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

Fernández-Mariño AI, Cidad P, Zafra D, Nocito L, Domínguez J, Oliván-Viguera A, Köhler R, López-López JR, Pérez-García MT, Valverde MÁ, Guinovart JJ, Fernández-Fernández JM - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratori de Fisiologia Molecular i Canalopaties, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

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.

Show MeSH

Related in: MedlinePlus

BK channels mediate both ERK1/2 phosphorylation and reduction of PDGF-stimulated proliferation induced by tungstate in human vascular smooth muscle cells.(A) Representative Western-blot showing phosphorylation of ERK1/2 using phospho-ERK-specific antibodies after 10 minutes incubation in control conditions (0% FBS), with PDGF (20 ng/ml), and with tungstate (1 mM) alone or in combination with iberiotoxin (IbTX 100 nM), as indicated. Beta-actin was used as loading control. (B) Protein expression and phosphorylation were quantified by densitometry of the corresponding Western blot signal and normalized to beta-actin. The ratio p-ERK/beta-actin in control conditions was taken as 1, so that fold-changes relative to control are shown. (C) VSMCs were serum starved for 48 hours and then incubated 30 hours with 20 ng/ml PDGF alone or in combination with the indicated compounds. During the last 6 hours of incubation, EdU was added to the media to detect the number of cells entering S-phase. Mean ± S.E.M. of 6–9 determinations from at least four different cultures. ***P < 0.001 (ANOVA followed by Tukey post hoc test).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4320054&req=5

pone.0118148.g005: BK channels mediate both ERK1/2 phosphorylation and reduction of PDGF-stimulated proliferation induced by tungstate in human vascular smooth muscle cells.(A) Representative Western-blot showing phosphorylation of ERK1/2 using phospho-ERK-specific antibodies after 10 minutes incubation in control conditions (0% FBS), with PDGF (20 ng/ml), and with tungstate (1 mM) alone or in combination with iberiotoxin (IbTX 100 nM), as indicated. Beta-actin was used as loading control. (B) Protein expression and phosphorylation were quantified by densitometry of the corresponding Western blot signal and normalized to beta-actin. The ratio p-ERK/beta-actin in control conditions was taken as 1, so that fold-changes relative to control are shown. (C) VSMCs were serum starved for 48 hours and then incubated 30 hours with 20 ng/ml PDGF alone or in combination with the indicated compounds. During the last 6 hours of incubation, EdU was added to the media to detect the number of cells entering S-phase. Mean ± S.E.M. of 6–9 determinations from at least four different cultures. ***P < 0.001 (ANOVA followed by Tukey post hoc test).

Mentions: We next analyzed whether tungstate also promoted ERK phosphorylation in human VSMCs and its relationship with the BK channel. Similar to that seen in HEKαβ1 cells (constitutively expressing the bovine α and β1 subunits of the BK channel), treatment of human VSMCs with tungstate (1 mM for 10 minutes) promoted the phosphorylation of ERK1/2 up to slightly lower levels than PDGF treatment although this difference did not reach statistical significance (P > 0.05, ANOVA followed by Tukey post hoc test). Such tungstate-induced ERK phosphorylation in human VSMCs was significantly reduced by pretreatment with the specific BK channel blocker iberiotoxin (IbTX, 100 nM) (Fig. 5A and 5B). As Gβγ-stimulated ERK1/2 phosphorylation has been reported to lead to differentiation of VSMCs to a contractile phenotype [36, 37], we also studied the effect of tungstate on PDGF-induced human VSMC proliferation. For this analysis, we lowered tungstate to 100 μM because we have previously reported that at this lower concentration, tungstate still promoted voltage-dependent activation of BKαβ1 channels and induced vasorelaxation in endothelin-1-pre-contracted mouse arteries [14]. Besides, such tungstate concentration is only slightly higher than the tungstate levels measured in the plasma of both experimentally treated rodent models and humans (~5–20 μM) [2, 42]. We found that 100 μM tungstate strongly reduced the proliferative action of PDGF (the effect was similar when using 1 mM tungstate). IbTX prevented significantly this inhibition. IbTX alone had no significant effect on PDGF-induced VSMC proliferation (Fig. 5C).


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

Fernández-Mariño AI, Cidad P, Zafra D, Nocito L, Domínguez J, Oliván-Viguera A, Köhler R, López-López JR, Pérez-García MT, Valverde MÁ, Guinovart JJ, Fernández-Fernández JM - PLoS ONE (2015)

BK channels mediate both ERK1/2 phosphorylation and reduction of PDGF-stimulated proliferation induced by tungstate in human vascular smooth muscle cells.(A) Representative Western-blot showing phosphorylation of ERK1/2 using phospho-ERK-specific antibodies after 10 minutes incubation in control conditions (0% FBS), with PDGF (20 ng/ml), and with tungstate (1 mM) alone or in combination with iberiotoxin (IbTX 100 nM), as indicated. Beta-actin was used as loading control. (B) Protein expression and phosphorylation were quantified by densitometry of the corresponding Western blot signal and normalized to beta-actin. The ratio p-ERK/beta-actin in control conditions was taken as 1, so that fold-changes relative to control are shown. (C) VSMCs were serum starved for 48 hours and then incubated 30 hours with 20 ng/ml PDGF alone or in combination with the indicated compounds. During the last 6 hours of incubation, EdU was added to the media to detect the number of cells entering S-phase. Mean ± S.E.M. of 6–9 determinations from at least four different cultures. ***P < 0.001 (ANOVA followed by Tukey post hoc test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4320054&req=5

pone.0118148.g005: BK channels mediate both ERK1/2 phosphorylation and reduction of PDGF-stimulated proliferation induced by tungstate in human vascular smooth muscle cells.(A) Representative Western-blot showing phosphorylation of ERK1/2 using phospho-ERK-specific antibodies after 10 minutes incubation in control conditions (0% FBS), with PDGF (20 ng/ml), and with tungstate (1 mM) alone or in combination with iberiotoxin (IbTX 100 nM), as indicated. Beta-actin was used as loading control. (B) Protein expression and phosphorylation were quantified by densitometry of the corresponding Western blot signal and normalized to beta-actin. The ratio p-ERK/beta-actin in control conditions was taken as 1, so that fold-changes relative to control are shown. (C) VSMCs were serum starved for 48 hours and then incubated 30 hours with 20 ng/ml PDGF alone or in combination with the indicated compounds. During the last 6 hours of incubation, EdU was added to the media to detect the number of cells entering S-phase. Mean ± S.E.M. of 6–9 determinations from at least four different cultures. ***P < 0.001 (ANOVA followed by Tukey post hoc test).
Mentions: We next analyzed whether tungstate also promoted ERK phosphorylation in human VSMCs and its relationship with the BK channel. Similar to that seen in HEKαβ1 cells (constitutively expressing the bovine α and β1 subunits of the BK channel), treatment of human VSMCs with tungstate (1 mM for 10 minutes) promoted the phosphorylation of ERK1/2 up to slightly lower levels than PDGF treatment although this difference did not reach statistical significance (P > 0.05, ANOVA followed by Tukey post hoc test). Such tungstate-induced ERK phosphorylation in human VSMCs was significantly reduced by pretreatment with the specific BK channel blocker iberiotoxin (IbTX, 100 nM) (Fig. 5A and 5B). As Gβγ-stimulated ERK1/2 phosphorylation has been reported to lead to differentiation of VSMCs to a contractile phenotype [36, 37], we also studied the effect of tungstate on PDGF-induced human VSMC proliferation. For this analysis, we lowered tungstate to 100 μM because we have previously reported that at this lower concentration, tungstate still promoted voltage-dependent activation of BKαβ1 channels and induced vasorelaxation in endothelin-1-pre-contracted mouse arteries [14]. Besides, such tungstate concentration is only slightly higher than the tungstate levels measured in the plasma of both experimentally treated rodent models and humans (~5–20 μM) [2, 42]. We found that 100 μM tungstate strongly reduced the proliferative action of PDGF (the effect was similar when using 1 mM tungstate). IbTX prevented significantly this inhibition. IbTX alone had no significant effect on PDGF-induced VSMC proliferation (Fig. 5C).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratori de Fisiologia Molecular i Canalopaties, Departament de Ciències Experimentals i de la Salut, Universitat Pompeu Fabra, Barcelona, Spain.

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.

Show MeSH
Related in: MedlinePlus