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.
Highlights
Discrete mechanisms that drive healthy aging, rather than disease progression, are largely unknown
These findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by the AMPactivated protein kinase (AMPK)-Forkhead box O3 (FOXO3) pathway and in some situations, but not all, by sirtuin 1 (SIRT1)
Overexpression of SIRT1 did not change cell shape but surprisingly, staining for SA-Gal and SIRT1 revealed that SIRT1 protein was localized within the cytosol of senesced cells (Fig. 1e red arrow), whereas it was localized in the nucleus of non-senesced cells (Fig. 1e gray arrow)
Summary
Discrete mechanisms that drive healthy aging, rather than disease progression, are largely unknown. Recent research has defined aging as a phenotype caused by epigenetic changes, e.g. DNA methylation, histone modification and aberrant miRNA expression, that increase chronologically in response to intrinsic and extrinsic stimuli [1,2]. Modulating these changes may slow or reverse the appearance of aging phenotypes. Histological analyses of human skin have shown that a younger biological age is associated both with fewer senescent cells and a higher proliferative index [3]. P16INK4A, an inhibitor of cyclin-dependent kinases and a key regulator of premature senescence, is both a biomarker for human epidermal aging in vivo and an inverse correlate of familial longevity [3,6,7]
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