Increased AMP:ATP ratio and AMP-activated protein kinase activity during cellular senescence linked to reduced HuR function.
Cytoplasmic export of the RNA-binding protein HuR, a process that critically regulates its function, was recently shown to be inhibited by the AMP-activated protein kinase (AMPK). In the present investigation, treatment of human fibroblasts with AMPK activators such as 5-amino-imidazole-4-carboxamide riboside, antimycin A, and sodium azide inhibited cell growth and lowered the expression of proliferative genes. As anticipated, AMPK activation also decreased both the cytoplasmic HuR levels and the association of HuR with target radiolabeled transcripts encoding such proliferative genes. HuR function was previously shown to be implicated in the maintenance of a "young cell" phenotype in models of replicative cellular senescence. We therefore postulated that AMPK activation in human fibroblasts might contribute to the implementation of the senescence phenotype through mechanisms that included a reduction in HuR cytoplasmic presence. Indeed, AMP:ATP ratios were 2-3-fold higher in senescent fibroblasts compared with young fibroblasts. Accordingly, in vitro senescence was accompanied by a marked elevation in AMPK activity. Evidence that increased AMPK activity directly contributed to the implementation of the senescent phenotype was obtained through two experimental approaches. First, use of AMPK activators triggered senescence characteristics in fibroblasts, such as the acquisition of senescence-associated beta-galactosidase (beta-gal) activity and increased p16INK4a expression. Second, infection of cells with an adenoviral vector that expresses active AMPK increased senescence-associated beta-gal activity, whereas infection with an adenovirus that expresses dominant-negative AMPK decreased senescence-associated beta-gal activity. Together, our results indicate that AMPK activation can cause premature fibroblast senescence through mechanisms that likely involve reduced HuR function.
Laboratory of Cellular and Molecular Biology, NIA Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224, USA.
Wengong
W
Wang
Xiaoling
X
Yang
Isabel
I
López de Silanes
David
D
Carling
Myriam
M
Gorospe
D055372:AMP-Activated Protein Kinases; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000954:Antigens, Surface; D000968:Antimycin A / Q000494:pharmacology; D001483:Base Sequence; D002460:Cell Line; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology*; D019925:Cyclin A / Q000235:genetics; D019926:Cyclin B / Q000235:genetics; D056744:Cyclin B1; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D016762:Genes, fos; D005822:Genetic Vectors; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D000954:Antigens, Surface; C534699:CCNB1 protein, human; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D019941:Cyclin-Dependent Kinase Inhibitor p16; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D000249:Adenosine Monophosphate; D000968:Antimycin A; D000255:Adenosine Triphosphate; D019810:Sodium Azide; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D001616:beta-Galactosidase; C031143:AICA ribonucleotide