Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes

Abstract

The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol's effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by the AMPK-FOXO3 pathway and in some situations, but not all, by SIRT1.

Fig 1. SIRT1 is involved in the attenuation of H₂O₂-induced senescence by resveratrol.

(a) Human primary keratinocytes (passage 4) showed a number of senescent cells positive for SA-Gal (dark blue-green color). These cells lacked SIRT1 immunostaining (red arrow), which normally appears in the nucleus (see brown color in adjacent cells). (b,c,d) Exposure of human primary keratinocytes to 50 µM H₂O₂ induced a 5–6 fold increase in SA-Gal expression. The effect of H₂O₂ was largely prevented by pre-treatment with 50 µM resveratrol for 30 min. Addition of Ex527, a SIRT1-specific inhibitor, prior to the addition of resveratrol did not alter AMPK activation but abrogated the effects of resveratrol on SA-Gal staining. For (d), numbers inside parenthesis denote n. (e) SIRT1 was ectopically expressed by recombinant lentivirus, which produced strong staining within the cytosol (red arrow), instead of nuclear staining in normal cells (gray arrow).

Fig 2. AMPK activation increases FOXO3 protein expression.

(a) Keratinocytes grown in a 6-well plate were treated with the indicated concentrations of resveratrol for 30 min. Cells were harvested and subjected to Western blot. 25–50 µM resveratrol induced a 3–fold increase in FOXO3 protein expression. * indicates p<0.05 compared to 0 µM resveratrol treatment. (b) Human keratinocytes and umbilical vein endothelial cells were cultured in 6-well plates until 80% confluence. The cells were then incubated with the indicated reagents for 30 min.-2 hrs. Keratinocytes and endothelial cells were harvested simultaneously for Western blotting. Keratinocyte blotting required only a 30-second exposure for FOXO3 and a 5-minute exposure for FOXO1. Endothelial cell blotting required a 5-minute exposure for FOXO3 and only a 30-second exposure for FOXO1. This suggests that FOXO3 is richer in keratinocytes than in endothelium. Various AMPK activators, in particular 2-deoxyglucose and resveratrol, increased FOXO3 and FOXO1 proteins in keratinocytes. This effect was less apparent in endothelium.

Fig 3. Resveratrol increases FOXO3 transcriptional activity and expression of FOXO3 target genes.

(a) FOXO3 transcriptional activity was assessed using the Fork Head Responsive Element (FHRE) reporter gene assay. Cells were grown in 24-well plates and transfected with the reporter plasmid at 40–50% confluence. The assay was performed 48 hrs. later. On the day of the assay, the cells were treated with H₂O₂ ± resveratrol for 6 hrs. after which they were harvested and luciferase activity was measured. Resveratrol induced a twofold increase in FOXO3 transcriptional activity. (b) The cells were incubated with the indicated concentrations of resveratrol for 30 minutes and harvested 16 hrs. later. mRNA levels were quantified by real-time PCR. Among the known FOXO3 target genes, expression of catalase (CAT) and BCL2-like 11 (BCL2L11) were significantly increased with 25 µM and 50 µM resveratrol and cell-cycle checkpoint protein cyclin G2 (CCNG2) and proapoptotic protein PUMA (BBC3) were increased by 50 µM resveratrol. * indicates p<0.05 compared to 0 µM resveratrol treatment.

Fig 4. Knockdown of FOXO3 attenuates the suppression of H₂O₂-induced senescence by resveratrol.

(a) Keratinocytes were cultured in 12-well plates and at 30% confluence, were infected with lentivirus expressing shRNA to non-targeting control (shNegative) and FOXO3 (shFOXO3). Three days later, cells were treated with H₂O₂ ± resveratrol, further cultured for an additional 64 hrs. and then fixed with paraformaldehyde for SA-Gal staining, an indicator of senescence. Infection of shFOXO3 lentivirus suppressed FOXO3 expression by 70% but not FOXO1 expression. (b,c) Resveratrol suppressed H₂O₂-induced SA-Gal positive staining in shNegative infected cells, but this effect was largely diminished in shFOXO3 infected cells. * indicates p<0.05 compared to control treatment (n = 6).

Fig 5. Effects of H₂O₂, insulin and resveratrol on ³H -thymidine incorporation in human primary keratinocytes.

Keratinocytes were grown in 12-well plates until confluency reached 40–60%, at which time they were incubated in growth factor-free basal media overnight. After the indicated treatment, media was changed to basal or basal media containing 1 nM insulin for 16 hrs. ³H -thymidine was added for the last 3 hrs. and cells were collected to measure ³H using a scintillation counter. (a) As observed previously, incubation with 50 µM H₂O₂ for 2 hrs, severely inhibited cell growth and diminished ³H—thymidine incorporation, an indicator of cellular proliferation, by about 50%. Lower concentrations of H₂O₂ had milder effects. * p<0.05 and ** p<0.01 compared to 0 µM H₂O₂ treatment (n = 4). (b,c) Cells were incubated with or without 25 µM resveratrol for 30 minutes prior to treatment with 20 µM H₂O₂ for 2 hrs. Media was then changed to a basal medium either without or with 1 nM insulin. Without insulin, resveratrol was not able to prevent H₂O₂-mediated suppression of ³H -thymidine incorporation (b)_. In the presence of insulin, resveratrol treatment significantly increased ³H -thymidine incorporation by 30% (p<0.001 compared to H₂O₂ treatment (c). For (b)-(c), numbers inside parenthesis denote n.

Fig 6. Effects of AMPK knockdown, AMPK inhibitor Compound C, SIRT1 inhibitor Ex527, and SIRT1 knockdown on ³H -thymidine incorporation.

(a) Keratinocytes were infected with lentivirus vector expressing non-targeting control shRNA (shNegative) or AMPK alpha1 (shAMPKa1). Cells were then treated with 20 µM H₂O₂, 1 nM insulin and resveratrol (Res) as described in Fig. 5. Knockdown of AMPK abrogated the effects of resveratrol (which are shown in Fig. 5c). (b) Non-infected cells were treated with H₂O₂ and resveratrol as in 6a, but incubated with 1 µg/ml Compound C (CC), an AMPK inhibitor, 30 min. prior to the addition of resveratrol. Compound C inhibited the effects of resveratrol on ³H -thymidine incorporation. (c) Keratinocytes were infected with lentivirus vector expressing non-targeting control shRNA (shNegative) or SIRT1 (shSIRT1) that reduced total SIRT1 by about 70%. Knocking down SIRT1 had no effect on resveratrol-induced changes in ³H -thymidine incorporation. (d) Non-infected cells were incubated with H₂O₂, insulin and resveratrol as in 6a, and 10 µM Ex527 was added 10 min. prior to the addition of resveratrol. Ex527 treatment did not alter ³H -thymidine incorporation suggesting that inhibition of SIRT1 did not modulate the effect of resveratrol. For (a) and (c), numbers inside parenthesis denote n.

Fig 7. Knockdown of FOXO3 suppresses resveratrol’s effects on ³H -thymidine incorporation.

Cells were infected with the same lentivirus targeting FOXO3 as described in Fig. 4. Compared to infection with shNegative, infection with shFOXO3 resulted in a 50% reduction in ³H-thymidine incorporation. While resveratrol increased ³H-thymidine incorporation in shNegative infected cells, the effects were diminished in shFOXO3 infected cells.

Fig 8. AMPK activation is decreased with aging in human skin.

Discarded skin was obtained from patients undergoing a surgical procedure. (a) The samples were processed for Western blot to evaluate AMPK activation levels using p-T172 AMPK and total AMPK (arrow) antibodies. GAPDH was used as a loading control. (b) The ratios of p-T172 to total AMPK were significantly lower in the samples from individuals over 50 years old (n = 6) compared to samples from individuals less than 20 years old (n = 6).