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* VA Medical Center, Long Beach, and the University of California, Irvine, California 92717, USA;
Third Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan; and
Division of Biochemistry, Biomedical Research Center, Osaka University Medical School, Osaka, Japan
1Correspondence: Department of Veterans Affairs Medical Center, 5901 E. 7th St., Long Beach, CA 90822, USA. E-mail: atarnawski{at}yahoo.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: RGM1 cells • receptor phosphorylation • c-Met/HGF receptor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
.
Such actions are particularly well documented for epidermal growth
factor (EGF) (1
2
3)
. It has been demonstrated that
hepatocyte growth factor (HGF), a peptide with potent hepato- and
renotrophic actions, also plays a role in gastric ulcer healing
(4
, 5)
. HGF is secreted by cells of mesodermal origin and
binds to the c-Met/HGF receptor on adjacent epithelial cells, thus
mediating epithelial–mesenchymal interactions during the healing
process (4)
. In addition to the mitogenic, motogenic, and
angiogenic properties, HGF also has unique morphogenic properties
reflected by branching and gland lumen formation during the late
remodeling phase of ulcer healing (4
5
6)
. Recent studies
demonstrated that expression of HGF mRNA is significantly up-regulated
at the margin of healing gastric ulcers (7
, 8)
.
The role of prostaglandins in the ulcer healing process is evidenced by
the fact that nonsteroidal antiinflammatory drugs (NSAIDs), by
inhibiting synthesis of prostaglandins, delay gastric ulcer healing and
worsen the quality of mucosal scars (9
, 10)
. Similarly,
the cell growth-promoting effect of prostaglandins has been
demonstrated directly in human and in experimental animal models by a
marked hyperplasia of the foveolar area of the gastric glands after
prolonged treatment with prostaglandins (11
, 12)
, and
indirectly by the inhibitory effect of some NSAIDs on development and
growth of colonic polyps (13)
. In some tissues, there is
evidence for an interaction between prostaglandins and HGF expression.
For example, it has been demonstrated that prostaglandins strongly
induce HGF expression in skin fibroblasts (14)
and, to a
lesser extent, in gastric fibroblasts (15)
. Conversely,
HGF mRNA expression was found to be down-regulated at the edge of
NSAID-induced gastric ulcers in humans (15)
. Based on the
latter observation, Takahashi et al. (15)
postulated that
prostaglandin deficiency causes decreased HGF induction and that this
effect may represent the key mechanism of NSAIDs interference with
ulcer healing. There is also evidence that HGF can induce prostaglandin
synthesis through up-regulation of cyclooxygenase in human gastric
carcinoma cells (16)
and in rat hepatocytes
(17)
. These findings suggest an interaction between HGF
and prostaglandin synthesis in mediating tissue growth and injury
healing.
COX-2 gene expression has recently been demonstrated in rat gastric
epithelial cells (18
19
20)
. We have demonstrated that
expression and translation of this gene can be activated in the rat
gastric epithelial cell line, RGM1, by epiregulin and basic fibroblast
growth factor (19)
. In vivo studies have
revealed that expression of the EGF and HGF receptors is localized to
undifferentiated epithelial cells at the margin of healing ulcers
(2
, 8)
and that expression of the COX-2 enzyme is
predominantly localized to adjacent nonepithelial cells
(21)
. This suggests the presence of a paracrine or
possibly an autocrine regulatory pathway between these growth factors
and prostaglandin synthesis.
The aims of this study were to determine whether HGF activates
expression of COX-2 mRNA and protein in rat gastric epithelial cells
and to investigate the possible signaling pathway(s) involved. We
focused our attention on the role of the extracellular signal-regulated
kinase (ERK) pathway, as we have recently demonstrated that MAP (ERK2)
kinase is activated during healing of gastric ulcers and that
interruption of this pathway with an inhibitor dramatically delays
ulcer healing (22)
. In addition, we examined the presence
of the c-Met/HGF receptor in RGM1 cells and whether HGF induces
phosphorylation of this receptor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
RGM1, an epithelial cell line derived from normal rat gastric
mucosa (established by Hirofumi Matsui, Institute of Clinical Medicine,
University of Tsukuba, Ibaraki, Japan), was obtained from Riken Cell
Bank (Tsukuba, Japan) and used at passages 20–28. The cells were
maintained in DMEM/F12 medium containing 20% FBS and 2 mM of
L-glutamine at 37°C with 5% CO2 and 95% air
in a humidified incubator. Cells were serum starved for 24 h prior
to experiments. Cells were incubated in medium containing vehicle
(controls) or the indicated concentrations of PD98059 (MAP kinase
kinase [MEK] inhibitor) or SB203580 (p38 MAP kinase inhibitor) for 50
min. The cells were then treated for the time indicated with either
vehicle (controls) or HGF (10 ng/ml). The dose of HGF was based on
studies demonstrating that this dose significantly induces cell
migration and cell proliferation in isolated epithelial cell lines,
including RGM1 cells (4
, 23)
.
RT/PCR
Total RNA was isolated using the guanidium
isothiocyanate-phenol-chloroform method (24)
. Reverse
transcription and polymerase chain reaction (RT/PCR) were performed as
described previously (25
26
27)
. The PCR amplification was
performed for 32 cycles of 1 min at 94°C for denaturing, 1 min at
55°C for annealing, and 2 min at 72°C for extension. The specific
primer set used for rat COX-1 was 5'-AGC CCC TCA TTC ACC CAT TT-3'
(forward) and 5'-CAC GGA CGC CTG TTC TAC GG-3' (reverse)
(26)
and for COX 2 was 5'-TGG TGC CGG GTC TGA TGA TG-3'
(forward) and 5'-GCA ATG CGG TTC TGA TAC TG-3' (reverse)
(28)
.
Rat ß-actin served as a positive control. The specific primer set
used for rat ß-actin (Clontech Laboratories Inc., Palo Alto, Calif.)
was 5'-TTGTAACCAACTGGGACGATATGG-3' (forward) and
5'-CAGGCTGCAGTCCTTTGATC-3' (reverse) (29)
. Nine microliter
aliquots of the products were subjected to electrophoresis on a 1.25%
agarose gel and DNA was visualized by ethidium bromide staining.
Location of the products (base pairs, or bp) was determined by using a
100 bp ladder (Gibco BRL, Gaithersburg, Md.) as standard size marker.
Competitive RT/PCR
Since COX-1 mRNA, in contrast to COX-2 mRNA expression, remained
unaffected with each of the various treatments, competitive RT/PCR was
performed to quantify only COX-2 mRNA. Competitive RT/PCR was used to
quantify the level of COX-2 mRNA by using one set of primers to amplify
both the target cDNA and another DNA fragment, whereby the second DNA
fragment competes with the target DNA for the same primers and thus
acts as an internal standard (30
, 31)
. Serial dilutions of
the competitor fragment were added to PCR amplification reactions
containing constant amounts of the target cDNA samples. By knowing the
amount of the competitor added to the reactions, the mRNA level can be
quantitatively determined. The competitor DNA fragment was constructed
using the MIMIC construction kit (Clontech Laboratories) according to
the manufacturer’s instructions. The primers used to construct the
competitor fragment were 5'-TGG TGC CGG
GTC TGA TGA TGC GCA AGT GAA ATC
TCC TCC G-3' (forward) and 5'-GCA ATG CGG
TTC TGA TAC TGT CTG TCA ATG CAG
TTT GTA G-3' reverse). The underlined portions of the above primers are
the target gene primer sequences; the remaining sequences were designed
to yield a competitive PCR product approximately twice the size (450
bp) of the target cDNA PCR product (253 bp). PCR amplification was
performed with the indicated concentrations of competitor fragment for
36 cycles of 1 min at 94°C for denaturing, 1 min at 55°C for
annealing, and 2 min at 72°C for extension. For quantitative
assessment of the PCR products, we used a video image analysis
(Image-1/FL, Universal Imaging Corp., Westchester, Pa.)
(32)
. The Image-1 system can distinguish density on a
scale of 0–255 units. Each measurement was standardized by subtracting
the background intensity in average.
Western blotting
To determine the effect of HGF on COX-2 protein synthesis, cells
were treated for 5 min with either HGF (10 ng/ml) or vehicle
(controls). Then the cells were washed and incubated in fresh
serum-free medium for an additional 3 and 6 h. To determine the
effect of MAP (ERK1, 2) kinase inhibition on COX-2 protein synthesis,
cells were incubated for 50 min with either vehicle (controls) or the
indicated concentrations of PD 98059 or SB203580. Cells were then
treated for 5 min with either HGF (10 ng/ml) or vehicle (controls),
washed, and incubated in fresh serum-free medium for an additional
3 h and lysed. Protein content of each lysate was determined as
described (33)
. Western blotting was performed as
described previously (25)
. Membranes were incubated with a
specific polyclonal antibody against rat COX-2 (Cayman Chemical Co.,
Ann Arbor, Mich.) diluted 1:1000 for 1 h. The bound
antigen-antibody complexes were detected with anti-rabbit
immunoglobulin G-horseradish peroxidase (Santa Cruz Biotechnology,
Inc., Santa Cruz, Calif.), using enhanced chemiluminescence (Amersham,
Arlington Heights, Ill.), and exposed to Amersham ECL detection film.
Quantification of COX-2 protein signals was performed by laser
densitometry (Pharmacia LBK Biotechnology, Uppsala, Sweden).
Determination of mitogen-activated protein kinase activity
ERK2 activity was determined as described previously
(22)
. Briefly, cells were incubated in medium containing
either vehicle (controls) or the indicated concentration of PD98059 or
SB203580 for 50 min, followed by treatment with either HGF (10 ng/ml)
or vehicle (controls). The cells were then lysed on ice; 30 µg of
total protein from each experimental sample was added to a conjugate of
protein A Sepharose and 1 µg anti-ERK2 antibody (Santa Cruz
Biotechnology) and mixed at 4°C for 2 h. The conjugates were
then pelleted by centrifugation and washed four times. After the final
wash, buffer was removed completely and 40 µl of MAPK assay mixture
(10 mM HEPES, pH 7.5; 10 mM MgCl2; 50 µM ATP;
30 µg myelin basic protein; and 4 µCi [32P]
ATP) was added to each sample. The samples were incubated at 30°C for
20 min and the reaction was terminated by the addition of sodium
dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer
(34)
. The samples were then electrophoresed on 15%
acrylamide gels. After electrophoresis, the gels were stained with
Coomassie brilliant blue and dried. The gels were autoradiographed; the
myelin basic protein bands were cut out and radioactivity was counted
in a scintillation counter.
Determination of c-Met/HGF receptor phosphorylation levels
Phosphorylation levels of the c-Met/HGF receptor were determined
as described for the EGF receptor (22)
. Briefly, cells
were incubated in medium containing either vehicle (controls) or
PD98059 (20 µM) for 50 min, followed by incubation with either HGF
(10 ng/ml) or vehicle (controls) for 5 min. The cells were then lysed
and the c-Met/HGF receptor was immunoprecipitated from 0.4 mg of total
cell protein for each condition by addition of 1 µg anti-c-Met/HGF
antibody (Santa Cruz Biotechnology) conjugated with protein
A-Sepharose. After immunoprecipitation, the immunoprecipitates were
electrophoresed on 7.5% polyacrylamide gels and transferred to
nitrocellulose membranes for immunoblot analysis (as described under
Western blotting) using anti-phosphotyrosine antibody (Santa Cruz
Biotechnology). The membranes were then ‘stripped’ and reprobed using
the anti-c-Met/HGF antibody to determine the amounts of c-Met/HGF
receptor immunoprecipitated under each condition. Phosphorylation
levels were determined by quantification of intensity measurements
using the Image-1 video image analysis system with normalization for
amounts of protein immunoprecipitated.
Statistical analysis
Student’s t test was used to compare data between
two groups (e.g., control and growth factor-treated group). One-way
ANOVA and Bonferroni correction were used to compare data between three
or more groups. Values are expressed as mean ± standard deviation
(SD). P values less than 0.05 were
considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. After 50 min treatment with
MEK (mitogen-activated protein kinase/ERK kinase) inhibitor (20 µM),
RGM1 cells had a viability of 93.8 ± 6%. There was no
statistical difference in viability compared to that of control cells.
COX-1 and COX-2 mRNA expression in RGM1 cells
RT/PCR was performed to determine whether RGM1 cells at baseline
express mRNA for COX-1 and COX-2. As shown in Fig. 1
and Fig. 2
, mRNAs for both isoenzymes were present in RGM1 cells at baseline. One
hour incubation with 10 ng/ml HGF did not affect COX-1 mRNA expression
(Fig. 1)
, but caused a marked increase in COX-2 mRNA expression (Fig. 2)
. COX-2 mRNA expression returned to baseline by 6 h (Fig. 2)
.
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Competitive RT/PCR was used to quantify PCR products for COX-2
(Fig. 3A
). The initial amount of COX-2 cDNA can be determined as equal to that
of MIMIC, when both products after PCR are in equal amount.
Accordingly, using 1 x 10-2 attomol/ml of
MIMIC, expression of COX-2 mRNA was compared (Fig. 3B, C
).
One hour incubation with 10 ng/ml HGF led to a significant increase in
COX-2 mRNA expression by 236 ± 56% (P<0.01 vs.
control). Pretreatment with the MEK inhibitor PD98059 (20 µM)
completely inhibited the HGF-induced increase in COX-2 mRNA expression.
However, PD98059 did not affect COX-2 mRNA expression when added alone
(Fig. 3B, C
).
|
Effect of HGF on COX-2 protein expression
Western blotting was performed to determine whether COX-2 protein
expression is affected by treatment with HGF. HGF at the concentration
10 ng/ml significantly increased COX-2 protein levels after 3 h
(159±19% vs. controls, P<0.003). As shown in Fig. 4
, HGF at this concentration resulted in a 175 ± 24% increase vs.
controls (P<0.02) at 6 h. Pretreatment with 20 µM of
PD98059 for 50 min completely prevented the HGF-induced increase in
COX-2 protein level. PD98059 did not affect COX-2 protein level at the
baseline (Fig. 4)
. Pretreatment for 50 min with SB203580 (a specific
inhibitor of the MAP kinase, p38), at concentrations of 10 µM to 40
µM, had no effect on the HGF-induced increase in COX-2 protein levels
(Fig. 5
).
|
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Effect of HGF on ERK2 activity
Five minute and 1 h incubations with 10 ng/ml HGF
significantly increased ERK2 activity by 684 ± 27% and 153 ± 6%, respectively (both P<0.01 vs. control).
Pretreatment with 20 µM PD98059 completely blocked the HGF-induced
increase in ERK2 activity (Fig. 6
). Maximal inhibition of the HGF-induced increase in ERK2 activity was
obtained by pretreatment with PD98059 at a concentration of between 10
µM and 25 µM (Fig. 7A
). The concentration of 20 µM PD98059 used throughout the
present study was chosen because it gave maximal inhibition without
affecting cell viability (see above). Pretreatment with SB203580 at
concentrations of 1 µM to 40 µM had no effect on the HGF-induced
increase in ERK2 activity (Fig. 7B
).
|
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Effect of HGF on c-Met/HGF receptor expression and phosphorylation
Five minute incubation of RGM1 cells with 10 ng/ml HGF triggered
more than a 13-fold increase in c-Met/HGF receptor phosphorylation
(P<0.005, Fig. 8
). Pretreatment with 20 µM PD98059 for 50 min, which abolished the
HGF-induced increase in ERK2 activity, had no effect on c-Met/HGF
receptor phosphorylation (Fig. 8)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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37
38)
. COX-1 is expressed constitutively in most
tissues, and prostaglandins produced in normal gastric tissue have been
claimed to be derived predominantly from COX-1 (36)
. The
COX-1 isoform is abundantly expressed in gastric mucosa and is
considered to exert, through generation of prostaglandins, a
‘housekeeping’ function essential for gastric protection. In
contrast, the COX-2 isoform is inducible and found predominantly in
inflammatory exudates in rheumatic disease (36
37
38)
. Its
activation occurs in certain cell types in response to proinflammatory
or mitogenic agents including cytokines, endotoxins, and tumor
promoters (39)
.
This study demonstrates that COX-2 mRNA, in addition to COX-1 mRNA, is
expressed in the gastric epithelial cell line RGM1, which is derived
from normal rat gastric mucosa. Furthermore, it clearly demonstrates
that HGF does not affect the constitutively expressed COX-1 gene but
significantly increases the expression of COX-2 mRNA and protein in
gastric epithelial cells. Moreover, our study shows for the first time
that COX-2 gene activation in gastric epithelial cells is mediated
through the MAP (ERK2) kinase signaling pathway. Although the antibody
used in the present study for the determination of ERK2 activity is
known to cross react with ERK1, we were unable to demonstrate growth
factor-induced ERK1 activity in RGM1 cells when using an anti-ERK1
antibody (data not shown). Therefore, although we cannot completely
rule out the possible involvement of ERK1 in the HGF-induced expression
of COX-2, our data suggest that ERK2 is the predominant mediator of
this induction. We have also demonstrated that gastric epithelial cells
express the c-Met/HGF receptor and that a dramatic activation of
c-Met/HGF receptor and ERK2 activity occurs within 5 min after
stimulation by HGF and lasts for at least 1 h. The latter findings
represent the first demonstration of c-Met/HGF receptor expression in
RGM1 cells and ERK2 activation in gastric epithelial cells by HGF.
Activation of the MAP (ERK) kinase pathway is responsible for the
mitogenic and motogenic properties of growth factors in other tissues
(40)
and also plays an important role in gastric ulcer
healing (22)
. Recently it was shown that COX-2 expression
induced by interleukin-1 in human fibroblasts and by bacterial
lipopolysaccharide in human monocytes involves p38 MAP kinase
(41
, 42)
. We used a specific inhibitor of p38 MAP kinase
in the present study to investigate the possibility that this kinase,
in addition to ERK, is involved in the HGF-induced expression of COX-2.
Inhibition of p38 MAP kinase did not affect induction of the COX-2 gene
by HGF, clearly indicating that HGF-induced expression of COX-2 in
gastric epithelial cells is mediated through the ERK signaling pathway
and is independent of p38 MAP kinase.
The presence of COX-2 in the gastric mucosa in vivo is
indicated by recent observations made from immunohistochemical studies
(21
, 43
44
45
46
47
48)
and by studies demonstrating expression of
COX-2 mRNA in gastric mucosal tissue (45
46
47
, 49
50
51
52
53)
. In
general, induction of COX-2 has been regarded as a mechanism by which
cells increase the capacity to synthesize prostaglandins in excess of
that provided by COX-1 (54
, 55)
. Recent studies of COX-1-
and-COX-2-deficient mice suggest that COX-1 and COX-2 use different
subcellular pools of arachidonic acid for synthesis of prostaglandins
(56
, 57)
. Large amounts of both COX-2 and cytosolic
phospholipase A2 have been found to be localized
to the nuclear envelope whereas COX-1 is also found in the endoplasmic
reticulum (58)
. Thus, MAPK-mediated activation of
cytosolic phospholipase A2 in the nuclear
envelope may lead to a preferential generation of prostaglandins by
COX-2.
The importance of COX-2 in ulcer healing is well established (21
, 52
, 53)
. First, COX-2-selective inhibitors delay ulcer healing;
second, there is a close correlation between the amount of COX-2
(assessed by immunoreactivity) present and the epithelial cell
proliferation rate at the ulcer margin (21)
. The role of
COX-2 in mucosal protection is also emerging from recent studies.
Animal experiments and limited clinical studies have shown that highly
selective COX-2 inhibitors do not damage the unchallenged gastric
mucosa (21
, 59
60
61)
. They abolish, however,
peptone-induced gastric mucosal protection against ethanol injury and
abolish mucosal protection induced by mild irritants, e.g., 20%
ethanol (62
, 63)
. Moreover, acute ischemia-reperfusion
induces (a significant) up-regulation of COX-2 mRNA in the rat gastric
mucosa to the same level as COX-1 mRNA, before induction of gastric
ulceration (51)
. The latter observations suggest that
COX-2 can, ‘on demand’, assist the housekeeping effect of COX-1 as a
second line of defense.
In summary, our present study has demonstrated that RGM1 (a cell line derived from normal rat gastric epithelium) cells express both COX-1 and COX-2, and that HGF triggers activation of COX-2 mRNA and protein in these cells. Furthermore, our study has demonstrated that RGM1 cells express the c-Met/HGF receptor protein and that HGF-triggered activation of COX-2 expression in these cells is mediated through phosphorylation of the c-Met/HGF receptor and activation of the MAP (ERK2) kinase signal transduction pathway. Since COX-2 is important to ulcer healing, the above actions of HGF provide a molecular basis for its participation in the ulcer healing process.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-phosphotyrosine). Lower panel: The same membrane as in the upper
panel was ‘stripped’ and the amounts of total c-Met/HGF receptor
contained in the immunoprecipitates were determined by reprobing with
the anti-c-Met/HGF receptor antibody used in the immunoprecipitations
(