small spheres, short rod-shaped structures, long filamentous entities, and networks. When cells proliferate, mitochondria also continuously move and change shape. In the intact pressurized resistance artery, mitochondria are largely immobile structures, except in a small number of cells in which motility occurred. When proliferation of smooth muscle was encouraged in the intact resistance artery, in organ culture, the majority of mitochondria became motile and the majority of smooth muscle cells contained moving mitochondria. Significantly, restriction of mitochondrial motility using the fission blocker mitochondrial division inhibitor prevented vascular smooth muscle proliferation in both single cells and the intact resistance artery.These results show that mitochondria are adaptable and exist in intact tissue as both stationary and highly dynamic entities. This mitochondrial plasticity is an essential mechanism for the development of smooth muscle proliferation and therefore presents a novel therapeutic target against vascular disease.

Project description:Objective: Abnormal proliferation or migration of vascular smooth muscle cells (VSMCs) can lead to vessel lesions, resulting in atherosclerosis and in stent-restenosis (IRS). The purpose of our study was to establish the role of miR-378a-5p and its targets in regulating VSMCs function and IRS. Methods: EdU assays and Cell Counting Kit-8 (CCK-8) assays were applied to evaluate VSMCs proliferation, wound healing assays and transwell assays were applied to assess cells migration. Furthermore, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to investigate the expression level of miR-378a-5p IRS patients and healthy individuals. Target genes were predicted using Target Scan and miRanda software, and biological functions of candidate genes were explored through bioinformatics analysis. Moreover, RNA-binding protein immunoprecipitation (RIP) was carried out to analyze the miRNAs interactions with proteins. We also used Immunofluorescence (IF) and fluorescence microscopy to determine the binding properties, localization and expression of miR-378a-5p with downstream target CDK1. Results: The expression of miR-378a-5p was increased in the group with stent restenosis compared with healthy people, as well as in the group which VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB) compared with NCs. MiR-378a-5p over-expression had significantly promoted proliferative and migratory effects, while miR-378a-5p inhibitor suppressed VSMC proliferation and migration. CDK1 was proved to be the functional target of miR-378a-5p in VSMCs. Encouragingly, the expression of miR-378a-5p was increased in patients with stent restenosis compared with healthy people, as well as in PDGF-BB-stimulated VSMCs compared with control cells. Furthermore, co-transfection experiments demonstrated that miR-378a-5p over-expression promoted proliferation and migration of VSMCs specifically by reducing CDK1 gene expression levels. Conclusion: In this investigatory, we concluded that miR-378a-5p is a critical mediator in regulating VSMC proliferation and migration by targeting CDK1/p21 signaling pathway. Thereby, interventions aimed at miR-378a-5p may be of therapeutic application in the prevention and treatment of stent restenosis.

Dataset Information

Tungstate-targeting of BK??1 channels tunes ERK phosphorylation and cell proliferation in human vascular smooth muscle.

Publications

Tungstate-targeting of BKαβ1 channels tunes ERK phosphorylation and cell proliferation in human vascular smooth muscle.

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