2 Veterans Affairs Medical Center, Departments of 1 Internal Medicine and 3 Biochemistry and Molecular Biology, and 4 Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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Although
aging enhances expression and tyrosine kinase activity of epidermal
growth factor receptor (EGFR) in the gastric mucosa, there is no
information about EGFR signaling cascades. We examined the age-related
changes in mitogen-activated protein kinases (MAPKs)
[extracellular signal-related kinases (ERKs), c-Jun
NH2-terminal kinases (JNKs), and p38], an
EGFR-induced signaling cascade, and activator protein-1 (AP-1) and
nuclear factor-B (NF-
B) transcriptional activity in the gastric
mucosa of 4- to 6-, 12- to 14-, and 22- to 24-mo-old Fischer 344 rats.
AP-1 and NF-
B transcriptional activity in the gastric mucosa rose
steadily with advancing age. This can be further induced by
transforming growth factor-
. The age-related activation of AP-1 and
NF-
B in the gastric mucosa was associated with increased levels of c-Jun, c-Fos, and p52, but not p50 or p65. Total and phosphorylated I
B
levels in the gastric mucosa were unaffected by aging. Aging was also associated with marked activation of ERKs (p42/p44) and JNK1.
In contrast, aging decreased p38 MAPK activity in the gastric mucosa.
Our observation of increased activation of ERKs and JNK1 in the gastric
mucosa of aged rats suggests a role for these MAPKs in regulating AP-1
and NF-
B transcriptional activity. These events may be responsible
for the age-related rise in gastric mucosal proliferative activity.
activator protein-1; nuclear factor-B; mitogen-activated protein
kinases; extracellular signal-related kinases; c-Jun
NH2-terminal kinases; p38; transforming growth factor-
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INTRODUCTION |
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RESULTS FROM THIS AND OTHER laboratories (2, 16, 17, 19, 29, 31, 32, 36, 37) have demonstrated that in the Fischer 344 rat model, aging is associated with increased mucosal proliferative activity in various tissues of the gastrointestinal tract, including the stomach. In the gastric mucosa, this is evidenced by increased labeling index, DNA synthesis, and thymidine kinase and ornithine decarboxylase (ODC) activities (29, 31, 32, 36). More recently, we (49) have demonstrated that aging is also associated with enhanced transition of G1 to S phase of the cell cycle in the gastric mucosa, as evidenced by increased expression and activation of cyclin-dependent kinase-2, accompanied by decreased expression of p21Waf-1. This suggests that the age-related rise in gastric mucosal proliferative activity could partly be the result of enhanced progression of cells through the G1 phase.
Although the responsible molecular mechanisms for the age-related rise
in mucosal proliferative activity are poorly understood, we (37, 46)
have observed that, at least in the gastric mucosa, aging is associated
with increased expression and activation of certain tyrosine kinases,
most notably the epidermal growth factor receptor (EGFR), the common
receptor for EGF and transforming growth factor- (TGF-
). Numerous
studies (4, 6, 19, 42) have demonstrated that the EGF family of
peptides, particularly EGF and TGF-
, stimulate mucosal proliferative
activity in much of the gastrointestinal tract, including the stomach.
Both EGF and TGF-
initiate their mitogenic action by activating the
intrinsic tyrosine kinase activity of their receptor, thereby
initiating the EGFR signaling process (41, 42, 47). In view of this, we
(37, 46) postulated that induction of the EGFR signaling pathway may
partly be responsible for the age-related rise in gastric mucosal
proliferative activity. Additionally, we (46) have observed that the
relative concentration of membrane-bound forms of TGF-
as well as
mRNA expression of the peptide are also markedly higher in the gastric
mucosa of aged rats than in young rats. This suggests that TGF-
,
which, unlike EGF, is synthesized in the gastric mucosa, may regulate
mucosal EGFR tyrosine kinase through an autocrine/juxtocrine mechanism
(46).
Activation of EGFR initiates a series of signaling events through phosphorylation of interacting proteins, which in turn transmit the signal to the nucleus (41, 42, 47). The details of the signaling events leading to stimulation in mucosal proliferative activity during advancing age are yet to be elucidated. However, a large body of evidence suggests that the mitogen-activated protein kinase (MAPK) signaling pathways, which regulate cellular growth and differentiation, respond to EGFR activation (18, 40, 43). At least three distinct families of MAPKs are present in mammalian cells: the p42/44 extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal/stress-activated kinases (JNK/SAPKs), and p38 (39). It has been suggested that ERKs are primarily responsive to cell proliferation signals, whereas JNKs and p38 respond to cellular stress (10, 21, 23). Once activated, MAPKs translocate to the nucleus, where they activate transcription factors (20).
Among the transcription factors, activator protein-1 (AP-1) and nuclear
factor-B (NF-
B) have been extensively investigated since they
appear to be critically involved in regulating the expression of a
variety of genes that participate in growth-related processes (3, 20,
24). NF-
B is thought to be involved in regulating the expression of
genes required for inflammatory responses, for suppression of apoptosis
as well as for controlling proliferation (4, 12). AP-1, which is also
involved in regulating cell proliferation, responds to many stimuli,
including growth factors and cytokines (1, 20). Furthermore, subunits
of AP-1 and NF-
B are able to cross talk, and both factors may play a role in cell transformation (7, 44). In view of the evidence indicating
a role for AP-1 and NF-
B in regulating cellular growth, we have
examined the age-related changes in AP-1 and NF-
B transcriptional activity in the gastric mucosa. The primary objective was to determine whether the age-related rise in gastric mucosal proliferative activity
would also be accompanied by increased activation of AP-1 and NF-
B
transcription factors. Because MAPKs play a key role in activating
transcription factors through phosphorylation, we also examined the
activity of different MAPKs in the gastric mucosa during aging. Our
current data show that aging is associated with a marked increase in
expression and activation of AP-1 and NF-
B in the gastric mucosa and
that these changes are accompanied by increased ERK and JNK1 activity,
but not p38 MAPK activity. In addition, we have also demonstrated that
in isolated gastric mucosal cells, TGF-
markedly stimulates the
transcriptional activity of AP-1 and NF-
B.
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METHODS |
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Reagents.
Double-stranded oligonucleotide probes containing the consensus
sequences of NF-B and AP-1 as well as the T4 polynucleotide kinase
were from Promega (Madison, WI). The sequences of the oligonucleotides were as follows: NF-
B, 5'-AGT TGA GGG GAC TTT CCC AGG
C-3', and AP-1, 5'-CGC TTG ATG AGT CAG CCG GAA-3'.
Poly(dI-dC) · poly(dI-dC) and protein G-Sepharose
were obtained from Pharmacia Biotech (Piscataway, NJ).
[
-32P]ATP (3,000 Ci/mmol) was from NEN Life
Science (Boston, MA). Polyclonal rabbit antibodies to NF-
B p50, p52,
and p65, JNK1, c-Jun, c-Fos, ERK1, and ERK2 as well as to c-Jun (amino
acids 1-79) substrate were from Santa Cruz Biotechnology (Santa
Cruz, CA). Polyclonal rabbit antibodies to p38, phospho-p38
(Thr180/Tyr182), phospho-ERK1, phospho-ERK2
(Thr202/Tyr204), I
B
, and phospho-I
B
(Ser32) were from New England Biolabs (Beverly, MA). Goat
anti-rabbit IgG conjugated with horseradish peroxidase and enhanced
chemiluminescence (ECL) were obtained from Amersham (Arlington Heights,
IL). Immobilon-P nylon membrane was from Millipore (Bedford, MA), and
X-Omat AR film was from Eastman Kodak (Rochester, NY). Concentrated
protein assay dye reagent was from Bio-Rad (Hercules, CA). Molecular
weight marker, myelin basic protein (MBP), and DMEM/F-12 medium were from GIBCO BRL (Grand Island, NY). Recombinant human
TGF-
was a product of Calbiochem (La Jolla, CA). All other reagents
were of molecular biology grade and were from either Sigma Chemical or
Fisher Scientific.
Animals and collection of gastric mucosal epithelial cells. Male Fischer 344 rats aged 4-6 (young), 12-24 (middle aged), and 22-24 (old) mo were used. The animals were obtained from the National Institute on Aging (Bethesda, MD), 2 mo before the experiment. During this period, they had access to Purina rat chow and water ad libitum. Animals were fasted overnight before the experiments.
All experiments were performed utilizing freshly isolated gastric mucosal cells. Cells were isolated from overnight fasted rats by a slight modification of the procedure described by Kinoshita et al. (22). Briefly, the contents of the stomach were washed out with PBS. After being transformed into inside-out gastric bags, they were filled with 5 ml of 3 mg/ml Pronase solution in buffer A (0.5 mM NaH2PO4, 1.0 mM Na2HPO4, 70 mM NaCl, 5.0 mM KCl, 11 mM glucose, 50 mM HEPES, pH 7.2, 20 mM NaHCO3, 2 mM EDTA, and 2% BSA). The filled gastric bags were incubated in Pronase-free buffer A at 37°C for 30 min. The gastric bags were then transferred into buffer B (containing 1.0 mM CaCl2 and 1.5 mM MgCl2 instead of EDTA in buffer A) and gently agitated by a magnetic stirrer at room temperature for 1 h. The epithelial cells, dispersed in buffer B, were collected by centrifuging at 500 g for 5 min. The cell pellets were immediately processed for nuclear or total cell extracts.Preparation of nuclear extracts.
Nuclear extracts were prepared by a slight modification of the method
described by Dignam et al. (8) and Gupta et al. (14). Briefly, cells
were resuspended in ice-cold hypotonic buffer [10 mM HEPES, pH
7.9, 1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethysulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 5 µg/ml of aprotinin, pepstatin A, and leupeptin] and incubated for 10 min at 4°C.
Swollen cells were homogenized with 10 or more slow up-and-down strokes in a glass Dounce homogenizer and centrifuged at 3,300 g for 15 min at 4°C. The pelleted nuclei were washed once with ice-cold low-salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM
MgCl2, 20 mM KCl, 0.2 mM PMSF, 1.0 mM DTT, 0.2 mM EDTA, and
5 µg/ml of aprotinin, pepstatin A, and leupeptin) by centrifuging at
10,000 g for 15 min at 4°C. The nuclei were resuspended in
ice-cold low-salt buffer, and nuclear protein was released by adding an
ice-cold high-salt buffer (same as the low-salt buffer, except that it contained 1.2 M KCl) drop by drop to a final concentration of 0.4 M
KCl. The samples were rotated at 4°C for 30 min. The nuclear extracts were recovered by centrifugation at 25,000 g for 30 min at 4°C and stored at 80°C in small aliquots.
Electrophoretic mobility shift assay.
Electrophoretic mobility shift assay (EMSA) was utilized to determine
the transcriptional activity of NF-B and AP-1 by assaying the extent
of binding of nuclear extracts to NF-
B and AP-1 consensus sequences
as described by Gupta et al. (14). Briefly, probes containing the
consensus sequences of NF-
B or AP-1 were labeled with
[
-32P]ATP, using T4 polynucleotide kinase
according to the protocol provided by Promega. Labeled oligonucleotides
were purified by chromatography through a Sephadex G-25 spin column.
For DNA-protein binding reactions, 5 or 10 µg of nuclear protein and
2 µg of poly(dI-dC) · poly(dI-dC) were preincubated
in 20 µl binding buffer (10 mM HEPES, pH 7.5, 4% glycerol, 1.0 mM
MgCl2, 50 mM KCl, 0.5 mM EDTA, and 1.0 mM DTT) for 15 min
at 4°C and then 300,000 counts/min of radiolabeled probe were
added. Reactions were further incubated for 30 min at room temperature.
In NF-
B EMSA, the binding buffer contained 0.05% Nonidet P-40. The
resulting products were separated by 6-7% native polyacrylamide
gel containing 0.25× TBE (89 mM Tris, pH 8.3, 89 mM boric acid,
and 2 mM EDTA) with 0.25× TBE as the running buffer. Gels were
dried and exposed to film at
80°C with intensifying screens.
Signals on the film were quantitated by densitometry using ImageQuant
image analysis system (Storm optical scanner, Molecular Dynamics,
Sunnyvale, CA). Competition was performed by adding the respective
nonradioactive oligonucleotide probes to the reaction mixture in a
50-fold molar excess. All assays were repeated at least three times
using nuclear extracts from different rats for each age group.
Supershift EMSA.
In supershift assays, EMSAs were performed as described above except
that the binding reaction contained 3 µg of polyclonal antibodies to
components of NF-B (p50, p52, or p65) or AP-1 (c-Jun or c-Fos).
Western immunoblot analysis. This was performed according to our standard protocol (35). In all analyses, protein concentration was standardized among the samples. Briefly, nuclear proteins (20 µg) were separated by SDS-PAGE and then electroblotted to Immobilon-P nylon membranes. The membranes were blocked overnight with 5% nonfat dried milk in TBS-T buffer (20 mM Tris, pH 7.6, 100 mM NaCl, and 0.1% Tween 20), followed by 3 h of incubation with the primary antibodies (1:1,000-1:1,500 dilution) in TBS-T buffer containing 5% nonfat dried milk or 1% BSA at room temperature. After being washed three times with TBS-T buffer, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000 dilution) for 1 h at room temperature. Protein bands were visualized using the ECL detection system and quantitated by densitometry. All of the Western immunoblots were performed at least three times using nuclear extracts or total cell lysates from different rats for each age group.
MAPK assay.
Immunocomplex kinase assays were performed as described previously (14,
48). Briefly, the cell pellets were washed with ice-cold PBS and lysed
at 4°C in lysis buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 2.5 mM
EDTA, 2.5 mM Na3VO4, 0.5% Triton X-100, 0.5%
Nonidet P-40, and 5 µg/ml of aprotinin, pepstatin A, and leupeptin).
Lysates were clarified by centrifuging at 11,000 g for 15 min
at 4°C, and the protein concentration in the supernatant was
measured by the Bio-Rad protein assay kit. MAPK/ERK1, MAPK/ERK2, or
JNK1 were immunoprecipitated from cell lysates containing the same
amount of proteins (1,000 µg) with polyclonal rabbit antibodies to
ERK1 and ERK2 or JNK1 and protein G-Sepharose for 3 h at 4°C under
constant stirring. Immunocomplexes were washed three times with TT
buffer (50 mM Tris · HCl, pH 7.6, 0.15 NaCl, and
0.5% Tween 20) and twice with kinase washing buffer [20 mM
HEPES, pH 7.5, 20 mM -glycerol phosphate, 10 mM
p-nitrophenyl phosphate (PNPP), 10 mM MgCl2, 1 mM
DTT, and 0.5 mM Na3VO4]. The kinase reactions were performed by incubating the beads with 25 µl kinase reaction buffer (20 mM HEPES, pH 7.5, 20 mM
-glycerol phosphate, 10 mM PNPP, 10 mM MgCl2, 1 mM DTT, 0.5 mM
Na3VO4, and 20 µM unlabeled ATP) containing 5 µCi [
-32P]ATP and 10 µg of MBP as
substrate for ERK1 and ERK2 or 3 µg of c-Jun as substrate for
JNK1. After 30 min at 30°C, the reactions were stopped
by the addition of 2× sample loading buffer (125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 4%
-mercaptoethanol, and 0.02% bromophenol
blue). The samples were boiled for 4 min and resolved on 12% SDS-PAGE.
The gels were dried and subjected to autoradiography. The extent of
phosphorylation was quantitated by densitometry.
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RESULTS |
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To determine whether aging affects AP-1 and NF-B transcriptional
activity in the gastric mucosa, we performed EMSA to examine the extent
of binding of nuclear extracts from isolated mucosal cells from rats
aged 4-6 (young), 12-14 (middle aged), and 22-24 (old)
mo to the consensus sequence of either AP-1 or NF-
B.
AP-1 DNA binding activity in the gastric mucosa of 12- to 14- and 22- to 24-mo-old rats was increased by ~60% and 190%, respectively, compared with their 4- to 6-mo-old counterparts (Fig.
1). No appreciable binding of nuclear
extracts to the AP-1 consensus was detected in the presence of 50-fold
molar excess of unlabeled oligonucleotide probe (Fig. 1). Because AP-1
is composed of members of the Jun and Fos families (20), supershift
EMSA was performed with antibodies against Jun and Fos to detect their
presence in the DNA-protein complexes. Antibodies against either Jun or
Fos completely supershifted the DNA complexes formed with these
proteins (Fig. 2). No such supershift
occurred in the presence of control rabbit serum (Fig. 2).
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Members of the Jun and Fos families of AP-1 associate to form a variety
of homo- and heterodimers that bind to a common site (20). To determine
whether the increase in AP-1 DNA binding activity in the gastric mucosa
during aging could be partly the result of increased levels of Jun
and/or Fos, the relative concentrations of these proteins were examined
by Western blot analysis. Jun and Fos levels in the gastric mucosa
increased steadily with advancing age (Fig.
3). We observed a 75% and 126% higher
concentration of Jun in 12- to 14- and 22- to 24-mo-old rats,
respectively, than in their 4- to 6-mo-old counterparts (Fig.
3A). Fos levels, on the other hand, rose abruptly with aging.
Whereas the concentration of this protein in the gastric mucosa of 12- to 14-mo-old rats increased by 88% over the 4- to 6-mo-old animals,
levels of mucosal Fos in 22- to 24-mo-old animals were found to be well
over 800%, compared with young rats (Fig. 3B).
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As has been observed for AP-1, there was also a progressive increase in
NF-B DNA binding activity in the gastric mucosa with aging,
revealing 120% and 650% higher values in 12- to 14- and 22- to
24-mo-old rats, respectively, compared with their 4- to 6-mo-old
counterparts (Fig. 4). Similar to what we
observed for AP-1, binding of nuclear extracts to the NF-
B consensus
sequence was also eliminated in the presence of excess unlabeled
oligonucleotide probe (Fig. 4). To detect the presence of
specific NF-
B protein in the DNA-protein complexes, we also
performed gel supershift assays with antibodies against different
components of NF-
B (p50, p52, and p65). As shown in Fig.
5, antibodies against these proteins completely supershifted the DNA complexes formed with different components of NF-
B. No such supershift occurred in the
presence of control rabbit serum.
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NF-B, a member of the Rel family of transcription factors, consists
of five members (c-Rel, p50, p65, p52, and RELB), which form homo- or
heterodimers (3). Different subunit combinations have different
functions in regulating transcription (3). However, the predominant
NF-
B activator of transcription is a p50/p65 heterodimer. To
evaluate the involvement of different members of the Rel family in
modulating NF-
B DNA binding activity in the gastric mucosa during
aging, levels of p50, p52, and p65 subunits in the gastric mucosa of 4- to 6-, 12- to 14-, and 22-to 24-mo-old rats were assayed by Western
blot analysis (Fig. 6). In the gastric mucosa, p52 levels rose steadily with advancing age, revealing 90% and
150% higher values in 12- to 14- and 22- to 24-mo-old rats,
respectively, compared with their younger counterparts. In contrast,
levels of p50 and p65 in the gastric mucosa were not significantly
affected by aging.
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IB
, the most widely studied member of the I
B protein family,
has been shown to play a key role in regulating nuclear import of
NF-
B (3). I
B
binds to NF-
B at a position that blocks nuclear translocation. However, phosphorylation-dependent degradation of I
B
produces dissociation of NF-
B from I
B
, resulting
in translocation of NF-
B to the nucleus (3). Therefore, to determine whether I
B
might be involved in regulating the age-related
changes in NF-
B activation in the gastric mucosa, the levels of
total and phosphorylated forms of I
B
were determined in isolated
gastric mucosal cells by Western immunoblot. As shown in Fig.
7, levels of the total or phosphorylated
form of I
B
in gastric mucosal cells were not affected by aging.
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Previously, we (46) demonstrated that the age-related rise in EGFR
tyrosine kinase activity in the gastric mucosa is accompanied by a
parallel increase in membrane-bound precursor forms of TGF-. In view
of this, we (37, 46) suggested that TGF-
might play a critical role
in regulating EGFR tyrosine kinase through an autocrine/juxacrine
mechanism. Results of our current investigation show that aging is also
associated with increased transcriptional activity of AP-1 and NF-
B
in the gastric mucosa. To determine whether conditions that activate
EGFR will also augment the transcriptional activity of AP-1 and NF-
B
during aging, we examined the effect of TGF-
on the DNA binding
activity of AP-1 and NF-
B in gastric mucosal cells from young and
aged rats. We have observed that exposure of freshly isolated gastric
mucosal cells from aged rats to 1 nM TGF-
for 20 min markedly
stimulates (~50%) the DNA binding activity of both AP-1 and NF-
B
over the corresponding controls (Fig. 8).
In contrast, the same dose of TGF-
stimulated the transcriptional activity of only NF-
B in mucosal cells from young rats, compared with the corresponding controls (Fig. 8).
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Because MAPKs play a key role in regulating the function of many
transcription factors, including AP-1 and NF-B, the next set of
experiments was performed to examine the relationship between the
activity of different MAPKs and AP-1 and NF-
B activation in the
gastric mucosa during aging. Activity of ERKs, as assessed by the
extent of phosphorylation of MBP by immunoprecipitated ERK1 and ERK2,
revealed a progressive rise with advancing age (Fig.
9). The enzyme activity was found to be
55% and 95% higher in 12- to 14- and 22- to 24-mo-old rats,
respectively, compared with their 4- to 6-mo-old counterparts (Fig. 9).
In addition, activation of ERKs was also determined by examining the
levels of phosphorylated ERKs by Western blot. Levels of phosphorylated ERKs in gastric mucosal cells from 22- to 24-mo-old rats were found to
be ~60% higher than in their younger counterparts (Fig. 9). The
relative concentration of ERKs (total) was also found to be ~30%
higher in the gastric mucosa of 22- to 24-mo-old rats compared with
young animals (Fig. 9).
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In the next set of experiments, the age-related changes in JNK1 and p38
activities in the gastric mucosa were examined. Changes in JNK1
activity in the gastric mucosa during aging were very similar to what
we have observed for ERKs. Activity of JNK1, as assessed by the extent
of c-Jun substrate phosphorylation by immunoprecipitated JNK1, was
increased by 100% and 230% in 12- to 14- and 22- to 24-mo-old rats,
respectively, compared with 4- to 6-mo-old animals (Fig.
10). In contrast, aging was associated
with decreased activation of mucosal p38 MAPK activity, as evidenced by
the decreased levels of phosphorylated p38 in 12- to 14- and 22- to
24-mo-old rats compared with 4- to 6-mo-old animals (Fig.
11). In 12- to 14- and 22- to 24-mo-old
rats, p38 MAPK activity in the gastric mucosa was found to be about
one-third of that observed in 4- to 6-mo-old animals (Fig. 11). On the
other hand, no significant difference in total mucosal p38 levels was
observed among different age groups (Fig. 11).
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Finally, it should be stated that although a representative autoradiograph from one experiment was presented, each analysis was repeated three to four times with mucosal cells isolated from three rats in each age group.
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DISCUSSION |
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The structural and functional integrity of the gastrointestinal mucosa are maintained by the constant renewal of cells. Therefore, a detailed knowledge of mucosal cell proliferation and the regulation of this process is essential for a better understanding of the normal aging process as well as many gastrointestinal diseases (including malignancy) that arise from dysregulation of growth (25). Accumulating data (19, 27, 28, 30) suggest that gastrointestinal epithelial cells undergo age-dependent changes in their proliferative rate. Previously, we (38) demonstrated that in rats, gastric mucosal proliferative activity remains elevated during the first 2 wk of life and then decreases dramatically over the next 2-3 wk. Conversely, morphological and biochemical studies have demonstrated that in Fischer 344 rats, aging is associated with increased mucosal proliferative activity in the stomach (2, 37, 36), small intestine (2, 16), and large intestine (2, 17).
Although the intracellular mechanisms responsible for the age-related
rise in gastrointestinal mucosal proliferative activity remain to be
fully elucidated, results from earlier studies (46) suggest a role for
EGFR in regulating this process. We (46) have demonstrated that the
expression and tyrosine kinase activity of EGFR as well as levels of
TGF- are higher in the gastric mucosa of aged rats than in young
rats. In addition, our recent preliminary data show that aging is also
associated with increased sensitivity of gastric mucosal EGFR to EGF
and TGF-
so that low doses of these ligands, which are ineffective
in young rats, can activate EGFR tyrosine kinase in the gastric mucosa
of aged animals (34).
Induction of intrinsic tyrosine kinase activity of EGFR triggers a
complex array of enzymatic events through activation of Ras converging
on MAPKs, which following translocation to the nucleus activate
transcription factors (41, 42, 47). Our current observation that aging
is associated with increased activation of ERKs and JNK1 as well as
AP-1 and NF-B suggests that induction of the EGFR signaling pathway
is partly responsible for the age-related rise in gastric mucosal
proliferative activity. This inference is further supported by the
observation that TGF-
, one of the primary ligands of EGFR, causes
further stimulation of NF-
B and AP-1 transcriptional activity in the
gastric mucosa of aged rats.
Overexpression of c-erbB-2/neu, the structural and functional homologue
of EGFR, which has been implicated in neoplastic transformation of
cells (5, 8, 26, 50), has also been associated with increased
activation of AP-1 and NF-B (11). Previously, we (46) demonstrated
that tyrosine kinase activity and mRNA levels of ErbB-2/neu are also
considerably higher in the gastric mucosa of aged animals than young
animals. Together, the results suggest that both EGFR and ErbB-2 are
involved in modulating the transcriptional activity of AP-1 and NF-
B
and in turn the proliferative activity in the gastric mucosa during aging.
Previous study (20) has demonstrated a relationship between activation of AP-1 and cell proliferation. However, the major components of the transcription factor AP-1 are encoded by two families of genes related to the protooncogenes c-fos and c-jun, the products of which associate to form a variety of homo- and heterodimers that bind to a common site, resulting in transcription of a number of genes involved in cell proliferation (45). Although several mechanisms are thought to be involved in inducing AP-1 activity, they can be broadly classified as those that stimulate the activity and those that increase the abundance of AP-1 (1). Our observation that the age-related rise in AP-1 activity, as evidenced by the increased binding of AP-1 to DNA, is associated with a concomitant rise in c-Jun and c-Fos levels suggests that aging affects the mechanisms that regulate phosphorylation and expression of AP-1 family members in the gastric mucosa. Whether the increased mucosal levels of c-Jun and c-Fos are the result of enhanced synthesis, however, remains to be determined.
Like AP-1, transcriptional activity of NF-B is also augmented in the
gastric mucosa of aged rats. This observation is similar to what has
been noted in the liver (15). Although the regulatory mechanisms for
the increase in mucosal NF-
B DNA binding activity remain to fully be
elucidated, our observation that protein levels of p52, but not p50 and
p65, in the nuclear fraction are increased in aged gastric mucosa
suggests that high p52 levels could enhance the formation of the
NF-
B complex with either p50 or p65. Additionally, alterations in
phosphorylation of p50 and p65 could also play a role in modulating the
binding activity of NF-
B in the gastric mucosa during aging.
However, the age-related rise in activation of NF-
B in the gastric
mucosa could not be attributed to decreased levels or increased
degradation of I
B
, an intracellular protein that functions as a
primary inhibitor of NF-
B (3). Our current data show that aging
produces no significant change in either the total or phosphorylated
form of I
B
. This observation is similar to what has been noted in
a number of other tissues in aged rats (15).
The three MAPKs (ERKs, JNKs, and p38) respond to different stimuli. Whereas ERKs are primarily responsible for responding to proliferation signals, JNKs and p38 respond to cellular stress (10, 21, 23). Our current observation that with aging ERKs and JNK1, but not p38, are induced in the gastric mucosa suggests that all three MAPKs are not equally affected by aging. Because ERKs, which are known to be activated by several growth factors, including the EGF family of peptides (41), are linked to cell proliferation, it is reasonable to assume that induction of ERKs is an essential intracellular event in the EGFR signaling pathway for the age-related rise in proliferative activity in the gastric mucosa. Although JNKs and p38 MAPKs are thought to respond primarily to stress signals, gastric and intestinal injuries that induce activation of EGFR also activate ERKs and JNKs (13, 33, 41). Thus our current observation of the age-related rise in JNK1 activity in the gastric mucosa could be partly the consequence of increased activation of EGFR. Although the detailed EGFR signaling events leading to JNK1 activation remain to be determined, it is possible that the age-related activation of JNKs is mediated by Ras, a key factor in the EGFR signal transduction pathway (41). We believe it is unlikely that the age-related induction of JNK1 is due to stress because the related stress kinase p38 is not activated in the gastric mucosa of aged rats. In fact, the levels of phosphorylated p38 are found to be considerably lower in the gastric mucosa of aged rats than young rats. This could not be attributed to decreased protein levels of p38, which were not significantly different among the three age groups.
In conclusion, our data demonstrate that aging is associated with a
marked induction of AP-1 and NF-B transcriptional activity in the
gastric mucosa, which could be further activated by TGF-
, one of the
primary ligands of EGFR. Aging is also associated with increased
activation of ERKs and JNK1, but not p38, suggesting a role for ERKs
and JNK1 in regulating AP-1 and NF-
B transcriptional activity in the
gastric mucosa during aging. These events may partly be responsible for
the age-related rise in proliferative activity in the gastric mucosa.
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ACKNOWLEDGEMENTS |
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This study was supported by National Institute on Aging Grant AG-14343 and by the Department of Veterans Affairs.
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FOOTNOTES |
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Z.-Q. Xiao is a visiting scientist from Hunan Medical University, Changsha, People's Republic of China.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. P. N. Majumdar, Research Service-151, VA Medical Center, 4646 John R, Detroit, MI 48201 (E-mail: a.majumdar{at}wayne.edu).
Received 13 September 1999; accepted in final form 22 January 2000.
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