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 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), 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.

Keywords: AMPKα2; Cellular senescence; HBP1; Reactive oxygen species; p16.

Fig. 1

Deficiency of AMPKα2, but not AMPKα1, leads to enhanced cellular senescence in MEFs. (A) Wild type (WT), AMPKα1^−/− and AMPKα2^−/− MEFs (two independent cell lines for each) were seeded with 0.5 × 10⁵ cells in 6-well plates. Cell number counting was taken at different time point. n=8, *p< 0.05 vs WT, †p< 0.01 vs WT. Representative Western blot of AMPKα1 and AMPKα2 was shown as an inset of bar graph. (B) Profile of cellular proliferation-associated proteins, including CDK2, Cyclin B1, and PCNA. n=6, *p< 0.01 vs WT, †p< 0.01 vs WT. (C) Representative images showing SA-β-galactosidase (SA-β-gal) activity in WT, AMPKα1^−/−, and AMPKα2^−/− MEFs. Scale bar = 50 μm. (D) Percentage of SA-β-gal-positive cells in cultured MEFs. n=10, *p< 0.05 vs WT. (E) HP1γ foci formation was increased in AMPKα2^−/− MEFs. Representative images showing HP1γ foci formation (red color), marking senescence. Scale bar = 2.5 μm. (F) Quantitative analysis of HP1γ foci per nucleus. n=20, *p< 0.01 vs WT. (G) Representative images showing staining of anti-H3k9me3. Scale bar = 20 μm.

Fig. 2

Transcriptional upregulation of p16 in AMPKα2^−/− MEFs. (A) (Upper) AMPKα2 deletion significantly upregulated p16. (Bottom) Quantification of Western blot data. n=8, *p< 0.001 vs WT. (B) (Upper) AMPKα2 deletion significantly down-regulated E2F1. (Bottom) Quantification of Western blot data. n=6, *p< 0.001 vs WT. (C) Proteasome inhibitor, MG132 (20 μM, 8 h) did not further increase p16 in AMPKα2^−/− MEFs. n=4, *p< 0.001 vs WT. (D) Quantitative RT-PCR analysis of p16 expression in MEFs. β-actin was used as endogenous loading control. Values are mean ± SEM of four independent experiments, *p< 0.001 vs WT.

Fig. 3

HBP1 is responsible for p16 induction in AMPKa2^−/− MEFs. (A) (Upper) AMPKα2 deletion significantly upregulated HBP1. (Bottom) Quantification of Western blot data. n=6, *p< 0.001 vs WT. (B) Neither AMPKa2 nor AMPKa1 deletion altered Bmi-1 and Ets1 protein levels. (C) (Upper) HBP1 knockdown by siRNA reversed p16 elevation in AMPKa2^−/− MEFs. (Bottom) Quantification of Western blot data. n=4, *p< 0.01 vs WT/Control siRNA, †p< 0.01 vs a2^−/−/Control siRNA. (D) HBP1 siRNA significantly downregulated p16 mRNA level in AMPKa2^−/− MEFs. n=6, *p< 0.01 vs WT/Control siRNA, †p< 0.01 vs a2^−/−/Control siRNA.

Fig. 4

Increased ROS in AMPKα2^−/− MEFs contributes to p16 upregulation. (A) AMPKα2 deletion elevated superoxide anion (O^{2 •−}) production. n=10, *p< 0.05 vs WT. (B) Anti-oxidants Tempol and Mito-Tempo inhibited p16 elevation in AMPKα2^−/− MEFs. (C) SOD2 and Catalase dampened p16 induction in AMPKα2^−/− MEFs. (D) Representative images indicated that Mito-Tempo blunted cellular senescence of AMPKα2^−/− MEFs demonstrated by SA-β-gal staining. (E) Percentage of SA-β-gal-positive cells in Mito-Tempo-treated MEFs. n=10, *p< 0.001 vs WT/Control, †p< 0.01 vs a2^−/−/Control. Scale bar = 50 μm.

Fig. 5

HBP1 is responsible for the cellular senescence in AMPKα2^−/− MEFs. (A) Representative images indicated that HBP1 knockdown by siRNA blunted cell senescence in AMPKα2^−/− MEFs. Scale bar = 50 μm. (B) Quantification of SA-β-gal staining. n=6, *p< 0.001 vs WT/Control siRNA, †p< 0.01 vs AMPKa2^−/−/Control siRNA. (C) Representative Western blot of HBP1.

Fig. 6

Increased senescent cells in the skin of AMPKα2^−/− mice compared with wild type (WT) mice. (A) Ki-67 staining (brown) was decreased in the skin of 24-month-old AMPKα2^−/− mice. (B) HBP1 (brown color) was increased in derma fibroblast and skin of 24-month-old AMPKα2^−/− mice. (C) p16 (brown staining) was increased in derma fibroblast and skin of 24-month-old AMPKα2^−/− mice. (D) Staining of fibroblast marker in skin of young and old wild WT and AMPKα2^−/− mice. All sections were counterstained with hematoxylin to detect nuclei (blue color). Arrows indicate the brown-stained cells. Scale bar = 50 μm. (E) Quantification of Ki-67-positive cells in dermis. n=10 in each group, # p<0.05 vs WT/5-month-old, * p<0.01 vs WT/5-month-old, †p<0.001 vs WT/24-month-old. (F) Quantification of HBP1-positive cells in dermis. n=10, # p<0.01 vs WT/5-month-old, † p<0.05 vs WT/5-month-old, * p<0.01 vs WT/24-month-old. (G) Quantification of p16-positive cells in dermis. n=10, # p<0.01 vs WT/5-month-old, † p<0.05 vs WT/5-month-old, *p<0.01 vs WT/24-month-old.

Fig. 7

Proposed mechanism underlying AMPKα2 deletion-stimulated cellular senescence. Absence of AMPKα2 will increase ROS production, which leads to p16 upregulation mediated by HBP1.