Absence of AMPKα2 accelerates cellular senescence via p16 induction in mouse embryonic fibroblasts

Emerging evidence suggests that activation of adenosine monophosphate-activated protein kinase (AMPK), an energy gauge and redox sensor, delays aging process. However, the molecular mechanisms by which AMPK alpha isoform regulates cellular senescence remain largely unknown. The aim of this study was to determine if AMPK alpha deletion contributes to the accelerated cell senescence by inducing p16(INK4A) (p16) expression thereby arresting cell cycle. The markers of cellular senescence, cell cycle proteins, and reactive oxygen species (ROS) were monitored in cultured mouse embryonic fibroblasts (MEFs) isolated from wild type (WT, C57BL/6J), AMPK alpha 1, or AMPK alpha 2 homozygous deficient (AMPK alpha(-/-), AMPK alpha 2(-/-)) mice by Western blot and cellular immunofluorescence staining, as well as immunohistochemistry (IHC) in skin tissue of young and aged mice. Deletion of AMPK alpha 2, the minor isoform of AMPKa, but not AMPK alpha 1 in high-passaged MEFs led to spontaneous cell senescence demonstrated by accumulation of senescence-associated-beta-galactosidase (SA-beta-gal) staining and foci formation of heterochromatin protein 1 homolog gamma (HP1 gamma). It was shown here that AMPK alpha 2 deletion upregulates cyclin-dependent kinase (CDK) inhibitor, p16, which arrests cell cycle. Furthermore, AMPK alpha 2 null cells exhibited elevated ROS production. Interestingly, knockdown of HMG box-containing protein I (HBP1) partially blocked the cellular senescence of AMPK alpha 2-deleted MEFs via the reduction of p16. Finally, dermal cells senescence, including fibroblasts senescence evidenced by the staining of p16, HBP1, and Ki-67, in the skin of aged AMPK alpha 2(-/-) mice was enhanced when compared with that in wild type mice. Taken together, our results suggest that AMPK alpha 2 isoform plays a fundamental role in anti-oxidant stress and anti-senescence. (C) 2015 Elsevier Ltd. All rights reserved.