2 Veterans Affairs Medical Center and 1 Departments of 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 is
associated with increased epidermal growth factor receptor (EGFR)
tyrosine kinase activity in Fischer 344 rat gastric and colonic mucosa,
the regulatory mechanisms for the age-related rise in EGFR tyrosine
kinase are poorly understood. Transmembrane transforming growth
factor- (TGF-
) may modulate EGFR function through an
autocrine/juxtacrine mechanism. The present study aimed to determine
the contribution of membrane-bound precursors of TGF-
in enhancing
EGFR activation in the gastric and colonic mucosa during aging. The
extent of EGFR tyrosine phosphorylation, a measure of EGFR activation,
was substantially higher (300-350%) in the gastric and colonic
mucosa of 23- (aged) vs. 4-mo-old (young) Fischer 344 rats. This was
accompanied by an increase (200-1,000%) in the relative
concentration of 18- to 20-kDa membrane-bound precursor forms of
TGF-
. The amount of TGF-
bound to EGFR was also higher
(150-250%) in the gastric and colonic mucosa of aged vs. young
rats. In vitro studies revealed that exposure of HCT 116 cells (a colon
cancer cell line) to TGF-
from gastric and colonic mucosal membranes
of aged rats caused a 200-250% higher activation of EGFR and
extracellular signal-related kinases (p42/44) compared with young rats.
Our data suggest that the membrane-bound precursor form(s) of TGF-
may partly be responsible for enhancing EGFR activation in the gastric
and colonic mucosa of aged rats, probably though an
autocrine/juxtacrine mechanism(s).
epidermal growth factor receptor signal transduction; tyrosine
kinase; autocrine/juxtacrine mechanism; transforming growth
factor-
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INTRODUCTION |
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OVER THE PAST SEVERAL
YEARS, results from this and other laboratories (1, 8, 9,
14, 16, 17, 26) have demonstrated that in Fischer 344 rats,
aging is associated with increased mucosal proliferative activity in
much of the gastrointestinal tract, including the stomach and
colon. This has, in part, been attributed (29, 31)
to the enhanced transition of mucosal cells through G1 to S
phase of the cell cycle. In addition, in the gastric mucosa, aging is
found to be associated with increased activation of the p42/44
extracellular signal-related kinases (ERKs) and c-Jun
NH2-terminal/stress-activated kinases and transcriptional
activity of activator protein-1 and nuclear factor-B
(30).
Although the responsible molecular mechanisms for the age-related
increase in gastrointestinal mucosal proliferative activity are poorly
understood, we (17, 25) have observed that 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-
). Basal EGFR tyrosine kinase activity in the
colonic mucosa of aged Fischer 344 rats is also found to be higher than
in young animals (19). Numerous studies (11,
23) have demonstrated that the EGF family of peptides, particularly EGF and TGF-
, stimulate proliferative activity in much
of the gastrointestinal tract, including the stomach and colon.
EGF and TGF- initiate their mitogenic action by activating the
intrinsic tyrosine kinase activity of their receptor, triggering the
EGFR signaling processes (21, 23, 27). However, TGF-
, as opposed to EGF, is synthesized in the mucosa of much of the gastrointestinal tract (2, 5, 23), suggesting that TGF-
may be physiologically more important than EGF in modulating mucosal proliferation. Results from cell surface immunocytochemical and biochemical characterization studies (4, 20, 22) have
demonstrated that the presence of the transmembrane TGF-
at the cell
surface is a normal consequence of TGF-
synthesis, and in most cases the peptide is present on the cell surface in its precursor form. Moreover, it has been reported (3, 4) that the
membrane-bound precursor form(s) of TGF-
can also activate the
intrinsic tyrosine kinase activity of EGFR, suggesting a role for the
precursors of the growth factor in regulating EGFR signal transduction
pathways, probably through an autocrine/juxtacrine mechanism
(3). Our (25) earlier observation that, in
the gastric mucosa, the age-related rise in EGFR tyrosine kinase
activity is associated with a concomitant increase in
membrane-associated precursor forms of TGF-
, suggests a plausible
role for the membrane-bound form(s) of TGF-
in modulating mucosal
EGFR function during aging. Whether the same phenomenon prevails in the
colonic mucosa has not been investigated. In addition, no information
is available as to whether the membrane-bound precursor form(s) of
TGF-
from the gastric and/or colonic mucosa will modulate EGFR
activation in vitro, and if so, whether this modulation will be
affected by aging. Therefore, to determine the role of the membrane-bound precursors of TGF-
in regulating EGFR function in the
gastric and colonic mucosa during advancing age, the present investigation examines 1) the relationship between EGFR
activation and the levels of membrane-associated precursor form(s) of
TGF-
in the gastric and colonic mucosa during aging and
2) whether aging alters the ability of the membrane-bound
precursors form(s) of TGF-
to activate EGFR and the signaling pathway.
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METHODS |
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Reagents.
Rabbit polyclonal antibodies to EGFR and monoclonal antibody against
TGF- were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Monoclonal antibody against phosphotyrosine (4G10) was from Upstate
Biotechnology (Lake Placid, NY). Polyclonal antibodies to phospho-ERKs
[p42/44; (Thr202/Tyr204)] were from New
England Biolabs (Beverly, MA). Enhanced chemiluminescence (ECL) protein
biotinylation module (RPN2202), goat anti-mouse or rabbit IgG
conjugated with horseradish peroxidase (HRP), and the ECL system were
products of Amersham (Arlington Heights, IL). Immobilon-P nylon
membrane was purchased from Millipore (Bedford, MA). Concentrated
protein assay dye reagent was from Bio-Rad (Hercules, CA). Molecular
weight marker was obtained from GIBCO-BRL (Grand Island, NY). All other
reagents were of molecular biology grade and were purchased from either
Sigma or Fisher Scientific.
Animal and collection of tissues.
In all experiments, male Fischer 344 rats aged 4 (young) and 23 mo
(old) were used. The animals were purchased from the National Institute
on Aging (Bethesda, MD) at least 1 mo before the experiment. During
this period, the rats had access to Purina rat chow and water ad
libitum. Four to five animals from each age group were utilized in this
investigation. In all experiments, animals were fasted overnight before
being killed. The stomach and entire colon were removed and used
immediately for isolation of cells. In experiments in which mucosa was
to be used, the stomach and colon were slit open and rinsed thoroughly
with cold PBS. Mucosa was obtained by scraping with glass slides.
Mucosal scraping were either processed immediately or frozen in small
aliquots in liquid nitrogen and stored at 90°C.
Isolation of membrane-bound TGF-.
Crude mucosal membranes (30,000 g pellet) were prepared as
described previously (26) and subsequently solubilized in
lysis buffer (10 mM HEPES, pH 7.2, 150 mM NaCl, 2.5 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM EDTA, 25 µg/ml of aprotinin, leupeptin, and pepstatin A, 0.5%
Triton X-100, and 0.5% Nonidet P-40). After clarification at 11,000 g for 15 min at 4°C, the supernatants were used for
immunoprecipitation of TGF-
. In all immunoprecipitation studies,
protein concentration was standardized among the samples.
Immunoprecipitation and Western immunoblot analysis.
This was performed according to our standard protocol
(29-31). Briefly, aliquots of the mucosal membranes
containing 2 or 3 mg proteins were incubated with either polyclonal
anti-EGFR or monoclonal anti-TGF- antibodies and Sepharose-protein G
or Sepharose-protein A beads at 4°C for 3 h. Immunoprecipitates
were resolved on a 7.5% or 15% SDS-PAGE. The electrophoresed proteins
were transferred to Immobilon-P nylon membranes. The membranes
containing EGFR were probed with anti-phosphotyrosine, and those
containing TGF-
were probed with TGF-
antibodies. Protein bands
were visualized by the ECL detection system and quantitated by
densitometry. All Western immunoblots were performed at least three
times using different rats from each age group.
Activation of EGFR in vitro.
This was performed using the colon cancer cell line HCT 116. Briefly,
aliquots of HCT 116 cells were incubated with 20 µl of TGF- eluted
from gastric or colonic mucosal membranes (containing 3 mg protein) as
described above. Cells were incubated for 8 min at 37°C. After
incubation, cells were lysed with 300 µl lysis buffer (50 mM Tris
buffer, pH 7.4, 150 mM NaCl, 2.5 mM EDTA, 2.5 mM
Na3VO4, 25 µg/ml of aprotinin, leupeptin, and
soybean trypsin inhibitor A, 0.5% Triton X-100, and 0.5% Nonidet
P-40). EGFR was immunoprecipitated from aliquots of cell lysates
containing 1 mg protein as described above. The immunprecipitates were
washed three times with TT buffer and twice with kinase buffer [25 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MnCl2, 0.5 mM
dithiothreitol (DTT), 0.5 mM Na3VO4, 5 mM
-glycerol phosphate, and 10 mM p-nitrophenyl phosphate],
and finally incubated in 25 µl kinase reaction mixture (25 mM HEPES,
pH 7.5, 100 mM NaCl, 5 mM MnCl2, 0.5 mM DTT, 0.5 mM
Na3VO4, 5 mM
-glycerol phosphate, 10 mM
p-nitrophenyl phosphate, and 20 µM ATP) containing 5 µCi
[
-32P]ATP for 30 min at 30°C. The reactions were
stopped by adding 2× 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 subjected to a 7.5% SDS-PAGE.
The gels were dried and autoradiographed. The extent of EGFR
phosphorylation was quantitated by densitometry.
Biotinylation of cell surface proteins in gastric and colonic
mucosal cells.
The presence of TGF- on gastric and colonic mucosal cell surface was
examined by biotinylation. For this purpose, mucosal cells from all
groups of rats were isolated by a slight modification of the procedures
described by Kinoshita et al. (12). Briefly, contents of
the stomach and colon were washed out by rinsing with PBS. The stomach
and colon were transformed into inside-out gastric or colonic bags,
respectively, and filled with 3 (stomach) or 1 mg/ml (colon) Pronase
solution in buffer A (0.5 mM
NaH2PO4, 1 mM Na2HPO4,
70 mM NaCl, 5 mM KCl, 11 mM glucose, 50 mM HEPES, pH 7.2, 20 mM
NaHCO3, 2 mM EDTA, and 2% BSA). The filled bags were
incubated in Pronase-free buffer A at 37°C for 30 min. The bags were then transferred into buffer B (containing 1 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 immediately utilized for biotinylation of protein using a
commercial ECL biotinylation kit (Amersham), according to the
manufacturer's suggested protocol. Briefly, freshly isolated gastric
or colonic mucosal cells were washed twice with cold PBS and
resuspended in 1 ml ice-cold 40 mM of bicarbonate buffer at a
concentration of 5 × 106 cells/ml. Next, 40 µl of
biotinylation reagent (biotinamidocaproate N-hydroxysuccinimide ester) were added per milliliter of
cell suspension and subsequently incubated for 30 min at 4°C under constant agitation. The labeled cells were washed three times with cold
PBS and lysed in lysis buffer and then centrifuged for 15 min at 4°C.
Aliquots of cell lysates containing 500 µg proteins were subjected to
immunoprecipitaton with 1 µg monoclonal anti-TGF-
antibodies and
protein A-Sepharose beads at 4°C for 3 h. The immunoprecipitates were subjected to 15% SDS-PAGE. The electrophoresed proteins were transferred to the Immobilon-P membranes, blocked with 5% blocking reagent in PBS containing Triton X-100 for 1 h and incubated with a 1:1,500 dilution of streptavidin-HRP for 1 h at room
temperature. The proteins were visualized with the ECL
detection system and quantitated by densitometry.
Statistical analysis. Results were statistically evaluated using Student's t-test for unpaired values.
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RESULTS |
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Although we (17, 25) have repeatedly demonstrated
that aging is associated with increased activation of EGFR in the
gastric mucosa, this phenomenon has not been investigated in detail in the colonic mucosa. Therefore, in the current investigation, we examined the changes in EGFR activation in the colonic mucosa of young
(4 mo) and aged (23 mo) rats by analyzing the extent of tyrosine
phophorylation of the receptor. The gastric mucosa was also included
for comparison purposes. Results revealed that in both gastric and
colonic mucosa, aging was associated with a marked increase in EGFR
activation. The levels of phosphotyrosine EGFR in the gastric and
colonic mucosa of 23-mo-old rats were found to be 300-350% higher
than the corresponding levels in 4-mo-old animals (Fig.
1).
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To determine whether the age-related increase in EGFR activation in the
gastric and colonic mucosa was associated with a concomitant increase
in membrane-bound precursor form(s) of TGF-, we analyzed the
relative concentration of this form(s) in the gastric and colonic
mucosa of 4- and 23-mo-old rats by both Western immunoblot and
biotinylation of cell surface proteins. The results of Western blot
analysis and biotinylation of cell surface proteins revealed the
presence of precursors of TGF-
in the gastric and colonic mucosal
membranes of both age groups; the molecular mass of the membrane-bound
forms of TGF-
ranged between 18-20 kDa (Fig.
2). The relative concentration of these
TGF-
forms was found to be substantially higher (200-1,000%)
in 23-mo-old rats, compared with the corresponding levels in 4-mo-old
animals (Fig. 2). It should also be noted that under both experimental
conditions, the TGF-
band from gastric and colonic mucosal membranes
from aged rats was broader than the corresponding band from young
animals (Fig. 2). This could due to the presence of several closely
related precursor forms of TGF-
in gastric and colonic mucosal
membranes of aged rats.
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Because ligand binding is one of the primary causes of activation of
EGFR, we sought to determine whether a part of the age-related rise in
EGFR activation in the gastric and colonic mucosa could be due to a
greater amount of TGF- bound to EGFR. Indeed, results of four
experiments consistently showed the proportion of TGF-
bound to EGFR
in the gastric and colonic mucosa of aged rats to be considerably
higher than in young animals. The results, depicted in Fig.
3, revealed that the proportion of
TGF-
bound to EGFR in the gastric and colonic mucosa of aged rats
was 150-250% higher than in young animals. The molecular
form(s) of TGF-
bound to EGFR in all samples was between 18 and 20 kDa, indicating that the major forms of TGF-
bound to EGFR are high
molecular mass precursor forms of the peptide.
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The next set of experiments was performed to compare the ability of
TGF- present in gastric and colonic mucosal membranes of young and
aged rats to activate EGFR. In vitro studies were performed utilizing
the colon cancer cell line HCT 116. We observed that exposure of HCT
116 cells to TGF-
, extracted from gastric and colonic mucosal
membranes of young and aged rats (TGF-
immunoprecipitated from 3 mg
membrane and subsequently eluted), stimulated EGFR activation as
evidenced by increased phosphorylation of the receptor compared with
the control (Fig. 4). However, whereas
TGF-
from mucosal membranes from 4-mo-old rats produced only a minor
(20-30%) increase, the peptide extracted from aged gastric and
colonic mucosal membranes caused a marked 175-250% stimulation in
EGFR activation compared with the control (Fig. 4). As a positive
control, we also measured the magnitude of stimulation of EGFR
phosphorylation in HCT 116 cells in response to 1 nM synthetic TGF-
,
which produced an ~400% stimulation in EGFR activation compared with
the control (Fig. 4).
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The last set of experiments was performed to determine whether the
observed induction in EGFR activation in HCT 116 cells by TGF- from
aged gastric and colonic mucosal membranes will also result in
activation of the downstream events of the EGFR signal transduction
pathways. We examined the extent of phosphorylation of ERKs (p42/44),
one of the mitogen-activated protein kinases, in HCT 116 cells after an
8-min exposure to TGF-
from gastric and colonic mucosal membranes
from young and aged rats. As observed for EGFR, TGF-
, extracted from
gastric and colonic mucosal membranes of aged rats, markedly stimulated
(100-200%) the levels of phosphorylated ERKs (p44/42), compared
with the control (Fig. 5). On the other hand, TGF-
from gastric and colonic mucosal membranes of young rats
caused only a small (20-40%) increase in phosphorylation of ERKs
over the control (Fig. 5).
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DISCUSSION |
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Accumulating evidence suggests that many gastrointestinal hormones
and growth factors, most notably EGF and its structural and functional
homologue TGF-, are critically involved in regulating mucosal growth
in various parts of the gastrointestinal tract, including the stomach
and colon (2, 11, 23). However, mucosal responsiveness to different gastrointestinal hormones and growth factors changes with aging. We (15, 18) have reported that doses of gastrin and bombesin, which stimulate gastric mucosal proliferative activity in young rats, have no effect on mucosal proliferation in aged rats. On the other hand,
pharmacological doses of either EGF or TGF-
have been found to
inhibit gastric mucosal proliferation in aged rats (14,
26). This has, however, been attributed to increased sensitivity
of the aged gastric mucosa to EGF and TGF-
, so that low doses are
stimulatory, whereas high doses inhibit proliferative processes
(26).
EGF and TGF- exert their mitogenic action by binding to their common
receptor, the EGFR, a 170-kDa transmembrane glycoprotein with intrinsic
tyrosine kinase activity (21, 27). On binding to ligands,
EGFR undergoes dimerization followed by activation of the intrinsic
tyrosine kinase with subsequent auto- and transphosphorylation of the
receptor leading to the induction of the signal transduction pathways,
resulting in stimulation in cell proliferation (7, 21,
27). Our current observation that activation of EGFR, as
assessed by the extent of tyrosine phosphorylaton of the receptor, is
higher in the gastric and colonic mucosa of aged than in young rats
supports our contention that stimulation of EGFR signaling pathways is
partly responsible for the age-related increase in gastric and colonic
mucosal proliferation. Although the regulatory mechanisms for EGFR
activation in the gastric and colonic mucosa of aged rats remain to be
elucidated, we postulated that TGF-
, one of the primary ligands for
EGFR, plays a key role in regulating this process. The basis for this
postulation comes from the fact that TGF-
, but not EGF, is
synthesized in much of the gastrointestinal tract (2, 5,
23), and specific binding sites for TGF-
are particularly
abundant in the stomach and colon (24). Additionally, we
(25) have reported that the age-associated rise in
tyrosine kinase activity and tyrosine phosphorylation of EGFR in the
gastric mucosa is also accompanied by a concomitant increase in the
levels of membrane-bound precursor form(s) of TGF-
. Our current
observation that the levels of membrane-bound precursors form(s) of
TGF-
are also higher in the gastric and colonic mucosa of aged than in young rats suggests that the membrane-bound TGF-
may partly be
responsible for activating EGFR in these tissues, probably through an
autocrine/juxtacrine mechanism(s). Additional support comes from the
observation that the fraction of TGF-
bound to gastric and colonic
mucosal EGFR is also higher in aged than in young rats. Because aging
is also associated with increased expression of EGFR protein in the
gastric mucosa (25), our finding of a greater amount of
TGF-
bound to EGFR in the gastric and colonic mucosa of aged than in
young rats could partly be due to an increased number of EGFR molecules.
TGF- is derived from a larger 20- to 22-kDa transmembrane precursor
(4, 6), and the presence of the transmembrane TGF-
at
the cell surface is a normal consequence of TGF-
synthesis (4,
20, 22). Although the precise mechanism(s) for accumulation of
precursor form(s) of TGF-
in cellular membranes has not been fully
elucidated, it has been demonstrated that release of mature TGF-
from pro-TGF-
is inefficient in most cells, which may cause accumulation of pro-TGF-
on the cell surface (20).
However, studies (4, 13) utilizing different cell lines
have demonstrated that the membrane-associated precursors of TGF-
are biologically active in that they activate intrinsic tyrosine
kinases on adjacent cells. Our current data are in complete agreement
with these observations. The results of our biotinylation of cell
surface proteins show the presence of 18- to 20-kDa transmembrane
precursor forms of TGF-
in both gastric and colonic mucosa,
indicating that normal gastrointestinal mucosal membranes, as has been
observed in malignant cells (20), also possess precursors
of TGF-
. The fact that the relative concentration of these
forms of TGF-
in gastric and colonic mucosal membranes is higher in
aged than in young rats suggests that they are partly responsible for
modulating the age-related increase in EGFR activation in the gastric
and colonic mucosa. In support of this postulation, we have observed that exposure of HCT 116 cells to TGF-
, extracted from gastric and
colonic mucosal membranes of aged rats, causes a substantially greater
stimulation in EGFR phosphorylation, compared with the levels achieved
with the corresponding TGF-
from young rats. Further support comes
from the observation that, in HCT 116 cells, TGF-
from gastric and
colonic mucosal membranes of aged rats produces a comparatively greater
stimulation in ERK activation than that achieved with the
membrane-associated peptide from young rats. ERKs, which are known to
be activated by several growth factors, including the EGF family of
peptides, are linked to cell proliferation (21). Our
observation of increased activation of ERKs (p42/44) in HCT 116 cells
by TGF-
from gastric and colonic mucosal membranes of aged rats
further suggests participation of the transmembrane TGF-
in
enhancing the EGFR signaling pathways, leading to induction of mucosal
proliferative activity during aging.
In conclusion, our current data demonstrate that aging is associated
with increased activation of EGFR in both gastric and colonic mucosa.
This is also accompanied by a concomitant increase in membrane-bound
precursor forms of TGF-. Moreover, the membrane-associated precursors of TGF-
from gastric and colonic mucosa of aged rats are
more active than those from young rats in inducing the EGFR signal
transduction processes.
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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
Address for reprint requests and other correspondence: A. P. N. Majumdar, Research Service-151, Veterans Affairs 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.
Received 29 August 2000; accepted in final form 31 January 2001.
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