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

Ana Isabel Fernández-Mariño,Miguel Ángel Valverde,María Teresa Pérez-García,Jorge Domínguez,Pilar Cidad,Laura Nocito,Ralf Köhler,Delia Zafra,Joan J Guinovart,Aida Oliván-Viguera,José M Fernández-Fernández,José R López-López

doi:10.1371/journal.pone.0118148

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 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 relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαβ1 channels potentiates the tungstate-induced ERK1/2 phosphorylation in a Gi/o protein-dependent manner. Tungstate activated BKαβ1 channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPβS or pertussis toxin. Moreover, analysis of Gi/o protein activation measuring the FRET among heterologously expressed Gi protein subunits suggested that tungstate-targeting of BKαβ1 channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and β1-knockout mice indicated that the presence of the regulatory β1 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 BKαβ1 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.

Highlights

Tungstate has antidiabetic and antiobesity actions in several animal models: 1) tungstate treatment normalizes hepatic carbohydrate metabolism [1, 2]; 2) stimulates insulin secretion and regenerates pancreatic β-cell population [3]; 3) mimics the effect of insulin on hepatocytes by increasing glycogen synthesis and deposition [4]; 4) increases the production and translocation of the insulin-regulated glucose transporterGLUT4 in muscle [5]; 5) favors thermogenesis and lipid oxidation in adipose tissue [6]; and 6)modulates hypothalamic gene expression by activation of the leptin-signaling pathway responsible for the regulation of food intake and energy expenditure [7]
BKαβ1 channels play a role in the Gi/o protein-dependent ERK1/2 phosphorylation induced by tungstate
Such enhanced extracellular signal-regulated kinases (ERK) phosphorylation induced by tungstate in HEKαβ1 cells was prevented by pretreatment with either the Gi/o protein inhibitor pertussis toxin (PTX, 100 ng/ml) (Fig. 1C, 1D) or the specific BK channel blocker iberiotoxin (IbTX, 100 nM) (Fig. 1E, 1F)

Summary

Introduction

Tungstate has antidiabetic and antiobesity actions in several animal models: 1) tungstate treatment normalizes hepatic carbohydrate metabolism [1, 2]; 2) stimulates insulin secretion and regenerates pancreatic β-cell population [3]; 3) mimics the effect of insulin on hepatocytes (but in an insulin receptor independent manner) by increasing glycogen synthesis and deposition [4]; 4) increases the production and translocation of the insulin-regulated glucose transporterGLUT4 in muscle [5]; 5) favors thermogenesis and lipid oxidation in adipose tissue [6]; and 6)modulates hypothalamic gene expression by activation of the leptin-signaling pathway responsible for the regulation of food intake and energy expenditure [7]. Tungstate reduces blood pressure in experimental animal models of both hypertension [8, 9] and metabolic syndrome [10]. Despite this knowledge on tungstate effects, our understanding of the underlying molecular mechanisms is incomplete. In this respect, it has been suggested that activation of several kinases (extracellular signal-regulated kinases (ERK) 1/2 and JAK2) by tungstate can lead to some of its antidiabetic and antiobesity actions [4, 5, 7]. Tungstate’s antihypertensive actions seem to be achieved by inhibition of endothelial xanthine oxidase [8] and by activation of the large-conductance voltage- and Ca2+-activated K+

Methods

Results

Conclusion