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Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy.

Armour SM, Baur JA, Hsieh SN, Land-Bracha A, Thomas SM, Sinclair DA - Aging (Albany NY) (2009)

Bottom Line: A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects.Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect.These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Resveratrol is a plant-derived polyphenol that promotes health and disease resistance in rodent models, and extends lifespan in lower organisms. A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects. Autophagy is a critical process by which cells turn over damaged components and maintain bioenergetic requirements. Disruption of the normal balance between pro- and anti-autophagic signals is linked to cancer, liver disease, and neurodegenerative disorders. Here we show that resveratrol attenuates autophagy in response to nutrient limitation or rapamycin in multiple cell lines through a pathway independent of a known target, SIRT1. In a large-scalein vitro kinase screen we identified p70 S6 kinase (S6K1) as a target of resveratrol. Blocking S6K1 activity by expression of a dominant-negative mutant or RNA interference is sufficient to disrupt autophagy to a similar extent as resveratrol. Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect. These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity.

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Resveratrol suppresses autophagy under TOR inhibition. (A) HEK293 cells stablyexpressing GFP-LC3 growing in complete media werepretreated with DMSO or 50 μM resveratrol (Res) for 1hour, prior to addition of DMSO or 200 nM rapamycinfor 4 hours. 40X magnification fields have beencropped and zoomed for ease of punctae visualization.(B) Quantification of punctae/cell from (A) of 10 fieldsper treatment are represented as a percentage of DMSOtreated cells. Error bars represent s.d.m. * (p < 0.0001) (C) HEK293 GFP-LC3 cells were pretreated for 1 hour with DMSO or resveratrol and subsequently treated with DMSO or 1 mM rapamycin in the presence or absence of 100 nM Bafilomycin A1 for 4 hours. A representative western blot of endogenous LC3 and tubulin are shown. Numbers represent the ratio of LC3-II to tubulin for each condition normalized to Rapamycin in the absence of BafA1.
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Figure 2: Resveratrol suppresses autophagy under TOR inhibition. (A) HEK293 cells stablyexpressing GFP-LC3 growing in complete media werepretreated with DMSO or 50 μM resveratrol (Res) for 1hour, prior to addition of DMSO or 200 nM rapamycinfor 4 hours. 40X magnification fields have beencropped and zoomed for ease of punctae visualization.(B) Quantification of punctae/cell from (A) of 10 fieldsper treatment are represented as a percentage of DMSOtreated cells. Error bars represent s.d.m. * (p < 0.0001) (C) HEK293 GFP-LC3 cells were pretreated for 1 hour with DMSO or resveratrol and subsequently treated with DMSO or 1 mM rapamycin in the presence or absence of 100 nM Bafilomycin A1 for 4 hours. A representative western blot of endogenous LC3 and tubulin are shown. Numbers represent the ratio of LC3-II to tubulin for each condition normalized to Rapamycin in the absence of BafA1.

Mentions: Rapamycin is an inhibitor of the nutrient sensingmTOR-Raptor complex and has been shown to induce autophagy [21,22]. In yeast,it had been shown that resveratrol can reverse some markers of autophagyinduced by rapamycin [23]. Consistent with these results and similar to the resultsseen under nutrient limitation, rapamycin-induced autophagy is almostcompletely abrogated by resveratrol treatment (Figure 2A and B). DecreasedGFP-LC3 punctae could be due either to increased flux or a block in autophagy. Todistinguish between these two possibilities, we examined LC3-II accumulationwith and without Bafilomycin A1, an inhibitor of lysosome degradation. Underboth conditions, resveratrol was able to block the accumulation of LC3-II, indicatinga suppression of autophagy rather than an enhancement of lysosomal clearance (Figure 2C).


Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy.

Armour SM, Baur JA, Hsieh SN, Land-Bracha A, Thomas SM, Sinclair DA - Aging (Albany NY) (2009)

Resveratrol suppresses autophagy under TOR inhibition. (A) HEK293 cells stablyexpressing GFP-LC3 growing in complete media werepretreated with DMSO or 50 μM resveratrol (Res) for 1hour, prior to addition of DMSO or 200 nM rapamycinfor 4 hours. 40X magnification fields have beencropped and zoomed for ease of punctae visualization.(B) Quantification of punctae/cell from (A) of 10 fieldsper treatment are represented as a percentage of DMSOtreated cells. Error bars represent s.d.m. * (p < 0.0001) (C) HEK293 GFP-LC3 cells were pretreated for 1 hour with DMSO or resveratrol and subsequently treated with DMSO or 1 mM rapamycin in the presence or absence of 100 nM Bafilomycin A1 for 4 hours. A representative western blot of endogenous LC3 and tubulin are shown. Numbers represent the ratio of LC3-II to tubulin for each condition normalized to Rapamycin in the absence of BafA1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2806030&req=5

Figure 2: Resveratrol suppresses autophagy under TOR inhibition. (A) HEK293 cells stablyexpressing GFP-LC3 growing in complete media werepretreated with DMSO or 50 μM resveratrol (Res) for 1hour, prior to addition of DMSO or 200 nM rapamycinfor 4 hours. 40X magnification fields have beencropped and zoomed for ease of punctae visualization.(B) Quantification of punctae/cell from (A) of 10 fieldsper treatment are represented as a percentage of DMSOtreated cells. Error bars represent s.d.m. * (p < 0.0001) (C) HEK293 GFP-LC3 cells were pretreated for 1 hour with DMSO or resveratrol and subsequently treated with DMSO or 1 mM rapamycin in the presence or absence of 100 nM Bafilomycin A1 for 4 hours. A representative western blot of endogenous LC3 and tubulin are shown. Numbers represent the ratio of LC3-II to tubulin for each condition normalized to Rapamycin in the absence of BafA1.
Mentions: Rapamycin is an inhibitor of the nutrient sensingmTOR-Raptor complex and has been shown to induce autophagy [21,22]. In yeast,it had been shown that resveratrol can reverse some markers of autophagyinduced by rapamycin [23]. Consistent with these results and similar to the resultsseen under nutrient limitation, rapamycin-induced autophagy is almostcompletely abrogated by resveratrol treatment (Figure 2A and B). DecreasedGFP-LC3 punctae could be due either to increased flux or a block in autophagy. Todistinguish between these two possibilities, we examined LC3-II accumulationwith and without Bafilomycin A1, an inhibitor of lysosome degradation. Underboth conditions, resveratrol was able to block the accumulation of LC3-II, indicatinga suppression of autophagy rather than an enhancement of lysosomal clearance (Figure 2C).

Bottom Line: A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects.Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect.These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Resveratrol is a plant-derived polyphenol that promotes health and disease resistance in rodent models, and extends lifespan in lower organisms. A major challenge is to understand the biological processes and molecular pathways by which resveratrol induces these beneficial effects. Autophagy is a critical process by which cells turn over damaged components and maintain bioenergetic requirements. Disruption of the normal balance between pro- and anti-autophagic signals is linked to cancer, liver disease, and neurodegenerative disorders. Here we show that resveratrol attenuates autophagy in response to nutrient limitation or rapamycin in multiple cell lines through a pathway independent of a known target, SIRT1. In a large-scalein vitro kinase screen we identified p70 S6 kinase (S6K1) as a target of resveratrol. Blocking S6K1 activity by expression of a dominant-negative mutant or RNA interference is sufficient to disrupt autophagy to a similar extent as resveratrol. Furthermore, co-administration of resveratrol with S6K1 knockdown does not produce an additive effect. These data indicate that S6K1 is important for the full induction of autophagy in mammals and raise the possibility that some of the beneficial effects of resveratrol are due to modulation of S6K1 activity.

Show MeSH
Related in: MedlinePlus