Increased AMP:ATP Ratio and AMP-activated Protein Kinase Activity during Cellular Senescence Linked to Reduced HuR Function*
Wengong Wang
,
Xiaoling Yang
,
Isabel López de Silanes
,
David Carling
¶ and
Myriam Gorospe
||
From the
Laboratory of Cellular and Molecular
Biology, NIA Intramural Research Program, National Institutes of Health,
Baltimore, Maryland 21224 and the
Medical
Research Council Clinical Sciences Centre, Imperial College School of
Medicine, London W12 0NN, United Kingdom
Received for publication, January 10, 2003
, and in revised form, April 14, 2003.
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ABSTRACT
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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 23-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
-galactosidase (
-gal)
activity and increased p16INK4a expression. Second, infection of
cells with an adenoviral vector that expresses active AMPK increased
senescence-associated
-gal activity, whereas infection with an
adenovirus that expresses dominant-negative AMPK decreased
senescence-associated
-gal activity. Together, our results indicate that
AMPK activation can cause premature fibroblast senescence through mechanisms
that likely involve reduced HuR function.
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INTRODUCTION
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The AMP-activated protein kinase
(AMPK),1 an enzyme
that is believed to serve as a metabolic sensor or a "low fuel warning
system" of the cell (1),
was discovered two decades ago. However, details of its molecular composition,
regulation, and function are only now beginning to be elucidated.
Structurally, mammalian AMPK is a heterotrimer of three subunits: one
catalytic (
) and two regulatory (
and
) subunits
(2,
3). AMPK activity is
ubiquitous, although different isoforms of AMPK subunits display
tissue-specific distribution, as well as preferential subcellular
localization. AMPK is activated directly by elevations in AMP and inhibited by
high concentrations of ATP. Conditions that elevate the AMP:ATP ratio in
cells, such as growth factor depletion, hypoglycemia, ischemia in heart
muscle, exercise in skeletal muscle, as well as treatment with arsenite,
azide, oxidative agents, and the pharmacological agent AICAR (which mimics the
effect of AMP), can cause activation of AMPK
(4,
5). In turn, AMPK inhibits
biosynthetic pathways, thus conserving energy while it activates catabolic
pathways, thereby generating more ATP (reviewed in Ref.
4).
AMPK has been shown to influence gene expression in a variety of ways
(1). AMPK-mediated
transcriptional regulation has been demonstrated in yeast, where the AMPK
homologue SNF1 is a pivotal regulator of glucose-related gene expression at
times of low fuel availability
(4,
6). The influence of AMPK on
gene transcription in mammalian cells has also been documented. Recently, AMPK
was shown to phosphorylate the transcriptional coactivator p300, thereby
modulating its ability to interact with many nuclear receptors
(7). AMPK has also been
proposed to influence the levels of transcription factor forkhead FKHR
(8) and to alter the
transcription of various genes, including GLUT4
(9). In addition, AMPK was
recently reported to have an inhibitory effect on protein synthesis,
associated with decreased activation of the mammalian target of rapamycin
signal transduction pathway and its effectors
(10). Finally, AMPK has been
shown to influence mRNA turnover by inhibiting the cytoplasmic export of the
RNA-binding protein HuR (11).
Because HuR is predominantly (>90%) localized in the nucleus of
unstimulated cells, it has been proposed that the mRNA-stabilizing influence
of HuR requires its translocation to the cytoplasm
(1217).
The AMPK-imposed inhibition of HuR transport to the cytoplasm blocks the
ability of HuR to stabilize and enhance the expression of target mRNAs,
including those that encode vascular endothelial growth factor, p21, cyclin A,
cyclin B1, c-Fos, tumor necrosis factor-
, and Glut-1
(13,
1720).
These HuR target mRNAs share the presence of AU-rich elements in their
3'-untranslated region (UTR), which modulate their half-life
(21,
22). AU-rich elements in many
labile mRNAs are also the targets of additional RNA-binding proteins
(20,
2329).
Among the cellular events that are influenced by HuR and its target mRNAs
is the process of in vitro senescence. Using two different human
fibroblast models of in vitro senescence, we recently reported the
influence of the RNA-binding protein HuR in regulating the expression of
several genes whose expression decreases during senescence
(30). We demonstrated that HuR
levels, HuR binding to target mRNAs, and the half-lives of such mRNAs were
lower in senescent cells. Importantly, overexpression of HuR in senescent
cells restored a "younger" phenotype, whereas a reduction in HuR
expression accentuated the senescent phenotype
(30). These studies
highlighted a critical role of mRNA turnover during the process of replicative
senescence and specifically implicated HuR in the regulation of such events.
In the present study, we set out to test the hypothesis that AMPK activity
might be elevated with senescence. Upon discovering that indeed AMP:ATP ratios
are higher in senescent cells, we found that AMPK activity was accordingly
elevated in senescent cells. Given the influence of AMPK on HuR function, we
further assessed whether differences in AMPK function could modulate the
process of in vitro senescence. Activation of AMPK led to an
enhancement of the senescent phenotype associated with reduced cytoplasmic HuR
levels, HuR binding to target mRNAs, and target mRNA half-life. By contrast,
reduced AMPK function brought about a young, proliferative phenotype. Our
findings strongly support a role for AMPK in the implementation of the
senescence phenotype and suggest that AMPK-regulated HuR may be a critical
factor in this process.
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MATERIALS AND METHODS
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Cell Culture, Transfection, Infection, and Assessment of
Senescence-associated
-Galactosidase ActivityHuman
IDH4 fibroblasts were generously provided by J. W. Shay
(31). Early passage
(
1520 population doublings (pdl)) and late passage (5053
pdl) human diploid IMR-90 fibroblasts as well as early passage (2530
pdl) and late passage (5458 pdl) human diploid WI-38 fibroblasts were
from Coriell Cell Repositories (Coriell Institute, Camden, NJ). All of the
fibroblasts were cultured in Dulbecco's modified essential medium (Invitrogen)
supplemented with 10% fetal bovine serum and antibiotics. Unless otherwise
indicated, IDH4 cell culture medium was further supplemented with 1
µM dexamethasone (Dex) for constitutive expression of SV40 large
T antigen to suppress senescence and stimulate proliferation
(31). To induce senescence of
IDH4 cells, Dex was removed from the culture medium, regular serum was
replaced with charcoal-stripped serum, and the cells were assessed at
different times thereafter. Actinomycin D, dexamethasone, AICAR, 5'-AMP,
antimycin A, and sodium azide were from Sigma.
Adenoviruses expressing either the control gene GFP (AdGFP), a
dominant-negative isoform of the
1 subunit of AMPK (Ad(DN)AMPK), or a
constitutively active isoform of the AMPK
1 subunit (Ad-(CA)AMPK)
(32) were amplified and
titered in 293 cells using standard methodologies. The infections were carried
out in serum-free Dulbecco's modified Eagle's medium for 4 h. Infection
efficiency was determined by infection with AdGFP at various plaque-forming
units (PFU)/cell, and assessment of the percentage of GFP-expressing cells 48
h later. For >90% infection of IDH4 and IMR-90 cells, 300 PFU/cell was
required.
Assessment of senescence-associated
-galactosidase (
-gal)
activity was carried out as previously described
(30,
33). Briefly, the cells were
seeded in 30-mm dishes, cultured in medium without Dex for different lengths
of time, and fixed with a 3% formaldehyde solution. The cells were then washed
and incubated with senescence-associated
-gal staining solution (1 mg/ml
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-gal), 40
mM citric acid/sodium phosphate buffer, pH 6.0, 5 mM
ferrocyanide, 5 mM ferricyanide, 150 mM NaCl, and 2
mM MgCl2) for 1218 h to visualize
senescence-associated
-gal activity
(33).
Northern and Western Blot Analysis and Subcellular
Fractionation Northern blot analysis was carried out as previously
described (34). An oligomer
complementary to 18 S (34) was
3' end-labeled using terminal transferase enzyme, whereas PCR-generated
fragments of cyclin A, cyclin B1, and
-actin cDNAs were random
primer-labeled using Klenow enzyme
(30); all of the labeling
reactions were carried out in the presence of [
-32P]dATP.
Signals on Northern blots were visualized and quantitated with a
PhosphorImager (Molecular Dynamics, Sunnyvale, Ca).
For Western blotting, whole cell (20 µg) or cytoplasmic (40 µg)
lysates were prepared as previously described
(17) and were
size-fractionated by SDS-polyacrylamide gel electrophoresis and transferred
onto polyvinylidene difluoride membranes. Monoclonal antibodies were used to
detect HuR (Molecular Probes, Eugene, OR), histone deacetylase 1, and
-actin (Santa Cruz Biotechnology, Santa Cruz, CA). Following secondary
antibody incubations, the signals were visualized by enhanced
chemiluminescence, quantitated by densitometry, and normalized against a
loading control (
-Actin for cytoplasmic and whole cell samples, histone
deacetylase 1 for nuclear protein samples). The quantitative values are
presented as the percentage of untreated cells or the percentage of
Ad(GFP).
Preparation of Radiolabeled TranscriptsFor in
vitro synthesis of radiolabeled transcripts, RNA from IDH4 cells was
reverse transcribed, and the cDNAs generated were used as templates in PCRs to
amplify the 3'-UTRs of cyclin A, cyclin B1, cyclin E, and c-Fos cDNAs,
as described (17,
35). All 5'
oligonucleotides contained the T7 RNA polymerase promoter sequence
CCAAGCTTCTAATACGACTCACTATAGGGAGA(T7). To prepare the cyclin A
3'-UTR template, oligonucleotides (T7)CCAGAGACACTAAATCTGTAAC
and GGTAACAAATTTCTGGTTTATTTC (region 14992718) were used. To prepare
the cyclin B1 3'-UTR template, oligonucleotides
(T7)GTCAAGAACAAGTATGCCA and CTGAAGTGGGAGCGGAAAAG (region
13691702) were used. To prepare the cyclin E 3'-UTR template,
oligonucleotides (T7)CACAGAGCGGTAAGAAGCAG and
GGATAGATATAGCAGCACTTAC (region 11691714) were used. PCR fragments
served as templates for the synthesis of corresponding RNAs
(36), which were used at a
specific activity of 100,000 cpm/µl (210 fmol/µl).
RNA Electrophoretic Mobility Shift Assay (REMSA) and Supershift
AssayReaction mixtures (10 µl) containing 1 µg of tRNA,
210 fmol of RNA, and 5 µg of protein in reaction buffer (15
mM Hepes, pH 7.9, 10 mM KCl, 10% glycerol, 0.2
mM dithiothreitol, 5 mM MgCl2) were incubated
for 30 min at 25 °C and digested with RNase T1 (500 units/reaction) for 15
min at 37 °C. The complexes were resolved by electrophoresis through
native gels (7% acrylamide in 0.25x Tris borate EDTA buffer). The gels
were subsequently dried, and radioactivity was visualized using a
PhosphorImager. For supershift analysis, 1 µg of antibody was incubated
with lysates for 1 h on ice before the addition of radiolabeled RNA; all of
the subsequent steps were as described for REMSA. For supershifts, anti-HuR
(Molecular Probes) and anti-p38 (Pharmingen, San Diego, CA) antibodies were
used.
AMPK Assay and Determination of AMP:ATP RatiosAMPK was
assayed as described (37).
Briefly, AMPK was immunoprecipitated from 5 µg of cell lysate using 1 µg
of anti-
1 and 1 µg of anti-
2 polyclonal antibodies in AMPK
immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150
mM NaCl, 50 mM NaF, 5 mM sodium
pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol, 0.1 mM benzamidine, 0.1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml soybean trypsin inhibitor) for 2 h
at 4 °C. Immunocomplexes were washed with immunoprecipitation buffer plus
1 M NaCl and then with a buffer containing 62.5 mM
Hepes, pH 7.0, 62.5 mM NaCl, 62.5 mM NaF, 6.25
mM sodium pyrophosphate, 1.25 mM EDTA, 1.25
mM EGTA, 1 mM dithiothreitol, 1 mM
benzamidine, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml soybean
trypsin inhibitor. AMPK activity in immunocomplexes was determined by
phosphorylation of peptide HMRSAMSGLHLVKRR (SAMS)
(37) in reaction buffer (50
mM Hepes, pH 7.4, 1 mM dithiothreitol, 0.02% Brij-35,
0.25 mM SAMS, 0.25 mM AMP, 5 mM
MgCl2, 10 µCi of [
-32P]ATP) for 10 min at 30
°C. The assay mixtures were spotted onto P81 filter paper and rinsed in 1%
(v/v) phosphoric acid with gentle stirring to remove free ATP. Phosphorylated
substrate was measured by scintillation counting. Measurement of AMP and ATP
was carried out as previously described
(38).
ImmunofluorescenceProliferating IDH4 cells were seeded on
coverslips and either left untreated or treated with AMPK activators. At the
end of the treatment period, HuR was detected by immunofluorescence as
previously described (11)
using anti-HuR (Santa Cruz Biotechnology). The nuclei were visualized using
Hoechst 33342 (Molecular Probes). The signals were detected using an Axiovert
200M microscope (Zeiss; 63x lens) using separate channels for the
analysis of phase contrast images, red fluorescence (HuR), and blue
fluorescence (Hoechst). Representative photographs from three independent
experiments are shown.
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RESULTS
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Treatment with AMPK Activators Causes Growth Arrest and Decreased
Expression of Proliferative Genes in Human IDH4 FibroblastsIn the
IDH4 human fibroblast cell model developed by Shay and co-workers
(31), constitutive, Dex-driven
SV40 large T antigen expression allows IDH4 cells to suppress senescence and
to continue to proliferate as young cells. Treatment of proliferating IDH4
cells with the AMPK activators AICAR, antimycin A, and sodium azide caused
up-regulation of AMPK activity (Fig.
1A), cessation of cell growth
(Fig. 1B), and a
marked reduction in the expression levels of proliferative proteins such as
cyclin A and cyclin B1 (Fig.
1C). These reductions in protein abundance were further
assessed by monitoring mRNA levels. As seen in
Fig. 2A, treatment
with either AICAR, antimycin A, or sodium azide led to a corresponding
reduction in the levels of mRNAs encoding cyclin A and cyclin B1. Given our
earlier findings that cyclin A and cyclin B1 mRNA turnover changed according
to the proliferative status of the cell
(35), we sought to determine
whether such gene expression differences were influenced by changes in the
stability of the corresponding mRNAs. We used an actinomycin
D-based approach to assess the relative mRNA half-lives following
treatment with AMPK activators. As shown in
Fig. 2B, IDH4 cells
showed significant differences in the half-lives of mRNAs encoding cyclin A
and cyclin B1. For these mRNAs, treatment with actinomycin D resulted in
faster transcript loss in the populations treated with AMPK activators,
revealing that their stability was lowered by the treatments. Such differences
in mRNA stability were specifically seen for cyclin A mRNA and cyclin B1 mRNA,
because the stability of other mRNAs studied, such as that encoding
-actin, was unchanged (Fig.
2B).

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FIG. 1. AMPK activity in IDH4 cells treated with AMPK activators AICAR,
antimycin A, and sodium azide. A, actively proliferating IDH4
cells were treated with either 2 mM AICAR, 5 µM
antimycin A, or 2 mM sodium azide for 5 h, and AMPK activity was
determined as explained under "Materials and Methods." B,
50,000 IDH4 cells/well were treated with either AICAR, antimycin A, or sodium
azide at the doses indicated above, and the cell numbers were assessed every
12 h using a hemacytometer. The data represent the means ± S.E.
C, Western blot analysis to monitor the expression of cyclin A and
cyclin B1 in whole cell lysates (20 µg) prepared from IDH4 cells that were
treated as described for A.
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FIG. 2. Effect of AMPK activators on the expression and half-life of mRNAs
encoding cyclin A and cyclin B1. A, Northern blot analysis to
assess the abundance of mRNAs encoding cyclin B1, cyclin A, and -actin
in IDH4 cells following treatment with 2 mM AICAR, 5
µM antimycin A, or 2 mM sodium azide for 5 h.
B, stabilities of mRNAs encoding cyclin A, cyclin B1, and
-actin in IDH4 cells that were cultured in the presence of AMPK
activators as indicated for A. Half-lives were assessed after adding
2 µg/ml actinomycin D during the continuous presence of the AMPK
activators, preparing RNA at the times indicated, measuring cyclin A, cyclin
B1, and -actin Northern blot signals, normalizing them to 18 S rRNA, and
plotting them on a logarithmic scale. Dashed horizontal lines, 50% of
untreated. The data represent the means ± S.E. of three independent
experiments.
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Treatment of IDH4 Cells with AMPK Activators Reduces Cytoplasmic HuR
Levels and Decreases Binding of HuR to Cyclin A, Cyclin B1, and c-Fos
3'-UTRsCyclin A and cyclin B1 are encoded by mRNAs
that are targets of HuR binding in proliferating cells
(35). To assess whether the
AMPK-triggered reduction in expression of cyclin A and cyclin B1 might be
linked to the ability of HuR to bind to the 3'-UTRs of these
transcripts, REMSA were carried out. IDH4 cells were either left untreated or
treated with AMPK activators, whereupon cytoplasmic lysates were prepared and
incubated with radiolabeled RNAs encompassing the 3'-UTRs of the cyclin
A and cyclin B1 mRNAs, as described
(30). REMSA was also carried
out using transcripts corresponding to the 3'-UTR of c-Fos, a
proliferative gene whose mRNA was previously reported to be a target of HuR
(30)
(Fig. 3). Remarkably, the
proteins present in cytoplasmic lysates prepared from untreated IDH4 cells
revealed much more extensive binding to radiolabeled transcripts than did
proteins present in cytoplasmic lysates from IDH4 cells treated with AMPK
activators. Evidence that HuR was part of the REMSA complexes and that HuR
abundance was indeed greater in the untreated populations was revealed through
use of an anti-HuR antibody to supershift the protein-RNA associations.
HuR-containing supershifted complexes (Fig.
3, arrowheads) were most abundant when lysates from
untreated populations were used and were markedly reduced following treatment
with AMPK activators. The specificity of the assay was tested using antibodies
directed to control proteins (such as p38MAPK, which lacks RNA
binding ability), which did not produce supershifts
(Fig. 3). For each transcript
and treatment group tested, nuclear lysates were assayed similarly and
revealed no change in binding patterns or signal intensities after treatment
with AMPK activators (not shown), in agreement with earlier findings
(11). Additional control
REMSAs using non-HuR target transcripts such as those corresponding to each
coding region (previously shown
(35) and therefore not shown
here) and that encompassing the cyclin E 3'-UTR
(Fig. 3) served to further
assess the specificity of these associations. Taken together, the reduced
association of HuR with cyclin A and B1 3'-UTR transcripts under
conditions that cause destablization of the encoded mRNAs is in keeping with
the reported ability of HuR to stabilize and enhance the expression of such
proliferative genes (30,
35).
Given our earlier observations in RKO colorectal carcinoma cells, where the
cytoplasmic localization of HuR was regulated by AMPK
(11), we set out to assess the
influence of AMPK activators on the subcellular distribution of HuR in IDH4
cells. As previously seen in RKO cells
(11), treatment of
proliferating IDH4 cells with AMPK activators AICAR, antimycin A, and sodium
azide also led to a time-dependent reduction in cytoplasmic HuR
(Fig. 4A). Also in
keeping with our previous findings using RKO cells, AMPK activators affected
the subcellular localization of HuR but did not modify the total or nuclear
levels of HuR, which remained unchanged throughout the time period examined
(Fig. 4A).
Confirmation of these findings was obtained using immunofluorescence
(Fig. 4B). Although
HuR was mostly nuclear in all treatment groups, as previously shown
(11,
30), the cytoplasmic signal
observed in control IDH4 cells (untreated) was further reduced in populations
treated with either AICAR, antimycin A, or Azide
(Fig. 4B). Given the
relatively low abundance of cytoplasmic HuR in untreated IDH4 cells, detection
by immunofluorescence was poor, and all subsequent assessments of HuR
subcellular localization were carried out by Western blotting, which allowed
use of sufficient cytoplasmic material for reliable analysis and
quantitation.

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FIG. 4. Effect of AMPK activators on the subcellular localization of HuR.
A, Western blot analysis of HuR levels in cytoplasmic (40 µg),
nuclear (20 µg), and whole cell (Total, 20 µg) lysates prepared
from IDH4 cells that were treated for the indicated times with either 2
mM AICAR, 5 µM antimycin A, or 2 mM sodium
azide. Hybridizations to detect either -actin (cytoplasmic and total
lysates) or histone deacetylase 1 (nuclear lysates) served to monitor the
differences in the loading of samples. B, detection of HuR in
proliferating IDH4 cells that were either left untreated or treated for 5 h
with AICAR, antimycin A, or sodium azide at the concentrations described for
A. Top row, phase contrast images; middle row, HuR
immunofluorescence; bottom row, Hoechst staining to visualize
nuclei.
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AMPK Activity Increases during Cellular SenescenceThe
observations described thus far indicate that AMPK activators were capable of
reducing cytoplasmic HuR levels in IDH4 cells. Such reductions in HuR
expression are reminiscent of those we previously observed in cellular models
of in vitro senescence
(30). We thus hypothesized
that changes in AMPK activity occurring during in vitro cellular
senescence may underlie the changes in HuR function described during this
process (30). First, we
examined AMPK activity during the process of in vitro senescence. To
this end, we employed the IDH4 model described above and compared AMPK
activity in young cells (cultured in Dex) with that in IDH4 cells induced to
undergo replicative senescence by removing Dex (and thereby inhibiting large T
antigen expression) (30) from
the culture medium. As shown, AMPK activity, which was measured as indicated
under "Materials and Methods," increased rapidly following the
removal of Dex to induce senescence, remaining elevated throughout the time
period studied (Fig.
5A). The cytoplasmic levels of HuR in IDH4 cells treated
with AMPK activators were comparable with those seen in IDH4 cells rendered
senescent by removing Dex for 5 days (Fig.
5B). We investigated the senescence-associated AMPK
activation in two other cell systems of replicative senescence: human diploid
fibroblasts IMR-90 and WI-38 that were cultured for either a low
(
2030) or a high (
5058) number of passages. As shown
in Fig. 5C, young, low
passage WI-38 and IMR-90 cells exhibited reduced AMPK activity, whereas
senescent, late passage WI-38 and IMR-90 cells displayed significantly
elevated AMPK activity. As shown in Table
I, measurement of AMP and ATP levels in two in vitro
senescence models revealed a 23-fold increase in AMP:ATP ratios in
senescent fibroblasts (IDH4 cells cultured without Dex and late-passage WI-38
cells) compared with young fibroblasts (proliferating IDH4 cells and
early-passage WI-38 cells).

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FIG. 5. Kinetics of AMPK kinase activity in IDH4, WI-38, and IMR-90 human
fibroblasts undergoing senescence. A, assessment of AMPK activity
in IDH4 cells following removal of Dex for the times indicated. B,
Western blot analysis of cytoplasmic HuR (40 µg of protein aliquots/lane)
in proliferating IDH4 cells (0 days) that were either left untreated or
treated with the indicated AMPK activators (2 mM AICAR, 5
µM antimycin A, or 2 mM sodium azide) for 5 h and in
IDH4 cells rendered senescent by removal of Dex for 5 days. C,
assessment of AMPK activity in WI-38 fibroblasts (left panel) and
IMR-90 fibroblasts (right panel) at the indicated population
doublings. The AMPK assays were performed as described under "Materials
and Methods." d, days; Sec., senescent.
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TABLE I Assessment of AMP:ATP ratios in cells undergoing senescence
AMP and ATP levels in young cultures (proliferating IDH4 cells, early-pdl
WI-38 cells) or senescent cultures (IDH4 cells cultured without Dex, late-pdl
WI-38 cells) were calculated
(38) and expressed as AMP:ATP
ratios. Representative values from two or more measurements are shown.
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Interventions to Increase AMPK Activity Accelerate Fibroblast
Senescence, whereas Reduction of AMPK Activity Delays SenescenceTo
assess whether up-regulation of AMPK activity could influence the
implementation of the senescent phenotype, young IDH4 and IMR-90 fibroblasts
were treated with AMPK activators, and the phenotypic features of senescence
were examined. As shown in Fig.
6A, neutral, senescence-associated
-gal
(33) was detected in treated
cultures but was largely absent from untreated cells. Senescence-associated
-gal activity is widely used as a biomarker for replicative senescence,
although the specific enzyme(s) involved remain poorly characterized
(33). In addition, expression
of p16INK4a, an inhibitor of cyclin-dependent kinases whose
expression and function are strongly linked to cellular senescence
(39), was markedly increased
in IDH4 cells treated with AMPK activators
(Fig. 6B).

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FIG. 6. Effect of AMPK activators on the senescent phenotype of IDH4 and IMR-90
fibroblasts. A, following treatment of IDH4 or IMR-90 cells with
AMPK activators AICAR (1 mM), antimycin A (1 µM), and
sodium azide (1 mM) for 7 or 3 days, respectively,
senescence-associated -gal activity was examined as described under
"Materials and Methods." B, Western blot analysis to
measure p16INK4a expression in IDH4 cells treated as explained in
the legend of Fig. 1.
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Further demonstration that AMPK contributed to modulating cellular
senescence came from experiments using adenoviral vectors to ectopically
express mutant forms of the AMPK
(catalytic) subunit: adenovirus
Ad(CA)AMPK, carrying
1312 (a constitutively active
1
mutant), and adenovirus Ad(DN)AMPK, which carries a dominant-negative mutant
of
1 (32). When
compared with infections using a control adenovirus that expresses the GFP
protein (AdGFP), infection with Ad(CA)AMPK led to an
2.7-fold increase in
AMPK activity in IDH4 cells (Fig.
7A) and a comparable increase in IMR-90 cells
(Fig. 8A), with
>90% cells infected at 300 PFU/cell (not shown). Importantly, such
intervention led to a marked decrease in cytoplasmic HuR in both IDH4
(Fig. 7B) and IMR-90
cells (Fig. 8B).
Conversely, infection with Ad(DN)AMPK led to a
40% reduction in AMPK
activity in IDH4 cells (Fig.
7A) and markedly elevated their cytoplasmic HuR abundance
(Fig. 7B). Neither
nuclear (not shown) nor total cellular HuR
(Fig. 7B) were altered
by the infections. Interestingly, senescence-associated
-gal activity
was elevated in cells displaying increased AMPK function, as shown for IDH4
and IMR-90 cells (Figs.
7C and
8C), whereas reduced
AMPK activity decreased the levels of senescence-associated
-gal in IDH4
(Fig. 7C); this
intervention could not be studied in IMR-90 cells, because its higher
constitutive AMPK activity could not be substantially reduced by Ad(DN)AMPK.
Taken together, our findings indicate that AMPK may play an important role
during the establishment of cellular senescence and points to HuR as a likely
mediator of gene expression changes observed during AMPK-triggered
senescence.

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FIG. 7. Effect of adenoviral infection to modulate AMPK activity on the
senescent phenotype of IDH4 fibroblasts. A, assessment of AMPK
activity in IDH4 cells 5 days after infection with 300 PFU/cell of a control
adenovirus (AdGFP) or with adenoviruses that express either a constitutively
active isoform (Ad(CA)AMPK or a dominant-negative isoform (Ad(DN)GFP) of the
AMPK catalytic subunit. The data represent the means ± S.E. of three
independent experiments. B, 5 days after infection of IDH4 cells as
described for A, HuR expression in cytoplasmic and whole cell
(Total) lysates was determined by Western blot analysis. C,
5 days after infection, senescence-associated -gal activity was assessed
as described under "Materials and Methods."
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FIG. 8. Effect of adenoviral infection to modulate AMPK activity on the
senescent phenotype of IMR-90 fibroblasts. A, assessment of AMPK
activity in IMR-90 cells 3 days after infection with 300 PFU/cell of a control
adenovirus (AdGFP) or with an adenovirus that expresses a constitutively
active isoform (Ad(CA)AMPK) of the AMPK catalytic subunit. The data represent
the means ± S.E. of three independent experiments. B, 3 days
after infection of IMR-90 cells as described for A, HuR expression in
cytoplasmic lysates was determined by Western blot analysis. C,
senescence-associated -gal activity was assessed 3 days after infection
of IMR-90 using 300 PFU/cell of a control adenovirus (AdGFP) or with
adenoviruses that express a constitutively active isoform (Ad(CA)AMPK), as
described under "Materials and Methods."
|
|
 |
DISCUSSION
|
---|
The present investigation originated at the point of convergence of two
earlier studies from our laboratory. In the first study, we identified AMPK as
an enzyme involved in regulating the cytoplasmic localization of HuR and
consequently HuR function in stabilizing target mRNAs and increasing their
expression (11). In the
second, we used several human fibroblast models of in vitro cellular
senescence to show that HuR abundance decreased as fibroblasts progressed
toward senescence (30); in
these models, we showed that reduced HuR levels effectively contributed to the
implementation of senescent phenotype. Here, we set out to test the hypothesis
that reductions in cytoplasmic HuR levels in human fibroblasts, brought about
by enhancing AMPK activity, were capable of inducing a senescent phenotype.
Indeed, interventions that induced AMPK activity, such as treatment with AMPK
activators and infection with an adenovirus that expresses a constitutively
active AMPK isoform, each caused premature senescence in IDH4 and IMR-90
cells. Conversely, reductions in AMPK activity caused delays in the onset of
cellular senescence.
However, it was somewhat unexpected to find that both AMP:ATP ratios, and
consequently AMPK activity, were naturally augmented in senescent
fibroblasts. To our knowledge, this constitutes the first report that AMP:ATP
ratios and AMPK activity increase with in vitro senescence. Although
elucidation of the precise mechanisms responsible for the senescence-related
increase in AMP:ATP ratios (Table
I) is of great interest, such analysis could be rather complex,
given the many possible means of altering the cellular concentration of ATP
and AMP, as discussed below. AMPK function can also be regulated by factors
other than AMP:ATP ratios. For instance, the AMPK 

heterotrimer composition may be influenced by in vitro senescence.
Although no acute changes in expression levels of specific subunits (
1,
2,
1,
2,
1,
2, and
3)
(2,
3) have been documented,
different heterotrimers have been shown to display tissue-specific
distribution, as well as restricted subcellular localization. In addition to
the particular
and
isoforms present in the complex, the degree
of AMPK activation can also depend on whether or not it is phosphorylated by
its upstream kinase, AMPK kinase (for review, see Ref.
1).
Generally speaking, AMPK is activated by stress situations that increase
the cellular AMP:ATP ratio. Physiological situations that cause a reduction in
ATP availability, such as exercise in skeletal muscle, have been shown to
induce AMPK function. Similarly, certain pathological conditions that cause
cellular hypoxia, hypoglycemia, oxidative stress, heat shock, and other
stresses also elevate AMPK function. In this regard, oxidative damage
accumulates in cellular components during normal mammalian aging, thereby
impairing the ability of the cell to adequately perform a variety of
functions. Such impairment in cellular functions, which has been linked to the
decreased ability of old cells to activate stress response pathways and their
diminished proliferative ability
(40), has also been associated
with the overall age-related decline in the expression of genes encoding
mitochondrial and energy metabolism proteins
(4143).
Age-related deficits and damage to cellular macromolecules involved in energy
production could therefore underlie the age-related lowered ATP production
(43,
44), elevated AMP:ATP ratio
(Table I), and consequently
increased AMPK function.
Previous studies demonstrated that activated AMPK was capable of causing
HuR to be predominantly nuclear and thereby blocked the ability of HuR to
stabilize target mRNAs (11).
In the present investigation, we sought to obtain more direct evidence that
HuR contributed to the establishment of a senescent phenotype when AMPK was
induced. We encountered technical limitations that precluded such an analysis.
Indeed, transient transfection experiments to test whether HuR overexpression
could block AICAR-, antimycin A-, or azide-triggered senescence were
unsuccessful because of the toxicity of the combined treatment (LipofectAMINE
plus the AMPK activator). Stable HuR overexpression was not attainable in
human diploid fibroblasts, as previously explained
(30). However, in light of the
influence of HuR on cellular senescence
(30), we propose that HuR
directly participates in the implementation of AMPK-triggered cellular
senescence.
In summary, we describe for the first time that replicative senescence is
characterized by elevations in AMP:ATP ratios and AMPK activity. Moreover,
modulation of AMPK activity via adenoviral vectors or chemicals was capable of
altering the senescent phenotype. Such modulations of AMPK activity
effectively altered the relative abundance of cytoplasmic HuR and consequently
the expression of HuR-regulated proliferative genes. Although AMPK activity
has not been previously studied in the context of cellular or organism aging,
it has been proposed to contribute to inhibiting cell proliferation. The
observation that active AMPK reduces the expression of cyclin A, cyclin B1,
and c-Fos (11) supports the
notion that AMPK could contribute to both acute growth arrest and growth
arrest associated with cellular senescence. Despite the controversy
surrounding the link between human aging and in vitro cellular
senescence, diminished proliferative capacity characterizes in vivo
aging. The discovery of the involvement of AMPK in cellular senescence
therefore warrants a careful look at AMPK in other models of aging.
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶ Supported by the Medical Research Council (UK). 
||
To whom correspondence should be addressed: Box 12, LCMB, NIA-IRP, National
Institutes of Health, 5600 Nathan Shock Dr., Baltimore, MD 21224-6825. Tel.:
410-558-8443; Fax: 410-558-8386; E-mail:
myriam-gorospe{at}nih.gov.
1 The abbreviations used are: AMPK, AMP-activated protein kinase; AICAR,
5-amino-imidazole-4-carboxamide riboside; Dex, dexamethasone; UTR,
untranslated region;
-gal,
-galactosidase; pdl, population
doublings; GFP, green fluorescent protein; PFU, plaque-forming units; REMSA,
RNA electrophoretic mobility shift assay. 
 |
ACKNOWLEDGMENTS
|
---|
We thank J. W. Shay for providing the IDH4 cells, the Coriell Institute for
Medical Research for supplying WI-38 and IMR-90 cells, and D. G. Hardie for
the anti-AMPK antibody. We appreciate the assistance of M. Wang with
photography and that of J. L. Martindale with critical reading of the
manuscript.
 |
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