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J. Biol. Chem., Vol. 275, Issue 27, 20685-20692, July 7, 2000
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From the Department of Microbiology, Showa University School of
Pharmaceutical Sciences, Hatanodai 1-5-8, Shinagawa-ku, Tokyo 142-8555, Japan
Received for publication, October 27, 1999, and in revised form, February 7, 2000
Two of mitogen-activated protein kinases (MAPK),
p44mapk/p42mapk extracellular
signal-regulated kinases (ERK1/2), translocate into nuclei following
activation and play critical roles in connecting the signal to gene
expression and allowing cell-cycle entry. Here we found that the
nuclear translocation of ERK1/2 in response to growth stimuli was
significantly inhibited in senescent cells that were irreversibly
growth arrested, compared with presenescent cells. The activation step
of these enzymes was not impaired, since ERK1/2 were phosphorylated and
activated in senescent cells as efficiently as in presenescent cells.
By elaborately localizing ERK2 in the nuclei of senescent cells, we
could restore c-fos transcriptional activity upon growth
stimuli, which was repressed in senescent cells. Furthermore, the
nuclear localization of ERK1/2 has been suggested to potentiate the
proliferative activity of the senescent cells in collaboration with
adenovirus E1A protein. More importantly, SV40 large T antigen, the
strong inducer of DNA synthesis, had the inherent ability to restore
nuclear relocalization of active ERK1/2 in senescent cells, which was
essentially required for the reinitiation of DNA synthesis. Thus,
manipulating the relocalization of ERK1/2 into nuclei was expected to
open the way to overcome some of the senescent phenotypes.
Cellular senescence is a postmitotic state which normal cells
reach after a finite number of cell divisions (1). Attention has
recently focused on the important roles of the INK4a locus (2-4) and
telomere shortening in this process (5, 6). Concerning the mechanisms
of the irreversible growth arrest of senescent cells, p21 and p16 are
believed to play critical roles in the persistent hypophosphorylation
of Rb and thereby inhibit cell-cycle entry of senescent cells (7, 8).
However, much remains to be resolved for the comprehension of
senescence. For example, the upstream signaling that accounts for
up-regulation of p21 and p16 has been less understood.
Mitogen-activated protein kinases (MAPK)1 are
serine/threonine kinases that play a
central role in a wide variety of signaling pathways in eukaryotic
organisms. They are activated at most downstream in the signaling
cascade, which are initiated by various extracellular stimuli,
including growth factors, hormones, and stresses. Subsequently, they
convert those stimuli to intracellular signals that control gene
expression, eventually leading to cell proliferation, differentiation, and programmed cell death (9, 10). In mammalian cells, MAP kinases have
been classified into three subfamilies: extracellular signal-regulated
kinase (ERK)1 and 2, JNK, and p38 kinase. The best studied MAP kinases,
ERK1/2, have been shown to play a pivotal role in processes such as
re-entry of fibroblasts into the cell cycle (11). These enzymes are
catalytically activated by MAPK kinase (MAPKK) through dual
phosphorylation at two key regulatory threonine and tyrosine residues
(12). The substrates of ERK1/2 distribute throughout cells from plasma
membrane to the nucleus. Thus, subcellular localization of ERK1/2 is an
important determinant of their functions. In response to extracellular
stimuli, ERK1/2 are activated and translocate into the nucleus, while
they are largely cytoplasmic in unstimulated cells (13-15).
In this study, we investigated activation and localization of ERK1/2 in
replicatively senescent human fibroblasts and tried to elucidate their
involvement in cellular senescence. Given the positive role of ERK1/2
in cell cycle entry, it was suspected that the signaling pathway
controlled by ERK1/2 should be down-regulated somehow in the senescent
cells that withdraw from cell cycle irreversibly. We show evidence that
nuclear localization but not activation of ERK1/2 is impaired in the
senescent cells and that relocalization of ERK1/2 in the nuclei of the
senescent cells is sufficient or required for overcoming some of the
senescent phenotypes.
Cell Culture--
TIG-3 and TIG-7, the human cell lines of
normal diploid fibroblasts, provided by the Japanese Cancer Resources
Bank were grown in a Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum (FCS) and kanamycin (50 µg/ml). The
cultures were divided 1:4; 1 passage was comparable to 2 PD (population doublings). We considered the cell population around 60 PD as the
senescent stage when less than 6% of the cells incorporated BrdUrd
after 72 h and more than 90% were senescence-associated Antibodies--
Monoclonal antibodies used in this study are
anti-HA epitope (12CA5, Roche Molecular Biochemicals), anti-SV40 large
T antigen (Pub101, Santa Cruz Biotechnology, Inc.), anti-pan ERK1/2
(ERK1 + ERK2, Zymed Laboratories Inc.), anti-activated
MAP kinase (Sigma), and anti-Myc tag (Invitrogen). Polyclonal
antibodies are anti-Sp-1 (sp-1(pep2)-G, Santa Cruz Biotechnology,
Inc.), anti-active ERK1/2 (the dual-phosphorylated p44/42MAPK,
Promega), anti-phospho-Elk-1 (Ser383) (New England
Biolabs), anti-HA (Zymed Laboratories Inc.), and anti-luciferase (Transformation Research, Inc.). Secondary antibodies, fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody and
tetramethylrhodamine B isothiocyanate-conjugated goat anti-rabbit IgG
antibody were purchased from DAKO.
Western Blot and Kinase Activity Assay--
Cells were
solubilized in lysis buffer (50 mM Hepes, pH 8.0, 150 mM NaCl, 0.02% sodium azide, 1% Triton X-100, 0.5%
deoxycholate, 0.1% SDS, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin) or
disrupted with Dounce homogenizer and fractionated into Triton
X-100-soluble and -resistant fractions in the buffer (10 mM
Tris, pH 8.0, 10 mM NaCl, 2 mM
MgCl2, 0, 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). The proteins were
quantified using Bio-Rad Protein Assay (Bio-Rad), and equal amounts
were loaded in each lane. After SDS-polyacrylamide gel electrophoresis,
resolved proteins were electrophoretically transferred onto a
nitrocellulose membrane (Amersham Pharmacia Biotech). The membranes
were then probed with a primary antibody followed by a secondary one.
The immune complexes were detected using enhanced chemiluminescence,
according to the manufacturer's protocol (NENTM Life
Science Products).
ERK kinase activity was measured with p44/42 MAP Kinase Assay Kit (New
England Biolabs) according to the manufacturer's instructions. Briefly, immune complex precipitated by anti-active ERK1/2 antibody was
incubated with a Elk-1 fusion protein in the presence of ATP, and
phosphorylation of Elk-1 at Ser383, which was a major
phosphorylation site by ERK1/2, was evaluated by Western blotting using
a phospho-Elk-1 antibody. The bands derived from phospho-Elk-1 was
quantified by Lightcapture (ATTO, Co., Tokyo) and normalized by total
amount of ERK1/2 existing in the starting material of cell lysate.
Expression Plasmids--
A series of HA-tagged ERK2 expression
vectors, including wild-type, nuclear localization signal (NLS)-added
(+NLS), and kinase-defective (kd) vectors, was constructed using a
pCG-HA vector (17). The open reading frame of ERK2 was polymerase chain
reaction-amplified and inserted into the vector with or without a NLS
sequence. The NLS sequence used here was derived from SV40 large T
antigen and expressed at the N-terminal of the resulting fusion protein
with ERK2. A kinase-defective ERK2 derivative (kd-ERK) mutated at
Lys44 to Arg and Lys45 to Arg (18) was created
by the Site-directed Mutagenesis System (TaKaRa Shuzoh, Co., Kyoto)
using a mutagenic oligonucleotide, 5'-GAGTAGCTATCAGGAGAATCAGCCCCTTTG-3'. The expression plasmids for Elk-1
(Myc tagged) (19) and constitutive active MEK1 (SDSE-MAPKK) (20) were
generously provided by Dr. Nishida (Kyoto University). The luciferase
reporters were based on the pGL3 basic luciferase reporter plasmid
(Promega). Promoter fragments of c-fos ( Microinjection--
TIG-7 cells were plated on glass coverslips.
After 24 h, plasmids were microinjected into the nuclei in the
culture medium buffered with 0.01 M Hepes for 0.3 s at
the constant pressure of 60 hPa. Effector and reporter plasmids were
diluted with sterile phosphate-buffered saline (PBS) to final
concentrations of 10 and 250 µg/ml, respectively. The injection
marker, mouse or rabbit normal IgG, was microinjected together with the
plasmids at a concentration of 1.25 mg/ml when needed. Injections were
performed using a Zeiss Axiovert 135, Eppendorf 5171 micromanipulater,
and Eppendorf 5242 injector.
Immunofluorescence, Luciferase, and BrdUrd-incorporation
Assay--
Cells, plated on glass coverslips, were washed with PBS and
fixed with 3.7% formaldehyde in PBS for 8 min at room temperature, followed by permeabilization in 0.2% Triton X-100/PBS for 2 min at
room temperature. Coverslips were washed with PBS and nonspecific sites
were blocked by incubation with PBS containing 3% bovine serum albumin
for 30 min at room temperature. The cells on the coverslips were then
incubated successively with the primary and secondary antibody diluted
in PBS/bovine serum albumin for 1 h at room temperature. Each
incubation was accompanied by four washes in PBS containing 0.1% Tween
20 for 15 min each. Subsequently, coverslips were incubated for 2 min
in a 4,6-diamidino-2-phenylindole (DAPI) solution (0.5 µg/ml DAPI in
PBS) to stain DNA, rinsed with PBS, and mounted with glycerol.
Fluorescence microscopy was carried out using an Axioskope microscope
(Carl Zeiss). As for the luciferase assay, the reporter and the
injection marker, mouse normal IgG (mIgG), or the effector plasmid were
co-microinjected into the cells. The injected cells were identified by
immunostaining using fluorescein isothiocyanate-conjugated goat
anti-mouse IgG antibody alone or in combination with anti-HA.
Luciferase positive cells were visualized by immunostaining using
anti-luciferase antibody and microscopically counted. A BrdUrd
incorporation assay was performed with a cell proliferation kit
(Amersham Pharmacia Biotech) according to the manufacturer's
instructions. The effector plasmids (10 µg/ml) were microinjected
with the injection marker, rabbit normal IgG (10 mg/ml), into the
senescent TIG-7 cells. After labeling with BrdUrd, the cells were fixed
and incubated with the anti-BrdUrd monoclonal antibody, followed by a
mixture of secondary antibodies to mouse IgG (fluorescein
isothiocyanate-labeled) and to rabbit IgG (tetramethylrhodamine B
isothiocyanate-labeled). The DNA was stained with DAPI to locate the nuclei.
Activation of ERK1/2 in Pre- and Senescent Cells--
We first
examined the activation of ERK1/2 upon growth stimuli in pre- and
senescent TIG-3 and -7 human normal diploid fibroblasts. The results of
Western blotting using the antibody that specifically recognizes the
dually phosphorylated ERK1/2 (anti-active ERK1/2) clearly showed that
both ERK1/2 were phosphorylated in senescent cells in response to 10%
FCS as efficiently as in presenescent cells with the same time course
(Fig. 1A). To verify and
compare the kinase activity of ERK1/2 quantitatively between pre- and senescent cells, in vitro kinase assay was performed using
Elk-1 as a substrate. In parallel with the increase in phosphorylation, kinase activity of ERK1/2 was remarkably elevated in the cell lysate
from the senescent cells as well as that from presenescent cells in
response to 10% FCS (Fig. 1B). Almost equal levels of activation were observed in pre- and senescent cells in a time- and
dose-dependent manner to the stimulus. Together, the
results clearly demonstrated that the signals were properly transmitted from cell surface receptors to ERK1/2 in the senescent cells.
Inhibition of Nuclear Relocalization of Activated ERK1/2 in the
Senescent Cells--
Despite the proper activation accompanying the
phosphorylation, which was reported to be sufficient to cause nuclear
translocation of ERK1/2 (14), we found that the phosphorylated forms of
ERK1/2 in the senescent cells were distributed exclusively in the 0.1% Triton-soluble fraction and not in the insoluble fraction containing nuclei either in the presence or absence of the stimulus. In the presenescent cells, the increase in the phosphorylated forms was observed not only in the soluble but also insoluble fractions. The
increase in the insoluble fraction appeared to be underestimated possibly because of redistribution of the nuclear proteins during fractionation of the cells (Fig. 1C). To investigate the
localization of ERK1/2 in the cells in further detail, we conducted
immunostaining. When the presenescent cells were immunostained with the
antibody to ERK1/2 after treatment with PDGF, strong nuclear staining
was observed with faint residual staining in the cytoplasm from 30 to
120 min after stimulation in all of the cells, as originally found by
Chen et al. (23) (Fig.
2A). In the senescent cells, however, the staining signals were not concentrated in the nuclei but
diffusely distributed within the cells at any time point from 1 to 120 min after the stimulation as shown in Fig. 2B. Essentially the same results were obtained using an antibody to active forms of
ERK1/2 (Fig. 2C). In this case, faint nucler staining was
sometimes observed in nearly but not completely senescent cells.
However, even in such cells, the nuclear staining was remarkably weak
compared with that in the presenescent cells. Therefore, it was most
likely that the nuclear localization of the activated forms of ERK1/2 was significantly depressed in the senescent cells. Subsequently, we
confirmed the above result using HA-tagged ERK2 exogenously expressed
by microinjection and visualized with the antibody to the tag. Nuclei
were barely stained in the senescent cells (Fig. 3B), while strong nuclear
signals were observed in the presenescent cells in response to serum
stimulation (Fig. 3A). Since Sp1 transcription factor was
detected in the nuclei of the senescent cells (Fig. 2B, right
panels), the accessibility of antibodies to the nuclei did not
appear to be generally hindered during preparation of the senescent
cells.
If the above notion was correct, it was reasoned that substrates of
ERK1/2 in the nuclei should remain unphoshorylated in the senescent
cells even when the cells were stimulated to activate the kinase. This
possibility was examined using a transcription factor, Elk-1, which was
a well established substrate of ERK1/2 in the nuclei (11, 30, 31). To
this end, the expression plasmid of Myc-tagged Elk-1 was introduced
into the cells by microinjection. After 24 h, the cells were
stimulated with PDGF, and the phosphorylation of Elk-1 was visualized
by immunostaining with the antibody to phospho-Elk-1. As predicted, the
nuclei of the senescent cells were hardly immunostained with the
anti-phospho-Elk antibody despite the successful expression of the
Elk-1 protein, which was proven by immunostaining with anti-Myc
antibody (Fig. 4A). Under the same condition, the nuclei of the presenescent cells were distinctively stained with the antibody in response to the stimulus (Fig.
4A). The fraction of the nuclei positive for phosphorylation
of Elk-1 was estimated microscopically, and the abolishment of Elk-1
phosphorylation in the senescent cells was evidently shown (Fig.
4B). Similarly, the phosphorylation of Elk-1 in the nuclei
was significantly inhibited in the senescent cells when the
constitutive active form of MAPKK was expressed to activate ERK1/2
instead of the PDGF treatment (Fig. 4C). Only in 7.0% of
the senescent cells, the phosphorylation of Elk-1 occurred, whereas
89.7% nuclei were positive in the presenescent cells. These
experiments provided further support for the conclusion that nuclear
localization was impaired in the senescent cells.
This phenomenon was observed not only in the senescent cells obtained
from both TIG-3 and -7 but also in quasi-senescent young cells that
were exposed to H2O2 and irreversibly ceased to
proliferate long before replicatively senescing (data not shown). In
addition, similar observations were reported in senescent and
terminally differentiated melanocytes (24). In this case, however, ERK2 was retained in the cytoplasm only because it was not phosphorylated and thus inactive. These results implied that the inhibition of nuclear
translocation was closely associated with the irreversible growth-arrested state and not merely a defect as a consequence of an
accumulative number of cell divisions of the senescent cells. In
accordance with this notion, ERK1/2 were found to be localized in the
nuclei in an immortalized TIG-3 derivative that had overcome the
growth-arrested state of senescence and undergone an infinite number of
cell divisions (data not shown).
The above results would seem to indicate that the nuclear transport
system of cellular proteins is generally impaired in the senescent
cells. However, this was not the case as demonstrated in Fig.
3C. We examined the localization of SV40 large T antigen whose nuclear translocation depends on its NLS (25). SV40 large T
antigen was found to be localized exclusively in the nuclei of the
senescent cells as well as in normal cells, suggesting that the
NLS-dependent nuclear transport system was not impaired in
the senescent cells. Since nuclear translocation of ERK1/2 was
independent of the NLS (13), the machinery specific to ERK1/2 transportation appeared to be inhibited. Concerning the mechanism by
which ERK1/2 are transported into nuclei, several possibilities have
been proposed (11, 13-15, 26); however, the details are still a matter
for discussion.
Inhibition of nuclear localization in senescent cells might result from
the nuclear export of ERK1/2 by a nuclear export signal-like sequence,
which predominated over their import. However, this is unlikely because
even in the presence of leptomycin B, which is a specific inhibitor of
the nuclear export signal function (27), ERK1/2 did not accumulate in
the nuclei of the senescent cells (data not shown). Another possibility
was that ERK1/2 were somehow masked or degraded so promptly in the
nuclei of the senescent cells that they escaped detection. This was
also unlikely, because +NLS-ERK2 (ERK2 carrying the NLS sequence of
SV40 large T antigen at its N-terminal) that was forced to be localized
in the nuclei by its NLS was clearly detected in the nuclei of the
senescent cells by a similar immunofluorescence technique (Fig.
3E).
Effect of Nuclear-targeted ERK2 on c-fos Transcriptional Activity
in the Senescent Cells--
The next question to be addressed here is
as to whether relocalization of ERK1/2 could restore any of the
senescent phenotypes. Although several transcription factors have been
known to be substrates for ERK1/2 in the nuclei (9), the biological
significance of the nuclear translocation of ERK1/2 had been unclear
until the recent work done by Brunet et al. (11) which
substantially showed that the inhibition of this translocation
repressed the gene expression as well as the re-entry of cells to the S
phase following growth factor stimuli. These results, together with our
above observations, suggested that inhibition of some nuclear events in
the senescent cells, including gene expression and DNA synthesis, was
partly due to the retention of ERK1/2 in cytoplasm under growth
stimuli, thereby blocking the signals to the nuclei.
To test this hypothesis, we first focused on the c-fos gene
expression in response to serum stimulation, which was repressed in the
senescent cells (28). In quiescent young cells, c-fos is
induced immediately after serum stimulation (29), which depends on the
relocalization of ERK1/2 into the nuclei (11). The efficient transcriptional response of c-fos to serum is mediated via
ternary complex formation on the serum response element (SRE) of the
gene (21). As proven by Gille et al. (30, 31), ERK1/2 played essential roles in this complex formation through phosphorylation of
Elk-1, one of the components of the ternary complex. In the present
study, transcriptional activity of the c-fos gene was monitored using the luciferase reporter that was placed under the
2.2-kilobase upstream sequence encompassing the c-fos
enhancer/promoter. Prior to the experiment, we tested whether this
reporter responded to serum appropriately. We microinjected the
reporter plasmid together with an injection marker, mouse IgG, into the
nuclei of pre- and senescent cells. Following 48 h of incubation
in a serum-starved medium, cells were stimulated with 10% FCS for
2 h and immunostained with the antibody to luciferase (Fig.
5B). In the quiescent
presenescent population, the number of luciferase positive cells
increased from 10% of the basal level to 40% by serum stimulation
(Fig. 5A, bar 1, pre/-FCS; 2, Pre/+FCS). In contrast, only 10% of the cells were
luciferase positive even after serum stimulation of the senescent
population (Fig. 5A, bar 3, Senes/-FCS;
4, Senes/+FCS). Thus, this assay was thought to
reflect the transcriptional behavior of the endogenous c-fos gene. In addition, the increase in reporter activity was completely abrogated when the kinase-defective/dominant-negative ERK2 was co-microinjected with the reporter plasmid in the presenescent cells
(data not shown), verifying the crucial role of ERK2 in the
transcriptional response of c-fos in our system, as
presented before (11, 30, 31).
Subsequently, we tried to recover c-fos inducibility in the
senescent cells by forcing ERK to localize in the nuclei. To this end,
we used the aforementioned +NLS-ERK2. The +NLS-ERK2 expression vector
was co-microinjected with the c-fos reporter plasmid into the nuclei of the senescent cells; the cells were incubated for 48 h under the continuous presence of serum to ensure that the expressed
+NLS-ERK2 was activated before being guided to the nuclei. The
transcriptional activity was visualized as shown in Fig. 5D, and the ratios of luciferase positive to injected cells are shown in
Fig. 5C. As expected, +NLS-ERK2 was localized in the nuclei as visualized with anti-HA (Fig. 5D, middle panel
(green)), and could recover c-fos transcriptional
activity in the senescent cells (Fig. 5, C, bar 4, +NLS-ERK/fos, and middle of D
(red)). Its efficiency in inducing c-fos
transcription was as high as that in the presenescent cells stimulated
with serum (Fig. 5, C, bar 1, Pre-mIgG/fos). In contrast, -NLS-ERK2, which remained in the
cytoplasm, could not stimulate c-fos transcriptional
activity even when overexpressed (Fig. 5, C, bar
3, -NLS-ERK/fos, and top of D).
This transcriptional activation of the reporter by +NLS-ERK2 was
considered to be relevant based on the following reasons. First,
+NLS-ERK2 did not affect the transcriptional activity of transglutaminase that was expressed specifically in keratinocytes (data
not shown). This suggested that the substrate(s) targeted by +NLS-ERK2
in the nuclei was specific to the c-fos enhancer/promoter. Second, kinase-defective +NLS-ERK2 (kd-ERK) did not induce an increase
in the c-fos transcription (Fig. 5C, bar
6, +NLS-kd-ERK/fos, and bottom of
D). Finally, the effect of +NLS-ERK2 was reduced to 40%
when the ternary complex formation on SRE, which was assumed to be the
nuclear target of ERK1/2, was specifically impaired by point mutations
(21) (Fig. 5C, bar 5, +NLS-ERK/mSREfos). Therefore, one of the key targets of
+NLS-ERK2 in the nuclei was thought to be Elk-1 in the ternary complex,
and not to be nonphysiological substrates. The residual activity of
40% was thought to be attributable to other transcriptional elements
such as cAMP response element adjacent to SRE and activated indirectly
by ERK1/2 (32). Altogether, these findings substantially supported our
hypothesis that the loss of c-fos inducibility in the
senescent cells was the consequence of cytoplasmic retention of ERK1/2,
and that nuclear targeting of ERK2 was sufficient for the
transcriptional reactivation of c-fos.
Previously, Atadja et al. (33) reported on
hyperphosphorylation of serum response factor (SRF), another component
in the ternary complex, resulting in the loss of SRE binding activity and transcriptional inhibition of c-fos in senescent cells.
They proposed an age-specific phosphatase activity as one of the
regulators of SRF phosphorylation, although neither the phosphorylation
sites nor the phosphatase have been identified. On the other hand,
phosphorylation at a specific serine residue of SRF has been
demonstrated to increase its binding ability to DNA (34). Among the
kinases that have been shown to phosphorylate the site of SRF are
pp90rsk and MAPKAP2, both of which are substrates of ERKs (34).
Therefore, to be compatible with the above results, it was speculated
that nuclear-targeted ERK2 could improve not only Elk-1 activity
directly but also the SRF activity indirectly through activation of
either of the two kinases, leading to complete recovery of the ternary complex formation on SRE. The c-fos gene is the first to be
activated in response to extracellular stimuli and thereafter affects
the expression pattern of genes in a transcription factor network. Keeping in mind other transcription factors targeted by ERK1/2 in the
nuclei, such as c-myc and NF-IL6 (9), whose activities also
appeared to be repressed in the senescent cells, restoration of nuclear
translocation of ERK1/2 could have profound effects on the expression
pattern of a large number of genes in the senescent cells, leading to
phenotypic rejuvenation of the cells.
Involvement of ERK in Reinitiation of DNA Synthesis in the
Senescent Cells--
Next, we tested whether nuclear-targeted ERK2
counteracts cell cycle arrest during senescence. We micoinjected the
expression plasmids of ERK2 into senescent cells and the effect on DNA
synthesis was examined. After microinjection, the cells were incubated
with BrdUrd in the presence of 10% serum for 48 h and then
analyzed for BrdUrd incorporation by an immunofluorescence technique.
In the senescent cells, +NLS-ERK2 alone could not induce DNA synthesis (data not shown). One of the reasons was thought to be a high expression of p16INK4a that was not suppressed by
+NLS-ERK2.2 If so, adenovirus
E1A protein could aid +NLS-ERK2 in stimulating DNA synthesis in the
senescent cells, since E1A is able to cancel the effect of
p16INK4a through binding to Rb. Actually, +NLS-ERK2 but not
-NLS-ERK2 could stimulate DNA synthesis in the presence of E1A in
25.6% of the cells, when the basal level of DNA synthesis was only
2.1% (Fig. 6A). This
potentiation of DNA synthesis by +NLS-ERK2 in collaboration with E1A
was reproducibly observed but the effect of +NLS-ERK2 seemed to be only
modest. Based on these observations, we changed the approach and
investigated the requirement of nuclear localization of ERK1/2 for
reprogramming the senescent cells to proliferate. Given that the
cellular senescent process comprises multiple stages and that the
irreversible growth arrested state is brought about by several kinds of
sequentially occurring events during the process (8), it seemed natural
that nuclear relocalization of ERK was not sufficient by itself to
release the senescent cells from the constraints of the growth arrest.
Instead, we hypothesized that nuclear relocalization of ERK could be
one of the essential requirements for reinitiation of DNA synthesis. As
natural sources to impose the senescent cells to re-enter cell cycle,
viral oncogenes are famous to have such activity. Among them, SV40
large T antigen was the strongest inducer of DNA synthesis. It could
induce DNA synthesis in 69.3% of the senescent cells in our system
(Fig. 6D, lane
In conclusion the growth-arrested state of senescent cells was
accompanied by inhibition of the translocation of ERK1/2 into nuclei,
which resulted in abrogation of nuclear activities such as gene
expression and DNA synthesis in response to growth stimuli. Further
investigation of the molecular mechanisms regulating this phenomenon
would not only lead to a better understanding of senescence but also
enable us to control the process pharmacologically or genetically.
We thank Dr. J.-I. Fujisawa (Kansai Medical
University) for pCG-HA vector, Dr. E. Nishida (Kyoto University) for
Elk-1 and SDSE-MAPKK expression plasmids, Dr. T. Ikuta (Saitama Cancer
Center) for NLS sequence, Dr. M. Yoshida (Tokyo University) for
leptomycin B, Dr. K. Oda (Science University of Tokyo) for E1A
expression plasmid, and Dr. K. Yamanishi (Kyoto Prefectural Medical
College) for transglutaminase reporter plasmid.
*
This work was supported by a grant-in-aid for scientific
research from the Ministry of Education, Culture, Science and Sports.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, March 27, 2000, DOI 10.1074/jbc.M908723199
2
J. K.-Kaneyama, K. Nose, and M. Shibanuma,
unpublished data.
The abbreviations used are:
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated
kinase;
FCS, fetal calf serum;
PDGF, platelet-derived growth factor;
SRE, serum response element;
SRF, serum response factor. PD, population
doubling;
BrdUrd, bromodeoxyuridine;
HA, hemagglutinin;
NLS, nuclear
localization signal;
PBS, phosphate-buffered saline;
DAPI, 4,6-diamidino-2-phenylindole.
Significance of Nuclear Relocalization of ERK1/2 in Reactivation
of c-fos Transcription and DNA Synthesis in Senescent
Fibroblasts*
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-galactosidase positive, which is a biomarker of cellular senescence (16).
2.2 or 0.74
kilobases) were subcloned into the vector to yield c-fos (2.2) and (0.74) luciferase reporter. The c-fos SRE
sequence was mutated using the mutagenic oligonucleotide
5'-CCCTCCCCCCTTACAACTGATGTCCATATTAGG-3' to yield mSRE/fos luciferase
reporter (21). SV40 large T antigen expression plasmid, SV0ri9-8-16,
was obtained from DNA Bank, Tsukuba Life Science Center, RIKEN. E1A
expression plasmid, pAd2-12S.E.1A (22), were kindly provided by Dr. Oda
(Science University of Tokyo).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Activation of ERK1/2 in the pre- and
senescent cells after growth stimuli. A, pre- (39 or 31 PD) and senescent (60 or 57 PD) cells of TIG-3 or -7 were stimulated
with 10% FCS for 0 ( ) or 1 h (+). Total proteins were analyzed
by Western blotting with the indicated antibody. In C, TIG-7
cells of the indicated PD were treated as in A and
fractionated as described under "Experimental Procedures."
Triton-soluble and -insoluble fractions were designated as S
and P, respectively. The upper and lower
bands corresponded to ERK1 and -2, respectively. Almost equal
amounts of total ERK/2 detected with anti-pan ERK/2 were present in
each set of extracts. B, TIG-7 cells of the indicated PD
that were serum starved for 48 h were stimulated with 10% FCS.
After the indicated times, cell lysate was prepared and ERK1/2 kinase
activity was determined in vitro according to the
manufacturer's instructions. First, active ERK1/2 kinase were
selectively immunoprecipitated by a phospho-antibody to the kinase and
then, the resulting immunoprecipitate was incubated with a Elk-1 fusion
protein as a substrate in the presence of ATP. The amount of
phosphorylated Elk-1 fusion protein was determined by Western blotting
with a phospho-Elk-1 antibody followed by quantification with
Lightcapture (ATTO Co., Tokyo, Japan). The values were shown as a ratio
to the control (time 0 of 38 PD presenescent cells) after normalization
with the total amounts of ERK1/2 in each cell lysate that were
estimated by Western blotting with anti-pan ERK1/2.
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Fig. 2.
Relocalization of ERK1/2 in the pre- and
senescent cells after growth stimuli. Pre- (A and
C) and senescent (B and D) TIG-3 cells
were incubated with PDGF (10 ng/ml) for 0 ( ) or 30 (+) min.
Immunofluorescence was performed as described under "Experimental
Procedures." The first antibodies used were against total ERK1/2
(pan-ERK1/2) (left panels of A and B)
and against active ERK1/2 (anti-activated MAP kinase from Sigma)
(left panels of C and D). DNA was
stained with DAPI. In A and B, Sp1 was
immunostained simultaneously and shown in the right panels.
The arrows in D indicated the location of the
same nuclei in both panels.
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Fig. 3.
Nuclear localization of ERK2 but not
NLS-dependent nuclear transport was inhibited in the
senescent cells. A and B, the expression
plasmid of HA-tagged ERK2 (wild-type) was microinjected into pre-
(A) or senescent (B) TIG-7 cells and the cells
were incubated in a serum-starved medium. After 24 h, the cells
were stimulated with 10% FCS for 0 ( ) and 1 h (+) and analyzed
by indirect immunofluorescence labeling with the antibody against the
HA epitope (left panel). DNA was stained with DAPI
(right). In 89.5% of the presenescent cells, ERK2 was
localized in the nuclei in response to serum, while 12.0% of the
senescent cells examined showed nuclear localization of ERK2. C,
D, and E, SV40 large T antigen (C),
HA-tagged /+NLS-ERK2 plasmids (D and E) were
microinjected into the pre- (D) or senescent (C
and E) cells. After 24 h, the cells were analyzed by
indirect immunofluorescence labeling using the antibody to SV40 large T
antigen (C) or HA epitope (D and E);
representative fields are shown in the left panels. DNA was
stained with DAPI (right).
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Fig. 4.
A nuclear substrate of ERK1/2, Elk-1,
remained unphosphorylated under the growth stimuli in the senescent
cells. A and B, after microinjection of the
Myc-tagged Elk-1 expression plasmid (100 µg/ml), the pre- and
senescent cells of TIG-7 were serum-starved in Dulbecco's modified
Eagle's medium supplemented with 0.1% bovine serum albumin for
24 h, followed by the stimulation with PDGF (10 ng/ml) for 1 h. Then, the cells were immunostained with anti-Myc tag monoclonal
antibody for the identification of Elk-1 expression and with
anti-phospho-Elk-1 polyclonal antibody simultaneously. DNA was stained
with DAPI. Among the cells stained with anti-Myc tag antibody, the
fraction of the cells with nuclei stained with anti-phospho-Elk-1 was
evaluated by microscopically and indicated in B with the
results of control experiments without the PDGF treatment. The number
of cells successfully microinjected were pre , 80; pre +, 210; senes
, 159; and senes +, 131. C, the pre- and senescent cells
of TIG-7 cells were microinjected with the Elk-1 expression plasmid (50 µg/ml) together with the SDSE-MAPKK plasmid (50 µg/ml) for
expression of a constitutive active form of HA-tagged MAPKK. After
24 h incubation in complete medium, the cells were processed to
immunostaining with anti-HA monoclonal and anti-phospho-Elk-1
polyclonal antibodies. The fractions of the cells whose nuclei were
stained with anti-phospho-Elk-1 antibody were 89.7% among the
presenescent cells expressing HA-MAPKK and only 7% among those of
senescent cells.
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Fig. 5.
c-fos transcriptional
activity was restored by the presence of +NLS-ERK2 in the nuclei of the
senescent cells. A and B, pre- or senescent
cells were microinjected with the c-fos ( 2.2) luciferase
reporter together with the marker, mouse IgG (mIgG), and
incubated in a serum-starved medium for 48 h. Subsequently, the
cells were treated with 10% FCS for 0 () or 2 h (+) and
immunostained as described under "Experimental Procedures." The
injected cells and their c-fos transcriptional activity were
labeled with fluorescein isothiocyanate (green, left
panels of B) and tetramethylrhodamine B isothiocyanate
(red, right), respectively. The luciferase
positive percentages are shown in A. The numbers of cells
successfully microinjected were 93, 69, 69, and 74, from bars
1 to 4, respectively. C and
D, senescent cells were co-microinjected with the reporter
and ±NLS-wild type or kinase dead (designated as kd) ERK2
effectors, and were cultured in a 10% serum-containing medium for
24-48 h. The percentages of luciferase positive cells (red
in right panels of D) among the cells expressing
the effectors (green in left panels of
D) are shown in C. The results were mean ± S.D. obtained from at least three independent experiments. The numbers
of cells successfully microinjected were 69, 69, 124, 151, 115, and
175, from bars 1 to 6, respectively. The basal
activities were examined in pre- (bar 1, Pre
mIgG/fos) and senescent cells (bar 2, mIgG/fos) in the same way as A and B.
The c-fos (2.2) and (0.74) reporters gave substantially
the same results. The basal activity of the mSRE reporter was almost
the same as the parental reporter.
), while E1A induced DNA synthesis in only
12.8% of them (Fig. 6A, lane). If nuclear localization of
ERK1/2 is indispensable for overcoming the senescence-associated cell
cycle arrest, it should accompany the effect of SV40 large T antigen in
restoring DNA synthesis. Actually, nuclear localization of endogenous
ERK1/2 (active forms) was detected in the senescent cells following
expression of SV40 large T antigen by microinjection (Fig. 6,
B and C). When the HA-tagged ERK2 was expressed
exogenously together with SV40 large T antigen and the cells were
stimulated with 10% FCS for 30 min, the signals visualized by either
of anti-HA or anti-active ERK1/2 were concentrated in the nuclei of
58% of the senescent cells as a maximal response (Fig. 6C, lanes
SV40). With or without ERK2 exogenously overexpressed, similar
results were obtained. E1A could also localize ERK1/2 in the nuclei to
some extent (Fig. 6C, lane E1A). However, it was less
effective compared with SV40 large T antigen, which was in good
agreement with the extent to which each protein could induce DNA
synthesis. More importantly, DNA synthesis induced by SV40 large T
antigen was significantly inhibited by co-microinjection of
+NLS-kinase-defective/dominant-negative-ERK2 (Fig. 6D). This
suggested that the ability of SV40 large T antigen to induce DNA
synthesis in the senescent cells was largely dependent on the
activities of ERK1/2 within nuclei. These results strongly support the
idea that localization of active ERK1/2 in the nuclei is one of the
critical requirements for overcoming senescence along with other
activities such as telomerase activity.
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Fig. 6.
Nuclear relocalization of ERK1/2 was restored
by SV40 large T antigen and required for DNA synthesis in the senescent
cells. A and D, the senescent cells of TIG-7
were co-microinjected with E1A (A) or SV40 large T antigen
(D) and ERK2 expression plasmids. After 12 h, BrdUrd
was added to the culture medium. Following an additional 48 h of
incubation in the presence of 10% serum, the cells were processed for
a BrdUrd assay as described under "Experimental Procedures." The
nuclei incorporating BrdUrd and those injected with the plasmid were
counted. BG means background incorporation of BrdUrd. The
values were mean ± S.D. The numbers of cells successfully
microinjected were 122, 96, 134, and 105, from bars
(black) 1 to 4, respectively, in
A, and 92, 94, 90, and 94, from bars 1 to
4, respectively in D. B and
C, the cells were microinjectecd with SV40 large T antigen
or E1A together with the injection marker or HA-tagged ERK2
(wild-type). After 48 h, the cells were stimulated with 10% FCS
for 30 min and then immunostained with anti-activated MAP kinase
(Sigma) alone or together with anti-HA. The representative fields in
which the active forms of endogenous ERK1/2 were stained are shown in
B. The percentages of the cells whose nuclei were labeled
with either of the antibodies (active; anti-MAP kinase,
activated, HA; anti-HA) are shown in C. The
values were mean ± S.D. obtained from at least three independent
experiments. The number of the cells successfully microinjected were
239, 75, and 106 (bars 1-3, respectively), 71 (a pair of
bars 4 and 5), and 128 (a pair of bars
6 and 7).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should addressed: Dept. of Microbiology,
Showa University School of Pharmaceutical Sciences, Hatanodai 1-5-8, Shinagawa-ku, Tokyo, Japan. Tel.: 81-3-3784-8209; Fax: 81-3-3784-6850;
E-mail: smotoko@pharm.showa-u.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1.
Hayflick, L.
(1965)
Exp. Cell Res.
37,
614-636
2.
Hara, E.,
Smith, R.,
Parry, D.,
Tahara, H.,
Stone, S.,
and Peters, G.
(1996)
Mol. Cell. Biol.
16,
859-867
3.
Serrano, M.,
Lee, H.-W.,
Chin, L.,
Cordon-Cardo, C.,
Beach, D.,
and DePinho, R. A.
(1996)
Cell
85,
27-37
4.
Kiyono, T.,
Foster, S. A.,
Koop, J. I.,
McDougall, J. K.,
Galloway, D. A.,
and Klingelhutz, A. J.
(1998)
Nature
396,
84-88
5.
Harley, C. B.,
Futcher, A. B.,
and Greider, C. W.
(1990)
Nature
345,
458-460
6.
Bodnar, A. G.,
Ouellette, M.,
Frolkis, M.,
Holt, S. E.,
Chiu, C.-P.,
Morin, G. B.,
Harley, C. B.,
Shay, J. W.,
Lichtsteiner, S.,
and Wright, W. E.
(1998)
Science
279,
349-352
7.
Alcorta, D. A.,
Xiong, Y.,
Phelps, D.,
Hannon, G.,
Beach, D.,
and Barrett, J. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13742-13747
8.
Stein, G. H.,
Drullinger, L. F.,
Soulard, A.,
and Dulic, V.
(1999)
Mol. Cell. Biol.
19,
2109-2117
9.
Davis, R. J.
(1993)
J. Biol. Chem.
268,
14553-14556
10.
Marshall, C. J.
(1995)
Cell
80,
179-185
11.
Brunet, A.,
Roux, D.,
Lenormand, P.,
Dowd, S.,
Keyse, S.,
and Pouyssegur, J.
(1999)
EMBO J.
18,
664-674
12.
Thomas, G.
(1992)
Cell
68,
3-6
13.
Fukuda, M.,
Gotoh, Y.,
and Nishida, E.
(1997)
EMBO J.
16,
1901-1908
14.
Khokhlatchev, A. V.,
Canagarajah, B.,
Wilsbacher, J.,
Robinson, M.,
Atkinson, M.,
Goldsmith, E.,
and Cobb, M. H.
(1998)
Cell
93,
605-615
15.
Lenormand, P.,
Brondello, J.-M.,
Brunet, A.,
and Pouyssegur, J.
(1998)
J. Cell Biol.
142,
625-633
16.
Dimri, G. P.,
Lee, X.,
Basile, G.,
Acosta, M.,
Scott, G.,
Roskelley, C.,
Medrano, E. E.,
Linskens, M.,
Rubelj, I.,
Smith, O. P.,
Peacocke, M.,
and Campisi, J.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
9363-9367
17.
Hirai, H.,
Suzuki, T.,
Fujisawa, J.,
Inoue, J.,
and Yoshida, M.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
3584-3588
18.
Robbins, D. J.,
Zhen, E.,
Owaki, H.,
Vanderbilt, C. A.,
Ebert, D.,
Geppert, T. D.,
and Cobb, M. H.
(1993)
J. Biol. Chem.
268,
5097-5106
19.
Kamakura, S.,
Moriguchi, T.,
and Nishida, E.
(1999)
J. Biol. Chem.
274,
26563-26571
20.
Fukuda, M.,
Gotoh, I.,
Adachi, M.,
Gotoh, Y.,
and Nishida, E.
(1997)
J. Biol. Chem.
272,
32642-32648
21.
Shaw, P. E.,
Schroter, H.,
and Nordheim, A.
(1989)
Cell
56,
563-572
22.
Nakajima, T.,
Nakamura, T.,
Tsunoda, S.,
Nakada, S.,
and Oda, K.
(1992)
Mol. Cell. Biol.
12,
2837-2846
23.
Chen, R. H.,
Sarnecki, C.,
and Blenis, J.
(1992)
Mol. Cell. Biol.
12,
915-927
24.
Medrano, E. E.,
Yang, F.,
Boissy, R.,
Farooqui, J.,
Shah, V.,
Matsumoto, K.,
Nordlund, J. J.,
and Park, H.-Y.
(1994)
Mol. Biol. Cell
5,
497-509
25.
Adam, S. A.,
and Gerace, L.
(1991)
Cell
66,
837-847
26.
Adachi, M.,
Fukuda, M.,
and Nishida, E.
(1999)
EMBO J.
18,
5347-5358
27.
Fukuda, M.,
Asano, S.,
Nakamura, T.,
Adachi, M.,
Yoshida, M.,
Yanagida, M.,
and Nishida, E.
(1997)
Nature
390,
308-311
28.
Seshadri, T.,
and Campisi, J.
(1990)
Science
247,
205-209
29.
Treisman, R.
(1985)
Cell
42,
889-902
30.
Gille, H.,
Sharrocks, A. D.,
and Shaw, P. E.
(1992)
Nature
358,
414-417
31.
Gille, H.,
Kortenjann, M.,
Thomae, O.,
Moomaw, C.,
Slaughter, C.,
Cobb, M. H.,
and Shaw, P. E.
(1995)
EMBO J.
14,
951-962
32.
Xing, J.,
Ginty, D. D.,
and Greengerg, M. E.
(1996)
Science
273,
959-963
33.
Atadja, P. W.,
Stringer, K. F.,
and Riabowol, K. T.
(1994)
Mol. Cell. Biol.
14,
4991-4999
34.
Heidenreich, O.,
Neininger, A.,
Schratt, G.,
Zinck, R.,
Cahill, M. A.,
Engel, K.,
Kotlyarov, A.,
Kraft, R.,
Kostka, S.,
Gaestel, M.,
and Nordheim, A.
(1999)
J. Biol. Chem.
274,
14434-14443
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