Regulation of TGF-
-induced activation of AP-1 in the aging gastric mucosa
Zhi-Qiang Xiao,2
Jianling Li,2 and
Adhip P. N. Majumdar1,2,3
1Veterans Affairs Medical Center,
2Department of Internal Medicine, and
3Karmanos Cancer Institute, Wayne State University
School of Medicine, Detroit, Michigan 48201
Submitted 19 February 2003
; accepted in final form 28 March 2003
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ABSTRACT
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Although the age-related activation of EGF receptor (EGFR) in the gastric
mucosa of Fischer 344 rats is associated with increased DNA binding activity
of activator protein-1 (AP-1), little is known about the EGFR signaling
cascades that regulate this process. The primary objective of this
investigation was to determine the role of signaling pathways initiated by
EGFR in regulating the transforming growth factor-
(TGF-
)-induced activation of AP-1 in the gastric mucosa in aged rats.
Freshly isolated gastric mucosal cells from male young (45 mo) and aged
(2224 mo) rats were used. We have observed that although exposure of
mucosal cells from young (45 mo) and old (2224 mo) rats to 1 nM
TGF-
for 20 min stimulates the DNA binding activity of AP-1 in both age
groups, the magnitude of stimulation is substantially higher in aged (131%)
than in young (35%) rats. This stimulation in the aged is associated with a
concomitant activation of MEKs and ERKs, but not JNKs and p38. The TGF-
induction of AP-1 transcriptional activity in gastric mucosal cells from aged
rats could be totally abrogated by either PD153035, a specific inhibitor of
EGFR tyrosine kinase, or PD98059, a specific inhibitor of MEKs, but not by
Wortmannin, which inhibits phosphatidylinositol kinase. PP2, a specific
inhibitor of Src kinase, produces a 50% inhibition of the TGF-
-induced
activation of AP-1 transcriptional activity. Our results suggest that the
TGF-
-induced stimulation of DNA binding activity of AP-1 in the gastric
mucosa of aged rats is primarily through a signaling pathway involving MEKs
and ERKs, whereas Src kinase pathways play a minor role.
epidermal growth factor receptor signaling processes; phosphatidylinositol kinase; Src kinase; transcription factor
OVER THE PAST SEVERAL YEARS, results from this and other
laboratories have demonstrated that in barrier-reared Fischer 344 rats, aging
is associated with increased mucosal proliferative activity in various tissues
of the gastrointestinal tract, including the oxyntic gland area of the stomach
(2,
11,
12,
21,
19,
22). In the oxyntic gland area
(referred to as gastric mucosa), the age-related rise in mucosal proliferative
activity was demonstrated by increased labeling index, DNA synthesis, and
thymidine kinase and orinithine decarcoxylase (ODC) activities
(6,
1921,
23) Moreover, progression of
gastric mucosal cells through G1 into S phase was also shown to be
increased in aged rats, as reflected by increased levels of cyclin E and the
activity of cyclin-dependent kinase (Cdk2)
(35)
Although the responsible mechanisms for the age-related rise in gastric
mucosal proliferative activity are poorly understood, we have suggested that
EGF receptor (EGFR)-induced signaling pathways may play a critical role in
regulating this process (22,
30). The basis for this
postulation comes from our observation that increased gastric mucosal
proliferative activity during aging in Fischer 344 rats is associated with a
concomitant activation of EGFR, as evidenced by increased tyrosine kinase
activity and tyrosine phosphorylation of the receptor
(30,
34). Aging is also found to be
associated with increased levels of membrane-bound precursor forms of
transforming growth factor-
(TGF-
), one of the primary ligands
of EGFR in the gastric mucosa
(34). In addition, sensitivity
of EGFR in the gastric mucosa to TGF-
and EGF is found to increase with
advancing age (31).
Activation of EGFR triggers a complex array of enzymatic events through
activation of Ras, converging on MAPKs, which, after translocation to the
nucleus, activate transcription factors and produce activation of genes that
promote growth (24,
27,
28,
32). We have demonstrated that
with aging, there is a marked activation of the ERKs and JNKs, and these
changes are accompanied by a concomitant stimulation of the DNA binding
activity of activator protein-1 (AP-1) and NF-
B
(33). AP-1, which is involved
in regulating cell proliferation, responds to many stimuli including growth
factors and cytokines (1,
14). In view of this, we also
examined the responsiveness of AP-1 to TGF-
in the gastric mucosa of
young and aged rats and demonstrated a comparatively greater activation of
AP-1 in aged than in young rats
(33). However, the
intracellular signaling events involved in mediating this process in the
gastric mucosa of aged rats remains to be delineated.
Numerous studies have demonstrated that activation of EGFR not only
stimulates Ras/Raf/MAP kinase signaling cascade but also phosphatidylinositol
(PI3) kinase and Src kinase pathways
(3,
13,
24), each of which is capable
of affecting transcriptional activity of AP-1. However, the signaling
pathway(s) involved in mediating the TGF-
-induced activation of AP-1 in
the gastric mucosa during aging remain to be determined. The present
investigation was, therefore, undertaken to examine the involvement of
different EGFR signaling pathways in regulating the TGF-
-induced
activation of AP-1 in the gastric mucosa of aged rats.
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METHODS
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Reagents. Double-stranded oligonucleotide probe containing the
consensus sequence of AP-1 (5'-CGC TTF ATG AGT CAG CCG GAA-3') and
the T4 polynucleotide kinase were purchased from Promega (Madison, WI), Poly
(dI-dC). Poly (dI-dC) was obtained from Pharmacia Biotech (Piscataway, NJ).
[
-32P]ATP (3,000 Ci/mmol) was from New England Nuclear Life
Science (Boston, MA). Polyclonal rabbit antibodies to EGFR, JNK, and ERK1/2
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal
rabbit antibodies to p38, phospho-p38 (Thr-180/Tyr-182), phospho-ERK1/2
(Thr-202/Tyr-204), phospho-JNK1/2 (Thr-183/Tyr-185), MEK1/2, and
phospho-MEK1/2 (Ser-212/221) were purchased from New England Biolaboratories
(Beverly, MA). Recombinant human TGF-
, PD153035, PD98059, PP2, and
Wortmaninn were obtained from Calbiochem (La Jolla, CA). 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). Concentrated protein assay dye
reagent was from Bio-Rad Laboratories (Hercules, CA). Molecular weight marker
and DMEM medium were from GIBCO-BRL (Grand Island, NY). All other reagents
were of molecular biology grade and were purchased from Sigma or Fisher
Scientific.
Animals and isolation of gastric mucosal epithelial cells. Male
Fischer 344 rats aged 45 (young) and 2224 (aged) mo were used.
The animals were purchased 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 using freshly isolated gastric mucosal
cells. Cells were isolated from overnight fasted rats by a slight modification
(33) of the procedure
described by Kinoshita et al.
(16). Briefly, the contents of
the stomach were washed out with PBS. The stomach was then ligated at the base
of the forestomach and the proximal end of antrum to obtain mucosal cells
primarily from the oxyntic gland area of the stomach. After being transformed
into inside-out gastric bags, they were filled with 5 ml of 3 mg/ml pronase
solution in buffer A (in mM: 0.5 NaH2PO4, 1.0
Na2HPO4, 70 NaCl, 5.0 KCl, 11 glucose, 50 HEPES, pH 7.2,
20 NaHCO3, and 2 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 and subsequently were resuspended in
45 ml serum-free DMEM. The yield ranged between 50 and 70 x
106 cells/stomach. After aliquots of cell suspension in 1 ml of
serum-free DMEM were washed twice with serum-free DMEM, they were exposed to
TGF-
for 20 min at 37°C. In experiments to study the effect of
inhibitors of EGFR signaling pathways, aliquots of cells in 1 ml serum-free
DMEM containing
10 x 106 cells were pretreated with
different concentrations of PD153035, PD98059, Wortmanin, or PP2 for 10 min at
37°C. After treatment of mucosal cells with TGF-
or different
inhibitors, nuclear extracts were immediately prepared and used for assessing
AP-1 activity by EMSA as described in EMSA. At the beginning and end
of each experiment, cell viability was monitored by the trypan blue exclusion
test.
Preparation of nuclear extracts. Nuclear extracts were prepared by
a slight modification (33) of
the method described by others
(4,
10). Briefly, freshly isolated
mucosal cells were resuspended in ice-cold hypotonic buffer (in mM: 10 HEPES,
pH 7.9, 1.5 MgCl2, 10 KCl, 0.2 PMSF, and 1 DTT, with 5 µg/ml of
aprotinin, pepstatin A, and leupeptin) and were 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 (in mM: 20 HEPES, pH 7.9, 1.5 MgCl2, 20 KCl, 0.2 PMSF, 1.0
DTT, and 0.2 EDTA with 25% glycerol 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.
EMSA. EMSA was used to determine the DNA binding activity of AP-1
by assaying the extent of binding of nuclear extracts to AP-1 consensus
sequence as described by Gupta et al.
(10). Briefly, probes
containing the consensus sequences of 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) 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 (cpm) of radiolabeled probe was added.
Reactions were further incubated for 20 min at room temperature. The resulting
products were separated by 6% native polyacrylamide gel containing 0.25x
89 mM Tris, pH 8.3, 89 mM boric acid, and 2 mM EDTA (TBE) with 0.25x 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 50-fold molar
excess. Signals on the blots were visualized by autoradiography and
quantitated by densitometry using ImageQuant image-analysis system. All assays
were repeated at least three times using nuclear extracts from different rats
for each age group.
Cell lysis and Western blot analysis. Freshly isolated gastric
mucosal cells from each rat were lysed in 0.2 ml lysis buffer (50 mM
Tris·HCl, pH 7.4, 100 mM NaCl, 2.5 mM EDTA, 2.5 mM
Na3VO4, 0.5% Triton X-100, 0.5% Nonidet P-40, 5 µg/ml
of aprotinin, pepstatin, and leupeptin). Western blotting was performed
according to our standard protocol
(34,
35). In all Western blot
analyses, protein concentration was standardized among the samples. Briefly,
100 µg proteins were separated on a 10% SDS-PAGE and subsequently
electroblotted to Immobilon-P nylon membranes. The membranes were blocked
overnight with 5% BSA or nonfat dried milk in buffer containing 20 mM
Tris·HCl, pH 7.6, 100 mM NaCl, and 0.01% Tween-20 (TBS-T) followed by 3
h of incubation with the primary antibodies (phospho-ERK1/2, phospho-MEK1/2,
phospho-JNK1/2, or phosphop38, each at 1:1,000 final dilution) in TBS-T buffer
containing 5% BSA at room temperature. After the membranes were washed three
times with TBS-T buffer, they were incubated with a horseradish
peroxide-conjugated goat anti-rabbit IgG (1:5,000 final dilution) as a second
antibody for 1 h at room temperature. Protein band(s) were visualized using
ECL detection system and quantitated by densitometry. After detection of
phospho-ERK1/2, MEK1/2, JNK1/2, or p38, the membranes were treated with
SDS/2-mercaptoethanol stripping buffer and reprobed with the corresponding
nonphosphorylated antibodies (1:1,000 final dilution). All Western blots were
performed at least three times using total cell extracts from different
rats.
EGFR tyrosine kinase activity. Immunocomplex assays were performed
as described previously (31).
Briefly, freshly isolated gastric mucosal cells were washed with ice-cold PBS
and lysed in lysis buffer. Lysates were clarified by centrifuging at 11,000
g for 15 min at 4°C. After determination of protein by the
Bio-Rad protein assay kit, aliquots of cell lysates containing 1.5 mg protein
were subjected to immunoprecipitation with EGFR antibodies and Sepharose G
under constant stirring at 4°C for 3 h. Immunocomplexes were washed three
times with TT buffer (50 mM Tris·HCl, pH 7.6, 0.15 M NaCl, and 0.5%
Tween-20) and twice with kinase buffer [20 mM HEPES, pH 7.5, 20 mM
-glycerol phosphate, 10 mM p-nitrophenyl phosphate (PNPP), 10
mM MnCl2, 1 mM DTT, and 0.5 mM Na3VO4]. The
kinase reactions were performed by incubating the immunoprecipitates with 25
µl kinase reaction buffer (20 mM HEPES, pH 7.5, 20 mM
-glycerol
phosphate, 10 mM PNPP, 10 mM MnCl2, 1 mM DTT, 0.5 mM
Na3VO4, and 20 µM unlabeled ATP containing 5 µCi
[
-32P]ATP). After 30 min at 30°C, the reactions were
stopped by adding 2x loading buffer (125 mM Tris·HCl, pH 6.8, 4%
SDS, 10% glycerol, 4%
-mercaptoethanol, and 0.02% bromophenol blue). The
samples were boiled for 4 min and resolved by 7.5% SDS-PAGE. The gels were
dried and subjected to autoradiography. The extent of EGFR phosphorylation was
quantitated by densitometry as described in EMSA. The kinase assays
were performed three times using cell extracts from different rats.
Statistical analysis. Where applicable, results were analyzed
using ANOVA followed by Fischer's protected least-significant differences or
Scheffé's test. A P value of <0.05 was designated as the
level of significance.
 |
RESULTS
|
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TGF-
-induced activation of AP-1 is greater in the
gastric mucosa of aged than in young rats. Our previous observation that
the age-related rise in EGFR activation in the gastric mucosa was accompanied
by a concomitant increase in membrane-bound precursor form(s) of TGF-
led us to postulate that TGF-
might play a critical role in regulating
gastric mucosal EGFR function during aging through an autocrine/juxtacrine
mechanism (34). To further
determine whether conditions that activate EGFR in aged gastric mucosa will
also stimulate the transcriptional activity of AP-1, we compared the effect of
TGF-
on DNA binding activity of AP-1 in freshly isolated gastric
mucosal cells from young (45 mo) and aged (2224 mo) rats. In
agreement with our earlier observation
(33), we noted that although
TGF-
stimulated DNA binding activity of AP-1 in both age groups, the
magnitude of stimulation was substantially higher in aged (130%) than in young
(35%) rats when compared with the corresponding controls
(Fig. 1). In this and all
subsequent experiments, no appreciable binding of nuclear extracts to the AP-1
consensus sequence was detected in the presence of 50-fold molar excess of
unlabeled oligonucleotide probe, indicating specificity of binding of AP-1 to
oligonucleotide probe containing the consensus sequence of the transcription
factor. Further support for this was derived from our earlier experiment,
which demonstrated that antibodies to either Jun or Fos completely
supershifted AP-1/DNA complex formed with these proteins from rat gastric
mucosal nuclear extracts
(33).
TGF-
-induced stimulation of AP-1 activity in aged
gastric mucosa is dependent on EGFR activation. To determine the role
EGFR plays in regulating the TGF-
-induced stimulation of DNA binding
activity of AP-1 in the gastric mucosa of aged rats, we exposed freshly
isolated gastric mucosal cells from 22- to 24-mo-old (aged) rats to either
TGF-
or PD153035, a specific inhibitor of EGFR
(8), or a combination of both
for 10 min and subsequently examined for changes in EGFR tyrosine kinase
activity and DNA binding activity of AP-1. As shown in
Fig. 2, whereas TGF-
by
itself caused a significant three- to fourfold stimulation in EGFR
phosphorylation, addition of PD153035, at a dose of either 1 or 5 µM,
totally abrogated this stimulation. PD153035 by itself caused no apparent
change in EGFR phosphorylation when compared with the control
(Fig. 2). A similar phenomenon
was also observed for the DNA binding activity of AP-1 in that TGF-
stimulated the DNA binding activity of AP-1, and this stimulation could be
completely blocked by the EGFR inhibitor PD153035
(Fig. 3). Taken together, the
data suggest that the TGF-
-induced stimulation of DNA binding activity
of AP-1 in the gastric mucosa of aged rats is through the EGFR signaling
cascades.
TGF-
activates ERKs but not JNKs and p38.
Activation by TGF-
of EGFR initiates a series of signaling events
through phosphorylation of interacting proteins, including ERKs, JNKs, and
p38, which, in turn, transmit signals to the nucleus by activating
transcriptional factors. To determine the involvement of MAPK signaling
processes in regulating TGF-
-induced activation of AP-1 in the aged
gastric mucosa, we examined the effect of TGF-
or PD153035, alone or in
combination, on activation of ERK1/2, JNK1/2, or p38 in freshly isolated
gastric mucosal cells from 22- to 24-mo-old rats. We observed that whereas
TGF-
caused a marked stimulation in ERK1/2 activation, as evidenced by
a close to threefold increase in the levels of phosphorylated ERK1/2
(Fig. 4), it produced no
significant change in activation of either JNK1/2 or p38
(Fig. 5). However, the
TGF-
-induced stimulation of ERK1/2 phosphorylation was totally
abrogated by 5 µM PD153035, a specific inhibitor of EGFR, and by
60%
with a 1 µM dose of the same inhibitor
(Fig. 4). PD153035 by itself
produced no apparent change in the levels of phosphorylated ERK1/2, JNK1/2, or
p38 (Figs. 4 and
5). The observed changes in the
levels of phosphorylated ERKs in response to TGF-
, PD153035, or
TGF-
plus PD153035 could not be attributed to differences in loading,
because the levels of total ERK1/2 were found to be the same among the samples
(Fig. 4).

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Fig. 5. Effect of EGFR tyrosine kinase inhibitor PD153035 on TGF- -induced
changes in the levels of total and phosphorylated forms of JNK1/2 and p38 in
freshly isolated gastric mucosal cells from 23-mo-old rats. The experimental
protocol was the same as that described in legend to
Fig. 4 with the exception that
polyclonal antibodies to phosphorylated and nonphosphorylated JNK1/2 or p38
were used. Relative changes in phosphorylated over the corresponding
nonphosphorylated JNK1/2 or p38, as determined by densitometric analysis, are
shown in the histogram. Densitometric value from control cells was taken as 1.
Values represent the means ± SE of 4 observations.
|
|
TGF-
-induced stimulation of DNA binding activity of
AP-1 is dependent on MEK1/2 and ERK1/2. The data from the foregoing
experiments (Figs. 3 and
4) demonstrate that the
PD153035-induced inhibition of TGF-
induction of EGFR phosphorylation
in gastric mucosal cells from aged rats leads not only to attenuation of ERK
activation but also DNA binding activity of AP-1. We hypothesize that the ERK
signaling pathway plays a key role in regulating the TGF-
-induced
activation of AP-1 in aged gastric mucosa. To test this hypothesis, we
examined the role of MEK1/2, an upstream regulator of ERK1/2, in modulating
ERKs and AP-1 activity in gastric mucosal cells from aged rats. We have
observed that exposure of freshly isolated gastric mucosal cells from 22- to
24-mo-old (aged) rats to TGF-
, which stimulates ERK1/2 activation
(Fig. 4), also causes a
significant twofold stimulation of activation of MEKs (as evidenced by the
increased levels of phosphorylated MEK1/2) without affecting the levels of
total MEK1/2 (Fig. 6). Again,
this induction could be completely inhibited by 5 µM PD153035 and by
36% with 1 µM of the same compound, indicating that inhibition of EGFR
activation leads to decreased activation of MEKs.
To determine whether inhibition of TGF-
-induced activation of MEKs
would lead to attenuation of ERK activation and, in turn, the DNA binding
activity of AP-1 in the gastric mucosa, isolated gastric mucosal cells from
aged rats were exposed to TGF-
or PD98053, a specific inhibitor of
MEKs, alone or in combination. The cells were assayed for the levels of
phosphorylated ERKs and DNA binding activity of AP-1. As expected, the levels
of phosphorylated ERK1/2 in gastric mucosal cells were greatly increased
(
2-fold) in response TGF-
when compared with the controls, and
this induction was completely inhibited by 5 µM PD98059
(Fig. 7). Neither TGF-
nor PD98059 caused any significant change in the levels of total ERK1/2
(Fig. 7). Similar to what has
been observed for ERKs, the DNA binding activity of AP-1 was also greatly
stimulated (
3-fold) by TGF-
over the corresponding control
(Fig. 8). Again, this
stimulation was totally abrogated by 5 or 25 µM PD98059
(Fig. 8).
TGF-
-induced stimulation of DNA binding activity of
AP-1 involves Src kinase but not PI3 kinase signaling pathway. Activation
of EGFR not only stimulates Ras-MAPK signaling pathway but also PI3 and Src
kinase signaling processes. The last set of experiments were therefore
performed to determine the involvement of PI3 and Src kinases in mediating the
TGF-
-induced activation of AP-1 in the gastric mucosa of aged rats. The
experimental protocol was the same as stated above, with the exception that
Wortmannin, a specific inhibitor of PI3 kinase, or PP2, a specific inhibitor
of Src kinase, was used either alone or in combination with TGF-
. The
cells were assayed for the DNA binding activity of AP-1. As expected,
TGF-
caused a significant three- to four-fold stimulation in DNA
binding activity of AP-1 (Fig.
9). However, Wortmannin at a dose of either 20 or 100 nM produced
no significant inhibition of the TGF-
-induced increase in DNA binding
activity of AP-1 (Fig. 9). In
contrast, the threefold stimulation of the DNA binding activity of AP-1 by
TGF-
was inhibited by
50% with 50 or 250 nM PP2
(Fig. 10).
 |
DISCUSSION
|
---|
The structural and functional integrity of different tissues of the
gastrointestinal tract, including that of the stomach, are maintained by
constant renewal of cells. Therefore, a detailed knowledge of mucosal cell
proliferation and regulation of this process is essential for a better
understanding of the normal aging process as well as many gastrointestinal
diseases that arise from dysregulation of growth. Although earlier
observations in the mouse suggest that with aging, proliferative activity of
the small intestine either decreases
(7,
18) or remains unchanged
(9), recent morphological and
biochemical studies from our own and other laboratories have demonstrated that
in barrier-reared Fischer 344 rats, aging is associated with increased mucosal
proliferative activity in various parts of the gastrointestinal tract,
including that of the stomach
(2,
11,
12,
19,
21,
22).
Although the responsible mechanism(s) for the age-related rise in
gastrointestinal mucosal proliferative activity in Fischer 344 rats still
remains to be elucidated, results from this laboratory suggest a role for EGFR
signaling pathways in regulating this process. The basis for this postulation
comes from the observation that increased gastric mucosal proliferative
activity during aging is accompanied by a concomitant increase in tyrosine
kinase activity and tyrosine phosphorylation of EGFR, leading to a marked
activation of ERKs and JNKs and stimulation of DNA binding activity of AP-1
and NF-
B (33).
Moreover, the fact that these changes are also associated with a marked rise
in the levels of membrane-bound precursor form(s) of TGF-
, one of the
primary ligands of EGFR that is synthesized in the gastric mucosa, suggests a
role for TGF-
in regulating the EGFR signaling processes during aging
(30,
34). Our current observation,
which is similar to what we noted earlier
(33), provides further support
for this postulation. We have observed that although TGF-
stimulates
the DNA binding activity of AP-1 in gastric mucosal cells from both young and
aged rats, the magnitude of this stimulation is considerably greater in aged
than in young rats. Whether this could partly be the result of increased
sensitivity of the gastric mucosa of aged rats to TGF-
remains to be
determined. This possibility arises from our earlier observation that lower
concentrations of EGF or TGF, which are ineffective in stimulating tyrosine
kinase activity of EGFR in the gastric mucosa of young rats, induce activation
of the same in aged rats
(31).
Induction of tyrosine kinase activity of EGFR is one of the essential
events for activating the receptor signaling processes. It is generally
accepted that after ligand binding, EGFR undergoes dimerization resulting in
activation of the intrinsic tyrosine kinase via auto- and
transphosphorylation. The receptor phosphorylation, in turn, leads to
activation of a number of signaling pathways, including PI3 and Src kinases
(13,
27,
32). Subsequently, the
activated signaling pathways converge on the nuclear transcription factor AP-1
as shown schematically in Fig.
11. Although the intracellular events regulating the
TGF-
-induced activation of AP-1 transcriptional activity in aged
gastric mucosa have not been fully elucidated, our observation that doses of
PD153035, which completely inhibited the TGF-
-induced phosphorylation
of EGFR in gastric mucosal cells from aged rats and also produced a similar
inhibition of DNA binding activity of AP-1, suggests that EGFR-dependent
signaling pathways are important in regulating the TGF-
-induced
stimulation of AP-1 transcriptional activity in the gastric mucosa of the
aged.

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Fig. 11. Schematic representation of ligand activation of EGFR signaling pathways
leading to stimulation in transcriptional activity of AP-1. SOS, son of
sevenless; SHC, Src homology collagen; GRB, growth factor
receptor-binding.
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|
Numerous studies have demonstrated that the MAPK signaling pathways, which
regulate cellular growth and differentiation, respond to EGFR activation
(24,
26,
29). At least three distinct
families of MAPKs are present in mammalian cells that include p42/44 ERKs,
JNK/SAPKs, and p38 (25). It
has been suggested that ERKs are primarily responsive to cell proliferation
signals, whereas JNKs and p38 respond to cellular stress
(5,
15,
17). Our current observation
that, in gastric mucosal cells from aged rats, TGF-
induces activation
of ERKs but not JNKs or p38 and that the activation of ERKs by TGF-
can
be totally abrogated by the EGFR tyrosine kinase inhibitor PD153035 suggests
that the downstream events of EGFR activation leading to stimulation of DNA
binding activity of AP-1 are through the ERKs signaling pathway. Further
support for this postulation comes from the observation that inhibition of MAP
kinase kinase (MEK1/2), an upstream regulator of MAPKs, by PD98059, a specific
inhibitor of MEKs, not only inhibits TGF-
-induced activation of ERKs
but also the DNA binding activity of AP-1 in gastric mucosal cells from aged
rats. Taken together, the results suggest that in aged gastric mucosa,
TGF-
-induced activation of the DNA binding activity of AP-1 is through
a signaling pathway involving MEK1/2 and ERK1/2.
EGFR activation is known to induce a number of signaling pathways,
including PI3 and Src kinases. As depicted in
Fig. 11, whereas activation of
Src kinase can stimulate transcriptional activity of AP-1 either directly or
through activation of the Ras-MAPK pathway, PI3 kinase-mediated activation of
AP-1 can occur independently of MAPK. Our current data suggest that the PI3
kinase pathway is not involved in TGF-
-induced activation of AP-1 in
the gastric mucosa of aged rats, because Wortmannin, a specific inhibitor of
PI3 kinase, failed to block the TGF-
-dependent AP-1 activation. In
contrast, inhibition of Src kinase by PP2 caused a partial inhibition of the
TGF-
-induced stimulation of the DNA binding activity of AP-1 in the
gastric mucosa of aged rats, indicating a minor role of this signaling pathway
in regulating AP-1 activation.
In conclusion, our data demonstrate that aging is associated with a marked
induction of the DNA binding activity of AP-1 in the gastric mucosa. This
could be further activated by TGF-
, one of the primary ligands of EGFR.
This stimulation of the DNA binding activity of AP-1 in the gastric mucosa of
aged rats is found to be primarily through a signaling pathway involving MEKs
and ERKs, whereas Src kinase pathways play a minor role in this process. The
PI3-kinase signaling pathway does not appear to be involved in regulating the
TGF-
-induced stimulation of AP-1 activation in the gastric mucosa
during aging.
 |
DISCLOSURES
|
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This study was supported by the National Institute on Aging AG-14343 and by
the Department of Veterans Affairs.
Z.-Q. Xiao is a visiting scientist from Human Medical University, Changsha,
Peoples Republic of China.
 |
FOOTNOTES
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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).
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.
 |
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