Inhibition of mammalian S6 kinase by resveratrol suppresses autophagy

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.

Keywords: Resveratrol; S6K1; autophagosome; autophagy; nutrient withdrawal; p70 S6 kinase.

Figure 1.. Resveratrol inhibits autophagy in mammalian cells.

(A) NIH/3T3 cells stably expressing the GFP-LC3 fusion protein were subjected to nutrient withdrawal by replacing growth media (Fed) with Earle's buffered saline solution (Starved) and treated with either DMSO or 50 μM resveratrol (Res) for 2 hours. Representative fields at 63X (oil immersion) magnification are shown. (B) Quantification of punctae/cell in (A) of at least 4 fields per treatment are represented as a percentage of the starved DMSO treated cells. (C) HEK293 cells stably expressing the GFP-LC3 fusion protein were subjected to starvation and either DMSO or 50 μM Res for 6 hours. Representative fields at 40X magnification are shown. (D) Quantification was performed on HEK293 cells as in (B). Error bars represent s.e.m. * (p < 0.0022).

Figure 2.. Resveratrol suppresses autophagy under TOR inhibition.

(A) HEK293 cells stably expressing GFP-LC3 growing in complete media were pretreated with DMSO or 50 μM resveratrol (Res) for 1 hour, prior to addition of DMSO or 200 nM rapamycin for 4 hours. 40X magnification fields have been cropped and zoomed for ease of punctae visualization. (B) Quantification of punctae/cell from (A) of 10 fields per treatment are represented as a percentage of DMSO treated 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.

Figure 3.. Resveratrol suppresses autophagy independently of SIRT1.

HEK293 cells stably expressing GFP-LC3 were transfected with either a control siRNA (A) or an siRNA directed against SIRT1 (B) for 72 hours. Subsequently, cells were subjected to nutrient starvation with or without 50 μM resveratrol (Res) treatment for 4 hours. 40X magnification fields have been cropped and zoomed for ease of punctae visualization. (C) Quantification of punctae/cell from (A) and (B) of 4 fields are represented as a percentage of fed DMSO treated control siRNA cells. Error bars represent s.d.m. (D) Representative western blot showing typical knockdown of SIRT1 by siRNA transfection in HEK293 GFP-LC3 cells.

Figure 4.. Resveratrol inhibits S6K1 in vitro.

(A) Structural similarity between resveratrol and quercetin, a known kinase inhibitor. (B) Kinase inhibition profile for resveratrol at 20 μM obtained using KinaseProfiler™ (Upstate). Dashed line represents 100% activity as compared to control. Black filled-in bar on the graph indicates S6K1. Complete data set is provided in Supplementary Table 1. Error bars represent s.d.m. (C) Phosphorylation of recombinant GST-tagged S6 by immunoprecipitated HA-S6K1 under increasing concentrations of resveratrol (Res). Autoradiograph depicts S6K1 phosphorylation of GST-S6. (D) Average of three separate kinase assay experiments as performed in (C). Densitometry was performed using NIH ImageJ. Error bars represent s.e.m.

Figure 5.. Resveratrol inhibits S6K1 in intact cells.

(A) NIH/3T3 or HEK293 cells were treated for 30 minutes with increasing doses of resveratrol and whole cell extracts were western blotted for the indicated proteins. (B) WT or two separate lines of SIRT1-/- MEFs (A and B) were treated with increasing doses of resveratrol for 30 minutes and analyzed by western blot.

Figure 6.. S6K1 is required for autophagy in mammalian cells.

(A) HEK293 cells stably expressing GFP-LC3 were infected with retrovirus encoding a dominant negative S6K1 (K100R) or with lentivirus encoding a specific shRNA directed against human S6K1 and subjected to nutrient withdrawal by replacing supplemented media (Fed) with EBSS for 4 hours (Starved). Representative fields at 63X (oil immersion) magnification are shown. (B) Efficiency of S6K1 knockdown and expression of HA-tagged S6K1 (K100R) in HEK293 GFP-LC3 cells. (C) Quantification of punctae/cell from (A) of at least 9 fields per treatment are represented as a percentage of the starved vector control cells. Error bars represent s.e.m. * (p < 0.0015) ** (p < 0.0002)

Figure 7.. Resveratrol does not affect autophagy in the absence of S6K1.

(A) HEK293 GFPLC3 cells were infected with shRNA S6K1 lentivirus or control virus and treated with EBSS (Starved) for 4 hrs ± 50 μM resveratrol (Res). Representative fields at 63X (oil immersion) magnification are shown. (B) Quantification of punctae/cell from (A) of at least 4 fields per treatment are represented as a percentage of DMSO treated starved vector control cells. Error bars represent s.e.m. N.S. = not significant.