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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 inhibits autophagy in mammalian cells. (A) NIH/3T3 cells stably expressing the GFP-LC3fusion protein were subjected to nutrient withdrawal byreplacing growth media (Fed) with Earle's buffered salinesolution (Starved) and treated with either DMSO or 50 μMresveratrol (Res) for 2 hours. Representative fields at63X (oil immersion) magnification are shown. (B) Quantificationof punctae/cell in (A) of at least 4 fields per treatmentare represented as a percentage of the starved DMSO treatedcells. (C) HEK293 cells stably expressing the GFP-LC3 fusionprotein were subjected to starvation and either DMSO or 50 μMRes for 6 hours. Representative fields at 40X magnificationare shown. (D) Quantification was performed on HEK293 cellsas in (B). Error bars represent s.e.m. * (p < 0.0022).
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Figure 1: Resveratrol inhibits autophagy in mammalian cells. (A) NIH/3T3 cells stably expressing the GFP-LC3fusion protein were subjected to nutrient withdrawal byreplacing growth media (Fed) with Earle's buffered salinesolution (Starved) and treated with either DMSO or 50 μMresveratrol (Res) for 2 hours. Representative fields at63X (oil immersion) magnification are shown. (B) Quantificationof punctae/cell in (A) of at least 4 fields per treatmentare represented as a percentage of the starved DMSO treatedcells. (C) HEK293 cells stably expressing the GFP-LC3 fusionprotein were subjected to starvation and either DMSO or 50 μMRes for 6 hours. Representative fields at 40X magnificationare shown. (D) Quantification was performed on HEK293 cellsas in (B). Error bars represent s.e.m. * (p < 0.0022).

Mentions: Autophagy is an importantcomponent of the cellular response to nutrient stress and growth factorwithdrawal. We therefore tested whether resveratrol treatment would influenceregulation of autophagy under these conditions. Autophagy was assessed bymonitoring the relocalization of a component of the autophagy machinery, LC3,from the cytoplasm to the forming autophagosome [19]. NIH/3T3 cells or HEK293cells stably expressing a GFP-LC3 fusion protein were generated and wereinduced to undergo autophagy in the presence or absence of resveratrol.Treatment with resveratrol resulted in a dramatic reduction in the number ofstarvation-induced GFP-LC3 punctae (Figure 1). Similar inhibitory effects onautophagy were observed in other cell lines, including human tumor cell lines (HeLa and U2OS), as well as mouseembryonic fibroblasts (MEFs) using monodansylcadaverine (MDC), a fluorescent compoundthat stains late autophagosomes (Supplementary Figures 1 and 3) [20]. Since previousstudies have described an increase in autophagy following 24 hours ofresveratrol treatment in nutrient richmedia, we also tested the effects of resveratrol under these conditions. Consistent withthese results, we observed an induction of autophagosome formation in cells treatedwith resveratrol for 24 hours in complete media containing serum(Supplementary Figure 2). Thus, the influence of resveratrol on autophagy is contextdependent, and in the case of autophagy induced by nutrient limitation,resveratrol is inhibitory.


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 inhibits autophagy in mammalian cells. (A) NIH/3T3 cells stably expressing the GFP-LC3fusion protein were subjected to nutrient withdrawal byreplacing growth media (Fed) with Earle's buffered salinesolution (Starved) and treated with either DMSO or 50 μMresveratrol (Res) for 2 hours. Representative fields at63X (oil immersion) magnification are shown. (B) Quantificationof punctae/cell in (A) of at least 4 fields per treatmentare represented as a percentage of the starved DMSO treatedcells. (C) HEK293 cells stably expressing the GFP-LC3 fusionprotein were subjected to starvation and either DMSO or 50 μMRes for 6 hours. Representative fields at 40X magnificationare shown. (D) Quantification was performed on HEK293 cellsas in (B). Error bars represent s.e.m. * (p < 0.0022).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Resveratrol inhibits autophagy in mammalian cells. (A) NIH/3T3 cells stably expressing the GFP-LC3fusion protein were subjected to nutrient withdrawal byreplacing growth media (Fed) with Earle's buffered salinesolution (Starved) and treated with either DMSO or 50 μMresveratrol (Res) for 2 hours. Representative fields at63X (oil immersion) magnification are shown. (B) Quantificationof punctae/cell in (A) of at least 4 fields per treatmentare represented as a percentage of the starved DMSO treatedcells. (C) HEK293 cells stably expressing the GFP-LC3 fusionprotein were subjected to starvation and either DMSO or 50 μMRes for 6 hours. Representative fields at 40X magnificationare shown. (D) Quantification was performed on HEK293 cellsas in (B). Error bars represent s.e.m. * (p < 0.0022).
Mentions: Autophagy is an importantcomponent of the cellular response to nutrient stress and growth factorwithdrawal. We therefore tested whether resveratrol treatment would influenceregulation of autophagy under these conditions. Autophagy was assessed bymonitoring the relocalization of a component of the autophagy machinery, LC3,from the cytoplasm to the forming autophagosome [19]. NIH/3T3 cells or HEK293cells stably expressing a GFP-LC3 fusion protein were generated and wereinduced to undergo autophagy in the presence or absence of resveratrol.Treatment with resveratrol resulted in a dramatic reduction in the number ofstarvation-induced GFP-LC3 punctae (Figure 1). Similar inhibitory effects onautophagy were observed in other cell lines, including human tumor cell lines (HeLa and U2OS), as well as mouseembryonic fibroblasts (MEFs) using monodansylcadaverine (MDC), a fluorescent compoundthat stains late autophagosomes (Supplementary Figures 1 and 3) [20]. Since previousstudies have described an increase in autophagy following 24 hours ofresveratrol treatment in nutrient richmedia, we also tested the effects of resveratrol under these conditions. Consistent withthese results, we observed an induction of autophagosome formation in cells treatedwith resveratrol for 24 hours in complete media containing serum(Supplementary Figure 2). Thus, the influence of resveratrol on autophagy is contextdependent, and in the case of autophagy induced by nutrient limitation,resveratrol is inhibitory.

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