3 Veterans Affairs Medical Center and Department of 1 Internal Medicine, 2 Pathology, 4 Biochemistry, and 5 Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan 48201
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ABSTRACT |
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Aging is associated with decreased reparative
ability of the gastric mucosa. Our recent data suggest a role for
epidermal growth factor receptor (EGFR) in the mucosal reparative
processes. Thus we examined changes in EGFR tyrosine kinase activity as
well as expression and subcellular localization of EGFR and its ligand transforming growth factor- (TGF-
) in the gastric mucosa of young
(4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early
reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion
of the neck region and initiation of foveolar cell migration could be
seen within 45 min of injury in young rats but not until 90 min in aged
rats. In young rats mucosal EGFR tyrosine kinase activity increased at
45 min after injury and subsequently fell to basal levels. Mucosal EGFR
mRNA increased throughout the reparative phase as did content of the
EGFR ligand TGF-
. In contrast, although the basal tyrosine kinase
activity and levels of EGFR mRNA and TGF-
were elevated in the
gastric mucosa of aged rats, injury did not cause increases in these
parameters. Immunofluorescent localization suggests that
internalization and/or degradation of EGFR may be higher in
aged than in young rats. We suggest that diminished induction of EGFR
tyrosine kinase activity and increased EGFR internalization after
injury may in part be responsible for the age-related decrease in the
reparative capacity of the gastric mucosa.
aging; epidermal growth factor receptor; tyrosine kinase; transforming growth factor-
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INTRODUCTION |
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A NUMBER OF IN VIVO and in vitro studies have demonstrated that the gastric mucosa of adult healthy animals possesses the remarkable capacity to repair promptly after a mild injury, (2, 5-8, 17, 25, 31, 32). Within minutes after injury the gastric mucosa begins to reconstitute its epithelial surface through migration of viable cells from gastric pits and glands, a process known as restitution (7, 17, 23, 25). In rats, this process is completed within about 2 h (24). Within several hours after injury, cell proliferation is increased to replace lost cells (24).
Results from this laboratory have demonstrated that aging is associated
with diminished reparative ability of the gastric mucosa (5, 10, 13,
14). Although the regulatory mechanisms for the age-related decline in
mucosal reparative ability remain to be fully elucidated, our recent
observations suggest a role for epidermal growth factor receptor (EGFR)
tyrosine kinase in regulating mucosal reparative processes (10, 11,
22). In evaluating the involvement of EGFR tyrosine kinase in the
gastric mucosal restitutional process, we have observed that in young, but not in aged rats, gastric mucosal EGFR tyrosine kinase activity is
greatly increased within 30 min after acute injury (22). This suggests
that, in aged gastric mucosa, diminished activation of EGFR tyrosine
kinase resulting in decreased activation of the EGFR signaling pathway
after injury may partly be responsible for attenuation of the
restitutional process (10, 22). However, the regulatory mechanism(s)
for the diminished activation of EGFR tyrosine kinase in aged rats
during the initial reparative period is poorly understood. We postulate
that transforming growth factor- (TGF-
), which is synthesized in
the gastric mucosa and is one of the primary ligands of EGFR (19, 30),
may play a critical role in regulating EGFR tyrosine kinase activity
during the initial mucosal reparative phase after acute injury. The
basis for this postulation comes from the observation that levels of
TGF-
in the gastric juice as well as in the gastric mucosa are
substantially elevated in young rats within 30 min after acute injury
(21). On the other hand, in aged rats, we found no significant change in mucosal TGF-
levels at 30 min after acute injury (22). To further
evaluate the role of EGFR tyrosine kinase in the age-related decline in
mucosal restitution, the current study examines changes in tyrosine
kinase activity of EGFR as well as expression and subcellular
distribution of the receptor in the gastric mucosa over a 240-min
reparative period after acute injury. The significance of
endogenous TGF-
in regulating EGFR tyrosine kinase during this
reparative phase is also examined.
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MATERIALS AND METHODS |
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Animals and collection of tissues.
Male Fischer 344 rats, aged 4 and 24 mo old, were used. The animals
were purchased from the National Institute on Aging (Bethesda, MD) at
least 1 mo before the experiment, during which period they were
supplied with Purina rat chow and water ad libitum. Gastric mucosal
injury was induced by orogastric administration of 2 M NaCl. Groups of
overnight-fasted rats (n = 5) were
given by stomach tube either 2 M NaCl (1.15 ml/100 g body wt) or an
equivalent volume of water (controls) and killed 45, 90, and 240 min
later. The stomach was removed, opened along the greater curvature, and rinsed thoroughly with cold normal saline. The oxyntic gland area was
dissected out, and two small portions of the tissue were fixed either
in 10% neutral buffered Formalin for histological evaluation or in
Bouin's fixative for immunofluorescence study. Mucosal scrapings, obtained from the rest of the oxyntic gland area (hereafter referred to
as gastric mucosa), were either processed immediately (for RNA
extraction) or immediately frozen in liquid nitrogen and stored at
90°C.
Mucosal histology. Formalin-fixed stomach tissues were dehydrated in graded alcohol and embedded in paraffin to yield full thickness sections of the mucosa. The paraffin-embedded tissues were serially sectioned at a thickness of 4 µm and were stained with hematoxylin and eosin or Periodic Acid Schiff stain and methylene blue for histological evaluation.
Immunofluorescent staining of EGFR.
Bouin's-fixed gastric tissue was embedded in paraffin and sectioned as
stated above. Sections were collected on
poly-L-lysine-coated slides.
After deparaffinization and rehydration, sections were blocked with
10% chick serum, 10% porcine serum, and 0.5% BSA in PBS for 1 h at
room temperature. After being washed twice with PBS, the slides were
incubated overnight at 4°C with rabbit anti-EGFR antibody
(polyclonal antibody, 2 µg/ml, Santa Cruz Biotechnology, Santa Cruz,
CA). Rabbit -globulin (2 µg/ml, Sigma Chemical, St. Louis, MO) in
PBS was used as a negative control. After an extensive washing in PBS,
the slides were incubated with FITC-conjugated F(ab')2 fragment of
anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) for 1 h at
room temperature (20 µg/ml). After being washed in PBS three to four
times (5 min each), the slides were mounted with Vectashield mounting
medium (Vector Laboratories, Burlingame, CA) and sealed with nail
polish. The slides were examined using a fluorescent microscope
(Olympus System, model BX60, Olympus America, Lake Success, NY).
Tyrosine kinase activity. The enzyme activity was determined as previously described (19, 26). Briefly, aliquots of mucosal scrapings were homogenized in lysis buffer [20 mM sodium phosphate, pH 7.4, 0.5% Triton X-100, 0.5% Nonidet P-40, 50 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na3VO4, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM 1,10-phenanthroline] using a Dounce homogenizer. The homogenate was stirred in a mechanical rotator for 30 min at 4°C and subsequently centrifuged at 10,000 g for 10 min at 4°C. The supernatant, after diluting with an equal volume of homogenizing buffer A (HEPES, pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM Na3VO4, 10 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM 1,10-phenanthroline), was used as the source for tyrosine kinase. Protein content was measured using the Bio-Rad protein assay kit (Bio-Rad, Richmond, CA).
For determination of tyrosine kinase activity of EGFR, aliquots of mucosal lysate containing 200 µg protein were diluted with an equal volume of lysis buffer containing 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate. The samples were incubated overnight at 4°C with 1 µg of polyclonal antibody to EGFR (Upstate Biotechnology, Lake Placid, NY). The immune complexes were precipitated with heat-denatured Pansorbin, resuspended in 30 µl of homogenizing buffer A containing 0.1% Triton X-100, and assayed for tyrosine kinase activity by measuring 32P incorporation from [Western blot analysis of TGF-.
For determination of the relative concentration of TGF-
in the
membrane, a 30,000 g pellet was prepared from aliquots of gastric mucosal scrapings as described previously (26). Aliquots of the
membrane fraction from all groups of rats containing the same amount of
protein (3 mg) were incubated overnight at 4°C with 0.3 µg
monoclonal antibody to TGF-
(Santa Cruz Biotechnology). The immune
complexes were precipitated with protein A-Sepharose, and the
immunoprecipitates were subjected to SDS-PAGE (14). After
electrophoresis proteins were transferred electrophoretically onto
nitrocellulose membranes (Bio-Rad) using a transfer cell (International, Mount Prospect, IL). Membranes were then incubated with
blocking buffer [PBS containing 5% nonfat dry milk (Sanalac), 0.25% BSA, and 0.1% Tween 20], washed extensively with PBS
containing 0.1% Tween 20, and incubated again with the same TGF-
antibodies as described previously (29). Finally, the protein bands
were visualized by an enhanced chemiluminescence detection system
(Amersham) according to the manufacturer's instruction. The density of
the protein bands on X-Omat films was analyzed by a digital image analysis system (Molecular Dynamics, Strom 860, Sunnyvale, CA). Molecular weight of the protein band(s) was calculated from protein markers (4-250 kDa, Novex) run concurrently.
Northern blot analysis.
Steady-state mRNA levels of EGFR and TGF- were determined using
poly(A)+ RNA as described
previously (29). Briefly, several aliquots of gastric mucosal scrapings
from each group of rats were pooled, and total RNA was extracted by
RNA-STAT solution (Tel-Test, Friendswood, TX) according to the
manufacturer's instructions.
Poly(A)+ RNA was then isolated
from total RNA by oligo(dT) cellulose chromatography according to the
instructions provided by GIBCO BRL (Gaithersburg, MD). Aliquots of
poly(A)+ RNA from each group were
denatured at 65°C for 15 min, subsequently mixed with an equal
volume of 5× formaldehyde gel loading buffer, and
size-fractionated by electrophoresis in a 1.5% agarose gel containing
2.2 M formaldehyde. After electrophoresis RNA was transferred to a
Nytran membrane (Schleicher & Schuell, Keene, NH) by capillary blotting. The membrane was ultravioletly cross-linked and subsequently prehybridized in 5× SSPE buffer (0.15 mM NaCl, 11.5 mM
NaH2PO4, and 1 mM EDTA) containing 50% formamide, 5× Denhardt's reagent, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA at 55°C for 2 h.
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RESULTS |
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The extent of gastric mucosal injury in young and aged rats by hypertonic saline and the degree of epithelial restitution were evaluated by light microscopy. Immediately after injury, the gastric mucosa of both young (4 mo old) and aged (24 mo old) rats showed a complete denudation of the surface with exposed capillaries (data not shown). Basement membrane and capillaries were found to be exposed and present at the mucosal surface (Fig. 1 ). However, neither bleeding nor extensive ulceration was observed in any of the animals. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. At this time exposed capillaries were difficult to identify in young rats (Fig. 1A), and the surface was mostly covered by attenuated epithelium (Fig.1A, arrows). In contrast, relatively little surface epithelium was present in the gastric mucosa of aged rats (Fig. 1B), and exposed capillaries (Fig. 1B, arrow) were easily identified. This and other evidence suggest that healing was minimal in the gastric mucosa of aged rats.
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Histological changes were further evaluated by examining the gastric mucosa at 45, 90, and 240 min after injury. Results are shown in Fig. 2. At 45 min after injury, the gastric mucosa from young rats showed expansion of the neck region zone and initiation of migration of foveolar cells toward the surface (Fig. 2, top, arrow). Further expansion of the neck region was observed at 90 min after injury, whereafter these changes were no longer evident (Fig. 2, bottom, arrow). Although the neck region of the gastric mucosa of noninjured aged rats (data from water-fed controls) appeared to be relatively more expanded than in their younger counterparts, further expansion of this region in aged rats was not evident until 90 min after injury. Only at 240 min after injury did we observe early migration of foveolar and parietal cells to the surface in aged rats.
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To determine whether EGFR may be involved in gastric mucosal restitution, the intrinsic tyrosine kinase activity of the EGFR protein as well as expression of its gene in the gastric mucosa were measured in young and aged rats during the 240-min reparative period. In young rats mucosal EGFR tyrosine kinase activity increased significantly by about 75% at 45 min after injury over the water-fed controls and remained elevated throughout the 240-min reparative period (Table 1). On the other hand, steady-state mRNA levels of EGFR increased steadily throughout the reparative period; at 240 min after injury the levels were 80% above the controls (Fig. 3). In aged rats basal EGFR tyrosine kinase activity as well as steady-state mRNA levels of the receptor in the gastric mucosa (data from water-fed controls) were found to be about 280 and 90% above, respectively, when compared with the corresponding levels in 4-mo-old animals (Table 1 and Fig. 3). However, in aged rats, injury produced no significant increase in either tyrosine kinase activity or steady-state mRNA levels of EGFR in the gastric mucosa (Table 1 and Fig. 3). In fact, at 240 min after injury, tyrosine kinase activity of EGFR in aged gastric mucosa was about 50% lower than in controls (Table 1). EGFR mRNA levels in the gastric mucosa of aged rats also remained 30-40% below the basal level throughout the reparative period (Fig. 3).
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To further determine whether the age-related decrease in gastric mucosal EGFR tyrosine kinase activity during mucosal restitution could in part be related to EGFR internalization and/or degradation, immunocytochemical studies were performed. In water-fed (noninjured controls) young rats, EGFR immunoreactivity was found primarily in the surface and neck regions of gastric mucosa (Fig. 4A). There was relatively weak staining in the gland area (Fig. 4A). In these animals EGFR was primarily localized to the apical surface membrane of foveolar-type mucus cells (Fig. 4, A and D). At 45 min after injury in young rats, we observed no apparent change in EGFR expression in the neck region. However, there was an increase in staining in the glandular portion (Fig. 4B). At 240 min after injury in young rats, the relative abundance of EGFR was decreased in the neck region with a concomitant increase in the glandular portion, when compared with the water-fed controls and at 45 min after injury (Fig. 4, A and C). At this time EGFR immunoreactivity appeared mostly in intracellular vesicles (Fig. 4F, arrowhead). In contrast to young control rats (water-fed), aged control rats showed intense EGFR staining in the glandular region (Fig. 4G). EGFR staining was strongest at the apical membrane (arrow) and was faintly present as cytoplasmic vesicles within glands (Fig. 4, G and J). At 45 min after injury in aged rats, there was a marked decrease in EGFR immunoreactivity in the entire glandular compartment with decreased apical membrane staining (Fig. 4H). At this time we noted intensely bright cytoplasmic and basally oriented vesicles coincident with the decrease in apical staining (Fig. 4K, arrowhead). The number and intensity of this vesicular staining increased throughout the period studied (240 min, Fig. 4L).
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Ligand binding to the extracellular domain of EGFR is one of the
primary causes for activation of intrinsic tyrosine kinase activity of
the receptor (30). Previously, we demonstrated that in young but not in
aged rats TGF- levels and EGFR tyrosine kinase activity in the
gastric mucosa were significantly elevated at 30 min after injury (22).
In view of the fact that TGF-
is a membrane-anchored peptide (19),
we hypothesized that the age-related changes in mucosal EGFR tyrosine
kinase activity during mucosal restitution could partly be due to
differential accumulation of the peptide in the membrane and thereby
modulating the enzyme activity through an autocrine-juxtacrine
mechanism. To test this hypothesis, we examined the changes in TGF-
levels in a gastric mucosal membrane preparation from young and aged
rats during the reparative period by Western blot analysis. The
antibody detected two precursor forms of TGF-
with molecular masses
of 14 and 15 kDa in all mucosal samples (Fig.
5). In young rats the
relative concentration of both forms of TGF-
increased steadily
throughout the reparative period, attaining a level of about 150%
above the water-fed controls at 240 min after injury (Fig. 5). Although the basal levels of TGF-
in gastric mucosal membranes were about 40% higher in aged than in young rats (data from water-fed controls), injury in aged rats produced no significant increase in the relative concentration of either 14 or 15 kDa TGF-
when compared with the
corresponding water-fed controls (Fig. 5).
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To further determine whether the age-related differences in TGF-
levels in gastric mucosal membranes during mucosal restitution are
under transcriptional control, mRNA levels of TGF-
were measured. Results are shown in Fig. 6. In noninjured
gastric mucosa of aged rats (data from water-fed controls), in which
proTGF-
levels in mucosal membranes were higher than in their
younger counterparts (Fig. 5), gastric mucosal TGF-
mRNA levels were
about 50% above the young animals (Fig. 6, data from water-fed
controls). In young rats, in which mucosal membrane concentration of
proTGF-
increased steadily during the reparative period (Fig. 5),
mRNA levels were appreciably elevated (50% above the controls) only at
240 min after injury (Fig. 6). In contrast, in aged rats, in which the levels of TGF-
in mucosal membranes were decreased after injury (Fig. 5), mucosal mRNA levels remained 40-50% below the basal levels throughout the 240-min reparative phase (Fig. 6).
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DISCUSSION |
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The mucosal lining of the gastrointestinal tract, especially the stomach, is not only exposed to the deleterious effects of luminal acid but also to a variety of exogenous injurious agents, including nonsteroidal anti-inflammatory drugs and ethanol. Each of these agents either alone or in combination with others may induce injury. However, a number of in vivo and in vitro studies have demonstrated that the gastric mucosa of healthy adult animals possesses the inherent capacity to repair promptly (often within 24 h) after mild injury (2, 5-8, 17, 25, 31, 32). At least two different mechanisms are thought to participate in full repair of the damaged gastric mucosa. The rapid repair process of mucosal restitution, which begins within minutes after injury, is accomplished by cell migration and is completed within 2 h. The subsequent slower process is replacement of lost cells by cell division (24), which in rats does not peak until 18-20 h after injury (5, 24).
Although the intracellular events that regulate the reparative processes remain to be fully elucidated, recent data from this laboratory suggest a role for EGFR tyrosine kinase in regulating these processes (10, 22). The basis for this postulation comes from our observation that repeated injections of a dose of tyrphostin 51 [a tyrosine kinase inhibitor (9)] during the 24-h reparative period, which inhibits tyrosine kinase activity of EGFR, but not pp60c-src, produces inhibition of mucosal proliferative activity as well as re-epithelialization of the mucosal surface (11). Others have demonstrated increased expression of EGFR and its ligands in the gastric mucosa during ulcer healing (26-28). Although these observations indicate a role for EGFR in gastric mucosal regeneration through cell proliferation, our recent finding that in young rats tyrosine kinase activity and expression of EGFR in the gastric mucosa are also augmented as early as 30 min after mucosal injury (22) suggests an involvement of EGFR in gastric mucosal restitution. Further support for this postulation comes from the observation that in aged rats, in which mucosal restitution is found to be attenuated (4, 5), the magnitude of stimulation of EGFR tyrosine kinase and expression of the receptor at 30 min after acute injury are considerably lower than what we noted in young rats, when compared with the corresponding water-fed controls (22). In addition, EGFR is also thought to have a role in the small intestinal restitutional process (20). Moreover, recent evidence suggests a role for EGFR in regulating polyamine-induced stimulation of cytoskeleton reorganization in IEC cells after injury (16). These and other relevant observations led us to suggest that decreased activation of the EGFR signaling pathway in aged gastric mucosa after injury may contribute to attenuation of mucosal restitution (10, 22). Our current data support this contention. We have observed that in young but not in aged rats gastric mucosal EGFR tyrosine kinase activity is greatly stimulated within 45 min after injury and remains elevated throughout the 240-min reparative period. This early rise in mucosal EGFR tyrosine kinase activity after injury in young rats is not associated with increased transcription of EGFR.
Although a number of factors, including expression of EGFR, are known
to modulate the intrinsic tyrosine kinase activity of the receptor,
ligand binding to its extracellular domain is considered to be one of
the primary causes for activation of the enzyme activity (3, 30).
Although both EGF and TGF- bind to EGFR with equal potency, the
observation that in rats levels of TGF-
but not EGF are
substantially elevated in the gastric mucosa and gastric juice shortly
after acute injury (21) suggests a role for endogenous TGF-
in
regulating EGFR tyrosine kinase activity during the early reparative
phase through an autocrine-paracrine mechanism. However, TGF-
is a
membrane-anchored peptide (19). Results from cell surface and
biochemical characterization studies have demonstrated that the
presence of the transmembrane TGF-
is a normal consequence of
TGF-
synthesis, and in most cases the peptide is present on the cell
surface in its precursor form (1, 15, 18). Moreover, it has been
demonstrated that the transmembrane TGF-
precursor form activates
EGFR (1). Taken together, these observations suggest that the
membrane-bound TGF-
may regulate EGFR tyrosine kinase activity
through an autocrine-juxtacrine mechanism. In support of this
postulation we have observed that the age-related rise in gastric
mucosal EGFR tyrosine kinase activity is associated with a concomitant
increase in the relative concentration of 18 and 14 kDa precursor forms
of TGF-
in mucosal membranes (29). The same mechanism may also be
responsible for the age-related changes in mucosal EGFR tyrosine kinase
activity during the current reparative period. We have observed that in
young rats, the relative concentration of 14 and 15 kDa precursor forms
TGF-
in gastric mucosal membrane fractions increased steadily during
the 240-min reparative period. In contrast, in aged rats, with
diminished gastric mucosal restitutional ability (4, 5), the relative concentration of both forms of TGF-
in mucosal membranes remained below the water-fed controls throughout the reparative period. The fact
that changes in TGF-
levels in mucosal membranes of aged rats are
also accompanied by parallel alterations in mucosal EGFR tyrosine
kinase further suggests that membrane-bound form(s) of TGF-
may have
a role in modulating EGFR tyrosine kinase activity during mucosal
repair. Therefore, decreased activation of the EGFR signaling pathway
in aged gastric mucosa after injury as a result of diminished
accumulation of proTGF-
can be considered an explanation for the
attenuation of mucosal restitution in these animals.
The decreased activation of EGFR tyrosine kinase in aged rats after
injury could also be the result of increased internalization and/or degradation of the receptor. Support for this
interpretation comes from the observation that at 45 min after mucosal
injury EGFR immunoreactivity associated with intracellular particles in
gastric mucosal cells is found to be higher in aged than in young rats.
Although the reasons for this are not fully understood, it is plausible
that increased binding of TGF- to EGFR in aged rats as a result of
higher basal levels of both TGF-
and EGFR in the gastric mucosa
(data from water-fed controls) may induce rapid internalization of the
receptor-ligand complex after the injury-induced stimulus. Recently, we
have also observed that aging enhances sensitivity of the gastric
mucosa to both EGF and TGF-
so that low doses activate mucosal EGFR,
whereas large doses inhibit this activation (12). Therefore, the
age-related decline in mucosal repair could be due to decreased
activation of EGFR tyrosine kinase resulting from increased sensitivity
of the receptor to it, in that doses of the ligands needed to activate
EGFR tyrosine kinase in the gastric mucosa of aged rats are lower than
those required for young animals. Further experiments are undoubtedly necessary to fully elucidate the role of EGFR in gastric mucosal reparative processes during aging.
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ACKNOWLEDGEMENTS |
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We are grateful to Amie Dozier for expert graphics assistance.
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FOOTNOTES |
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This work was supported by National Institute on Aging Grant AG-14343 to A. P. N. Majumdar and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-K08-02503 and Fund for Medical Research and Education of Wayne State University to J. R. Turner.
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: A. P. N. Majumdar, Research Service-151, VA Medical Center, 4646 John R, Detroit, MI 48201.
Received 31 March 1998; accepted in final form 15 July 1998.
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