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

Abstract

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 AMPKα isoform regulates cellular senescence remain largely unknown. The aim of this study was to determine if AMPKα deletion contributes to the accelerated cell senescence by inducing p16INK4A (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), AMPKα1, or AMPKα2 homozygous deficient (AMPKα1−/−, AMPKα2−/−) mice by Western blot and cellular immunofluorescence staining, as well as immunohistochemistry (IHC) in skin tissue of young and aged mice. Deletion of AMPKα2, the minor isoform of AMPKα, but not AMPKα1 in high-passaged MEFs led to spontaneous cell senescence demonstrated by accumulation of senescence-associated-β-galactosidase (SA-β-gal) staining and foci formation of heterochromatin protein 1 homolog gamma (HP1γ). It was shown here that AMPKα2 deletion upregulates cyclin-dependent kinase (CDK) inhibitor, p16, which arrests cell cycle. Furthermore, AMPKα2 null cells exhibited elevated ROS production. Interestingly, knockdown of HMG box-containing protein 1 (HBP1) partially blocked the cellular senescence of AMPKα2-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 AMPKα2−/− mice was enhanced when compared with that in wild type mice. Taken together, our results suggest that AMPKα2 isoform plays a fundamental role in anti-oxidant stress and anti-senescence.

AMP-activated protein kinase reduces inflammatory responses and cellular senescence in pulmonary emphysema

“…It has been labeled as an anti-aging and anti-inflammatory molecule [5, 6]. Our preliminary findings show the reduction of AMPKα1 phosporylation at Thr-172 in lungs of COPD pateints as compared to nonsmokers (data not shown).…”

Activation of AMP‐activated protein kinase by metformin ablates angiotensin II‐induced endoplasmic reticulum stress and hypertension in mice in vivo

“…Sections were successively incubated in endogenous peroxidase and alkaline phosphatase block buffer (Dako, Glostrup, Denmark), protein block buffer and primary antibodies, which were incubated with the sections overnight at 4°C. After rinsing in wash buffer, sections were incubated with labelled polymer–horseradish peroxidase anti‐mouse or anti‐rabbit antibodies and 3,3'‐diaminobenzidine (DAB) chromogen as described previously (Ding et al , ). After a final wash, the sections were counterstained with haematoxylin.…”

AMPKα1 deletion in fibroblasts promotes tumorigenesis in athymic nude mice by p52-mediated elevation of erythropoietin and CDK2

“…Oncogene H-RasV12-transformed WT MEFs dramatically develops tumors in nude mice [ 5 ], which is contrast to the results reported by Phoenix et al [ 8 ]. H-RasV12-transformed AMPKα1α2 double KO MEFs fails to form tumors in mice [ 5 ], which may be due to the increased cellular senescence in AMPKα2-KO MEFs [ 38 ]. Here, we showed that AMPKα1 deletion stimulated anchorage-independent MEF growth, which is consistent with a previous report that AMPKα1 deletion triggers MEF hyperproliferation and DNA damage [ 39 ].…”