Acute activation of AMP-activated protein kinase prevents h 2o 2-induced premature senescence in primary human keratinocytes

Abstract

We investigated the effects of AMPK on H 2O 2-induced premature senescence in primary human keratinocytes. Incubation with 50 μM H 2O 2 for 2 h resulted in premature senescence with characteristic increases in senescence-associated ß-galactosidase (SA-gal) staining 3 days later and no changes in AMPK or p38 MAPK activity. The increase in SA-gal staining was preceded by increases in both p53 phosphorylation (S15) (1 h) and transactivation (6 h) and the abundance of the cyclin inhibitor p21 CIP1 (16 h). Incubation with AICAR or resveratrol, both of which activated AMPK, prevented the H 2O 2-induced increases in both SA-Gal staining and p21 abundance. In addition, AICAR diminished the increase in p53 transactivation. The decreases in SA-Gal expression induced by resveratrol and AICAR were prevented by the pharmacological AMPK inhibitor Compound C, expression of a DN-AMPK or AMPK knock-down with shRNA. Likewise, both knockdown of AMPK and expression of DN-AMPK were sufficient to induce senescence, even in the absence of exogenous H 2O 2. As reported by others, we found that AMPK activation by itself increased p53 phosphorylation at S15 in embryonic fibroblasts (MEF), whereas under the same conditions it decreased p53 phosphorylation in the keratinocytes, human aortic endothelial cells, and human HT1080 fibrosarcoma cells. In conclusion, the results indicate that H 2O 2 at low concentrations causes premature senescence in human keratinocytes by activating p53-p21 CIP1 signaling and that these effects can be prevented by acute AMPK activation and enhanced by AMPK downregulation. They also suggest that this action of AMPK may be cell or context-specific. © 2012 Ido et al.