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.