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(Received for publication, March 14, 1995; and in revised form, June 7,
1995) From the
Epidermal growth factor (EGF) acutely inhibits acid secretion;
however, prolonged administration of EGF has been reported to increase
acid production. We undertook these studies to examine whether the
physiological effects of EGF on acid secretion are mediated by
regulation of gastric H
Epidermal growth factor (EGF) ( In the stomach EGF affects acid
secretion in a divergent fashion. Under acute conditions, EGF has a
long recognized inhibitory effect on gastric acid
secretion(14, 15, 16, 17) , whereas
prolonged administration of EGF increases both basal and maximal acid
secretion in vivo(18) and acid production in
isolated parietal cells invitro(19) . In
order to gain further insight into the mechanisms of EGF action on
gastric acid secretion, we examined the peptide's effects on
expression of the gene encoding the
H
Relative quantification of a gene-specific mRNA was
achieved by digital densitometry on a Loats Image Analysis System
(Westminster, MD) as described previously(22) . Levels of
H
Figure 1:
Effects of EGF on
Figure 2:
Effect of EGF on parietal cell
H
Employing a
luciferase reporter construct (HK-619+34) containing the first
exon and 619 bp of the 5`-flanking region of the
H
Figure 3:
Effect of EGF on
H
Figure 4:
Effect
of genistein on H
Figure 5:
Effects of EGF on expression of luciferase
following transfection of parietal cells with vectors constructed with
deletion mutants of the H
Figure 6:
Effects of EGF on the
H
To examine
whether a sequence-specific DNA-binding protein can bind to the ERE, we
carried out electrophoretic mobility shift assays using a
Figure 7:
Electrophoretic mobility shift assays with
an ERE probe and parietal cell nuclear extracts. The wild type
We noted that the
5`-GACATGG-3` sequence of the ERE is homologous to the 3` half-site of
the c-fos serum responsive element (28) to which the
DNA-binding proteins SRE-ZBP, rE12, and rNFIL-6 bind (29, 30, 31) (Fig. 8), suggesting that
the ERE-binding protein might be one of these. Accordingly, we utilized
antibodies specific for SRE-ZBP, E12, NFIL-6, and SRF in
electrophoretic mobility shift assays and observed, as shown in Fig. 9, that they did not shift or reduce the intensity of the
DNA-protein complex band obtained with the ERE-WT probe and parietal
cell nuclear extracts. Moreover, E12 and NFIL-6 consensus
oligonucleotides did not inhibit the formation of the ERE-protein
complex (Fig. 10). These data indicate that neither E12, NFIL-6,
SRE-ZBP, nor SRF is the transcriptional factor that specifically binds
to the ERE sequence and mediates the EGF responsiveness of the
H
Figure 8:
Structure of the c-fos serum
response element and its DNA-binding sites. The ERE of the
H
Figure 9:
Effects of antibodies for DNA-binding
proteins on the formation of the ERE-protein complex. Antibodies for
various DNA-binding proteins were tested for their ability to alter the
formation of the ERE-protein complex by electrophoretic mobility shift
assays performed with the
Figure 10:
Effects of consensus oligonucleotides on
the ERE-protein binding. Consensus oligonucleotides were tested for
their ability to bind to the ERE sequence by competition analysis in
electrophoretic mobility shift assays performed with
To examine whether
the ERE-binding protein is expressed in a cell- or species-specific
manner, we performed electrophoretic mobility shift assays utilizing
the ERE-WT probe and nuclear extracts obtained from nonparietal cells
such as canine gastric chief cells, MDCK cells derived from canine
kidney and L cells derived from mouse fibroblasts (Fig. 11). The
nuclear proteins prepared from chief cells and MDCK cells formed one
common band (complex 3), the formation of which was competitively
inhibited with the cold ERE-WT probe. This band corresponds to the
major DNA-protein complex generated with the ERE-WT probe and parietal
cell nuclear proteins. Since nuclear proteins obtained from
GH
Figure 11:
Electrophoretic mobility shift assays
with nuclear proteins obtained from various cells. Nuclear proteins
obtained from parietal and nonparietal cells were tested for their
ability to bind to the ERE sequence by electrophoretic mobility shift
assays performed with the
Figure 12:
Effects of EGF on the
H
EGF, when administered acutely, exerts potent inhibitory
effects on gastric acid
secretion(14, 15, 16, 17) . However,
since the half-maximal dose for its inhibition of acid secretion
(2-10 nM) is 10-100 times higher than the plasma
concentration of EGF (0.2-0.6 nM) (32, 33, 34) and lumenal administration of
EGF does not affect gastric acid secretion except at exceedingly high
doses, its inhibitory actions are considered to be pharmacological and
not physiological. In contrast to its acute inhibitory effects,
prolonged administration of EGF has been reported to increase both
basal and maximal acid secretion in vivo(18) , and
acid production in parietal cells in vitro(19) . In
the present study, we have confirmed that EGF exerts chronic
stimulatory effects on acid production by isolated canine gastric
parietal cells at physiological concentrations. Our data also suggest
that this increase in gastric acid secretion may result from enhanced
expression of the H Gastric
H We have observed in the present studies that EGF induces
H The ERE sequence
differs from previously reported EGF response elements found in genes
encoding gastrin(11) , prolactin(12) , tyrosine
hydroxylase(13) , transin(35) , and pS2 (36) but is homologous to the 3` half-site of the c-fos SRE(28, 31, 37) . The function of the 3`
c-fos SRE half-site has been the subject of considerable
investigation. Rivera et al.(28) reported that the
inner core of the c-fos SRE excluding the half-sites binds to
SRF and is, itself, sufficient to mediate both the induction and
termination of serum-stimulated transcription. However, they also
demonstrated that the sequences of the 3` and 5` arms of the SRE can
modulate the degree of serum inducibility. Boulden and Sealy (37) reported that maximal serum stimulation of the c-fos SRE requires SRF as well as SRE-BP, which binds to the 3`
half-site of the c-fos SRE. Moreover, they demonstrated that a
mutated enhancer factor III element, which binds to SRE-BP but not to
SRF, has serum responsiveness as well. rNFIL-6, another nuclear protein
that binds to the 3` half-site of the c-fos SRE, has been
reported to be involved in adenylate cyclase-dependent signal
transduction in PC12 cells. To date, rNFIL-6(30) ,
rE12(30) , SRE-ZBP(29) , and SRE-BP (37) have
been reported to bind the 3` half-site of the c-fos SRE.
Although one or more of these proteins may represent the parietal cell
nuclear protein that binds to the ERE, electrophoretic mobility shift
assays obtained with selective antibodies and competitive
oligonucleotides suggest that there may be a novel protein to account
for EGF responsiveness of the parietal cell
H
Volume 270,
Number 31,
Issue of August 04, pp. 18637-18642, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
,K
-ATPase
-Subunit Gene Expression through an Element Homologous to the 3`
Half-site of the c-fos Serum Response Element (*)
,K
-ATPase, the
principle enzyme responsible for acid secretion. EGF in concentrations
equivalent to those in plasma increased
H
,K
-ATPase
-subunit mRNA levels.
Using H,K
-ATPase-luciferase
constructs transfected into primary cultured parietal cells, a
significant step up in EGF inducibility was observed between bases
-162 and -156 (5`-GACATGG-3`) relative to the cap site.
This EGF response element (ERE) conferred EGF inducibility when linked
to homologous and heterologous promoters. The ERE is homologous to the
3` half-site of the c-fos serum response
element to which rNFIL-6, rE12, and SRE-ZBP bind. Electrophoretic
mobility shift assays using an ERE probe and parietal cell nuclear
extracts revealed a specific DNA-protein complex, the formation of
which was changed by neither E12 and NFIL-6 consensus oligonucleotides
nor antibodies for NFIL-6, SRE-ZBP, and E12. Our studies indicate that
EGF induces gastric H
,K
-ATPase
-subunit gene expression via an interaction between a specific ERE
and a novel transcriptional factor and that this may be a physiologic
mechanism by which EGF regulates acid secretion.
)is the prototypic
member of a large family of peptide growth factors that have biological
actions on the function of many organs(1, 2) . In
addition to its well known growth-promoting properties, EGF has many
physiologic non-growth-related actions including modulation of
pituitary hormone production(3) , amylase
secretion(4) , insulin synthesis(5) , and intestinal
electrolyte transport(6) . These diverse effects of EGF on
organ function are accompanied at the cellular level by induction of
immediate early response genes such as
c-fos(7, 8) , c-myc(9) , and
c-jun(10) as well as non-growth-related genes such as
those encoding gastrin(11) , prolactin (12) , and
tyrosine hydroxylase(13) .,K
-ATPase
-subunit, which is
the principle enzyme responsible for gastric acid
secretion(20) .
Cell Isolation
Gastric parietal cells were prepared from canine fundic
mucosa as described previously(21, 22) . Briefly,
cells were dispersed from stripped fundic mucosa by sequential exposure
to collagenase I (0.35 mg/ml, Sigma) and EDTA (1 mM). After
washing with Hanks' balanced salt solution, parietal cells were
enriched by centrifugal elutriation. For further purification,
elutriated parietal cells were centrifuged through density gradients
generated with 50% Percoll (Pharmacia Biotech Inc.). The cell fraction
banding at = 1.05 consisted of more than 95% parietal
cells, as determined by hematoxylin and eosin and by periodic
acid-Schiff reagent staining.
Northern Blot Analysis
Purified parietal cells were suspended in Earle's
balanced salt solution containing 0.1% bovine serum albumin and
incubated with or without various concentrations of human recombinant
EGF (Collaborative Biochemical Products, Bedford, MA) for 1 h at 37
°C in 95% O
, 5% CO. The cells were lysed
with TRIzol (Life Technologies, Inc.), and RNA pellets were obtained by
isopropyl alcohol precipitation. Aliquots (10 µg) of total RNA were
electrophoresed on a 1.25% formaldehyde-agarose gel, blotted to a nylon
membrane (maximum strength NYTRAN, Schleicher & Schuell), and
hybridized to an H,K
-ATPase
-subunit cDNA probe that was labeled with P by random
priming. The blot was then rehybridized to a
P-labeled
ubiquitin carboxyl-terminal precursor (UBCP) cDNA probe after washing.
The cDNAs used as probes for Northern blots were the 2247-bp BamHI fragment of the canine
H
,K
-ATPase
-subunit cDNA cloned
in our laboratory (23) and the AccI-PstI
fragment of the human UBCP cDNA lacking ubiquitin sequences (24) (a gift from Dr. P. Kay Lund, University of North
Carolina).,K
-ATPase
-subunit mRNA (3.5
kb) were normalized by comparison with the levels of UBCP mRNA (600 bp)
that have been established as suitable controls in this system.Luciferase Analysis
Construction of Luciferase Plasmids
Using a 6-kb
segment of the canine H,K
-ATPase
-subunit gene as a template for polymerase chain reaction we
synthesized DNA fragments, consisting of 34 bp of exon 1 and various
lengths of the 5`-flanking region, which were inserted into a pGL-2
basic luciferase vector (GeneLight plasmid; Promega,
Madison, WI). All deletion mutants included the 5`-GCTCCGCCTC-3`
sequence (bases -54 to -45 relative to the cap site)
through which Sp1 confers basal transcriptional activity to the
H
,K
-ATPase
-subunit
gene(25) . In separate experiments the putative EGF response
element (ERE, bases -162 to -156 relative to the cap site)
of the H,K
-ATPase
-subunit gene
was linked to the H,K
-ATPase
-subunit minimal promoter (bases -54 to +34 relative to
the cap site) and the thymidine kinase promoter, and these constructs
were inserted into the pGL-2 basic luciferase vector as well.Luciferase Assays
Primary cultured parietal cells
were transiently transfected with a test luciferase vector, and
luciferase assays were performed as described previously (26) after 3 h of incubation with 10M EGF. Protein concentrations of the cell lysates were measured
using a Bio-Rad protein measurement system (Bio-Rad), and luciferase
activities were normalized by comparison with protein concentration.
Samples in each experiment were analyzed in duplicate or triplicate.
Aminopyrine Uptake
The accumulation of 
C-aminopyrine was used as an
indicator of acid production by parietal cells(27) . Parietal
cells were cultured in Ham's F12/Dulbecco's modified
Eagle's medium (1:1) with or without various concentrations of
EGF for 18 h. The cells were washed with Earle's balanced salt
solution and preincubated with 0.1 µCi of C-aminopyrine (Amersham Corp.) for 30 min and then
stimulated with carbachol (Sigma) or histamine (Sigma) for 30 min.
Parietal cells were lysed with 500 µl of 1% Triton X-100 (Sigma),
and the radioactivity of lysate was quantified in a liquid
scintillation counter.Electrophoretic Mobility Shift Assay
Nuclear proteins were prepared from isolated gastric parietal
cells and other cells for electrophoretic mobility shift assays.
Briefly, isolated test cells were rinsed with phosphate-buffered saline
and incubated on ice for 10 min in 5 volumes of a solution consisting
of 10 mM HEPES (pH 7.9), 1.5 mM MgCl, 10
mM KCl, 0.5 mM dithiothreitol. After centrifugation
for 5 min at 250 g the cells, resuspended in 3 volumes
of the same solution, were homogenized with a tight fitting Dounce
homogenizer to release the nuclei. The nuclei were then centrifuged at
250
g for 10 min, resuspended, and incubated on ice in
a solution consisting of 25% glycerol, 20 mM HEPES (pH 7.9),
1.5 mM MgCl
, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride (Boehringer Mannheim), 1 µg/ml
pepstatin (Boehringer Mannheim), and 10 units/ml aprotinin (Boehringer
Mannheim). After centrifugation at 250 g for 15 min,
the supernatants were dialyzed overnight in a solution consisting of
20% glycerol, 20 mM HEPES (pH 7.9), 100 mM KCl, 0.2
mM EDTA, 0.5 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, and 10 units/ml
aprotinin. Electrophoretic mobility shift assays were carried out with
a 10-µl reaction mixture containing 10 mM Tris-HCl (pH
7.5), 0.5 mM dithiothreitol, 1 mM MgCl
,
50 mM NaCl, 0.5 mM EDTA, 4% glycerol, 250 ng of
poly(dI-dC), 10 fmol (
30,000 cpm) of P-labeled probe,
and 10 µg of nuclear protein. Following 15 min of incubation at
room temperature, the reaction mixtures were loaded onto 4% native
polyacrylamide gels (acrylamide/bis ratio of 29:1). The gels were
electrophoresed at 10 V/cm in a buffer containing 45 mM Tris
borate and 1 mM EDTA. In some experiments, nuclear proteins
were preincubated with 1 µl of antibody at 0 °C for 2 h and
then incubated with a
P-labeled probe. Antibodies specific
for SRF, SRE-ZBP, NFIL-6, E12, and Sp1 were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). To generate probes for
electrophoretic mobility shift assays, oligonucleotides were annealed
by heating (75 °C, 5 min) in a volume of 100 µl containing 1
nmol of each strand, 40 mM Tris-HCl (pH 7.5), 50 mM
NaCl, and 20 mM MgCl
followed by sequential
cooling to 25 °C. Annealed oligonucleotides (10 pmol) were
radiolabeled with 40 µCi of [P]dCTP and
10 units of Escherichia coli DNA polymerase 1 Klenow fragment
(Boehringer Mannheim) for 30 min at 25 °C. Free
[
-P]dCTP was removed with a Nuc-trap
(Stratagene, La Jolla, CA). As competitors for binding to the proteins,
we used consensus oligonucleotides as follows: Sp1,
5`-ATTCGATCGGGGCGGGGCGAGC-3`; AP2, 5`-GATCGAACTGACCGCCCGCGGCCCGT-3`;
E12, 5`-GATCCCCCCAACACCTGCTGCCTGA-3`; NFIL-6,
5`-TGCAGATTGCGCAATCTGCA-3`.
Statistics
Statistical analysis was performed using Student's t test. p values <0.05 were considered to be
significant. The half-maximally effective concentrations
(EC) in dose response studies were estimated using a
curve-fitting program (CA-Cricket graph III).
Effects of EGF on Secretagogue-stimulated Acid
Secretion by Gastric Parietal Cells
EGF has acute inhibitory
effects on gastric acid secretion in vivo and in
vitro; however, prolonged EGF treatment (5 days) increases both
basal and stimulated gastric acid output in unweaned rats(18) .
Recently, long term incubation with EGF has been reported to enhance
gastric acid production by isolated rabbit parietal cells(19) .
In order to confirm the chronic acid stimulatory effect of EGF in our
canine system, we isolated and cultured gastric parietal cells for 18 h
with or without EGF and examined basal and secretagogue-stimulated acid
production. Preincubation with EGF significantly increased histamine-
(0.1 mM) and carbachol- (0.1 mM) stimulated
aminopyrine uptake by gastric parietal cells in a dose-dependent manner (Fig. 1). Maximal induction of parietal cell acid production was
achieved at an EGF concentration of 1 nM. EC values for EGF effects on histamine- and carbachol-stimulated
aminopyrine uptake were 70 and 90 pM, respectively, roughly
10-100-fold less than the reported half-maximal concentrations
for its acute inhibitory effects(17) . Basal levels of
aminopyrine uptake were slightly increased by EGF preincubation. These
results support the notion that, in contrast to its acute inhibitory
effects, EGF has a chronic effect to up-regulate gastric acid
secretion.
C-aminopyrine uptake by cultured canine parietal cells.
Parietal cells cultured with or without EGF for 18 h were stimulated
with 0.1 mM of histamine or carbachol for 30 min.
Preincubation with EGF dose-dependently increased histamine- and
carbachol-stimulated C-aminopyrine uptake when compared
with cells cultured without EGF (means ± S.E., n = 6).
Effects of EGF on Parietal Cell
H
To gain further insight into the role of EGF in
gastric acid secretion, we examined whether EGF influences the
expression of the gene encoding the
H,K
-ATPase
-subunit Gene
Expression,K
-ATPase
-subunit, the
principle enzyme responsible for gastric acid secretion. By Northern
blot analysis (Fig. 2A), we observed that EGF induced
H,K
-ATPase
-subunit mRNA in
isolated gastric parietal cells. A linear transformation of the
densitometric analysis of the Northern blots after correction with UBCP
mRNA levels indicated that EGF significantly increased
H,K
-ATPase
-subunit mRNA levels
in a dose-dependent manner (Fig. 2B). Maximal induction
by EGF (253 ± 18% of basal, mean ± S.E., n = 5) was achieved at 10M, and
EC
was estimated to be 5
10
M, a concentration of EGF equivalent to the EC
for its chronic stimulatory effect on acid secretion but not the
IC
for its acute inhibitory effect.
,K
-ATPase
-subunit mRNA levels. A, EGF increased H,K
-ATPase
-subunit mRNA in this representative Northern blot. B, a
linear transformation of the densitometric analyses of the Northern
blots for H,K
-ATPase mRNA levels
stimulated by EGF, corrected by UBCP mRNA levels. Changes in mRNA
levels were expressed as percentage of basal (means ± S.E., n = 5).
,K
-ATPase
-subunit gene, we
examined the transcriptional effects of EGF. EGF significantly
stimulated H,K
-ATPase
-subunit
promoter activity in a dose-dependent manner similar to that obtained
by Northern blot analysis (Fig. 3). EGF achieved maximal
induction of luciferase activity (174 ± 11% of basal, mean
± S.E., n = 7) at 10M, and EC
was estimated to be 5
10
M. These data indicate that
physiological concentrations of EGF are capable of inducing
H
,K
-ATPase
-subunit gene
expression at the transcriptional level. Preincubation with genistein,
a tyrosine kinase inhibitor, significantly reduced
H,K
-ATPase
-subunit promoter
activity induced by EGF but had no effect on 8-Br-cAMP-induced activity (Fig. 4). Thus, EGF appears to induce
H,K
-ATPase
-subunit gene
expression through its well characterized receptor tyrosine kinase
activity.
,K
-ATPase
-subunit gene
transcription. Primary cultured parietal cells transiently transfected
with an H,K
-ATPase
-subunit
gene-luciferase vector (HK-619+34) were stimulated with various
concentrations of EGF for 3 h. EGF increased luciferase activity in a
dose-dependent manner similar to the increase obtained by Northern blot
analysis. Luciferase activity (RLU) was expressed as
percentage of basal (means ± S.E., n =
7).
,K
-ATPase
transcriptional regulation. Primary cultured canine parietal cells
transfected with HK-619+35 were preincubated with (hatchedbar) or without (solidbar) a specific
tyrosine kinase inhibitor genistein (2
10
M) for 30 min and stimulated with or without
10
M EGF and 10
M 8-Br-cAMP (8BcAMP) for 3 h (means ± S.E., n = 6).
Characterization of the EGF Response Element of the
H
To identify a sequence motif that mediates EGF
responsiveness of the H,K
-ATPase
-Subunit
Gene,K
-ATPase
-subunit gene, deletion mutants of its 5`-flanking region were
inserted into a luciferase reporter construct and transfected into
cultured parietal cells (Fig. 5). There was a decrease in
EGF-mediated induction of luciferase activity upon deletion of
sequences between -162 and -156 bp upstream of the cap site
(5`-GACATGG-3`), suggesting that an ERE is present in this region. In
order to confirm that the element located between bases -162 and
-156 confers EGF responsiveness, we placed the ERE upstream of
both homologous and heterologous thymidine kinase promoters. The
homologous promoter consisted of a minimal
H,K
-ATPase
-subunit promoter
element (-55 to +34 bp relative to the cap site). The ERE
conferred significant EGF inducibility to both promoters as compared
with the enhancerless control promoters (Fig. 6).
,K
-ATPase
-subunit gene. Deletion mutants of the
H,K
-ATPase
-subunit gene coupled
to a luciferase reporter gene were transfected into primary cultured
canine parietal cells. The transfected cells were then incubated with
or without 10M EGF for 3 h. EGF induction
of transcriptional activity generated through the
H
,K
-ATPase
-subunit gene
promoter was expressed as percentage of basal (means ± S.E., n = 6).
,K
-ATPase EGF response element
linked to H
,K
-ATPase
-subunit
gene minimal promoter- and thymidine kinase promoter-luciferase vectors
in gastric parietal cells. Primary cultured parietal cells were
transiently transfected with
H,K
-ATPase
-subunit gene minimal
promoter- and thymidine kinase promoter-luciferase vectors with
(ERE-HK-54+34-LUC, ERE-TK-LUC) or without (HK-54+34-LUC,
TK-LUC) the ERE of the H,K
-ATPase
-subunit gene. The cells were stimulated with or without
10M EGF for 3 h. The ability of EGF to
induce transcriptional activity was expressed as percentage of basal
(means ± S.E., n = 5). Similar results were
obtained in transfected MDCK cells (data not
shown).
P-labeled DNA probe (ERE-WT) that has the native ERE
sequence (5`-GACATGG-3`) in the center and 6-bp random sequences on the
3` and 5` ends (Fig. 7). Since these random sequences could
generate artificial DNA-binding sites distinct from the ERE, we
constructed a series of probes in which the ERE sequence itself or the
3` and 5` random sequences were mutated (ERE-M1, ERE-M2 and ERE-M3,
respectively, Fig. 7) for use in electrophoretic mobility shift
assays. The assays performed with the
P-labeled ERE-WT
probe and nuclear extracts obtained from parietal cells showed a
distinct band indicating a DNA-protein complex, the formation of which
was completely inhibited by competition with a 100-fold excess of
unlabeled ERE-WT probe as well as with both the ERE-M2 and ERE-M3
probes. In contrast, the ERE-M1 probe (mutated in the ERE sequence) and
other consensus oligonucleotides (Sp1 and AP2) did not competitively
inhibit the formation of the DNA-protein complex.
P-labeled ERE probe (ERE-WT) and parietal cell nuclear
extracts formed a DNA-protein complex (lane2), the
formation of which was inhibited with a 100-fold excess of unlabeled
ERE-WT probe (lane3) as well as the ERE-M2 and
ERE-M3 probes mutated in the region of the random sequences on the 3`
and 5` ends (lanes4 and 5, respectively).
In contrast, the ERE-M1 probe (lane6) mutated in the
ERE sequence and other consensus oligonucleotides (Sp1 and AP2; lanes7 and 8, respectively) were unable to
inhibit the formation of the DNA-protein
complex.
,K
-ATPase
-subunit gene. To
examine whether the ERE-binding protein is able to bind to the 3`
half-site of the c-fos SRE, we also utilized the SRE consensus
oligonucleotides (SRE-1 and SRE-2) shown in Fig. 10. The SRE-2
probe possessing the 3` half-site of the SRE appeared to inhibit the
formation of the ERE-protein complex more effectively than the SRE-1
probe without the 3` half-site. Thus, the ERE-binding protein appears
to be able to bind the 3` half-site of the c-fos SRE
as well. It is of note that the formation of the ERE-protein complex
was not changed by applying nuclear proteins from parietal cells
stimulated with EGF (Fig. 9), suggesting that the expression of
the ERE-binding protein is not induced by EGF.
,K
-ATPase
-subunit gene is
homologous to the 3` half-site of the c-fos SRE. The reported
binding sequences of rNFIL-6, rE12, SRE-ZBP, SRE-BP, and SRF overlap
with this region of the SRE.
P-labeled ERE-WT probe and
parietal cell nuclear extracts.
P-labeled ERE-WT probe and parietal cell nuclear
extracts.
C rat pituitary tumor cells and AGS human
gastric cancer cells also formed complex 3 (data not shown), the
parietal cell ERE-binding protein appears to be expressed in a wide
variety of cells from different species. However, it is of note that
nuclear proteins from L cells formed additional ERE-protein complexes
(complexes 1 and 2) distinct from complex 3 but not complex 3 itself.
Complex 2 was also formed with MDCK (Fig. 11) and AGS cell (data
not shown) nuclear proteins. In view of these observations, we examined
whether the ERE of the H,K
-ATPase
-subunit gene is active in nonparietal cells as well. In MDCK
cells, which demonstrate the ERE-nuclear protein complex predominantly
expressed in parietal cells (complex 3), the ERE conferred EGF
inducibility to homologous and heterologous thymidine kinase promoters
in the same manner observed in parietal cells (Fig. 6). In
contrast, in L cells that do not appear to express the parietal cell
ERE-binding protein, the ERE did not confer EGF inducibility (Fig. 12).
P-labeled ERE-WT probe with or
without a 100-fold excess of unlabeled ERE-WT
probe.
,K
-ATPase EGF response element
linked to the H
,K
-ATPase
-subunit gene minimal promoter- and thymidine kinase
promoter-luciferase vectors in L cells. L cells were transiently
transfected with H,K
-ATPase
-subunit gene minimal promoter- and thymidine kinase
promoter-luciferase vectors with (ERE-HK-54+34-LUC, ERE-TK-LUC) or
without (HK-54+34-LUC, TK-LUC) the ERE of the gastric
H,K
-ATPase
-subunit gene. The
cells were stimulated with or without 10M EGF for 3 h. The ability of EGF to induce transcriptional activity
was expressed as percentage of basal (means ± S.E., n = 5).
,K
-ATPase
-subunit gene since the concentrations of EGF required for both
effects are similar and in the range observed in the circulation under
physiological conditions.,K
-ATPase, a member of the
phosphorylating ion-motive ATPase family, is expressed in parietal
cells as a heterodimer of the catalytic
- and
-subunits(20) . Inasmuch as gastric
H,K
-ATPase is the principle enzyme
responsible for H
formation by gastric parietal cells,
the level of gastric H
,K
-ATPase gene
expression is a critical determinant of gastric acid secretion. Indeed,
we have observed previously that the major gastric acid secretagogues,
histamine, carbachol, and gastrin, increase
H
,K
-ATPase
-subunit mRNA levels
in gastric parietal cells(22) . In the present studies we have
demonstrated that EGF induces
H,K
-ATPase
-subunit gene
expression in gastric parietal cells, and the maximal induction with
EGF is comparable with that obtained with histamine, carbachol, or
gastrin (22) . The fact that the EGF-induced expression of
H,K
-ATPase can be reversed with the
tyrosine kinase inhibitor genistein is consistent with the known
tyrosine kinase activity of the EGF receptor. Our observations suggest
that EGF confers a physiologically important chronic stimulatory effect
on gastric acid secretion by inducing expression of the
H
,K
-ATPase gene, the principle enzyme
responsible for acid production. However, since the gastric acid
secretory event is a process integrated with many others in the
parietal cell, we cannot rule out the possibility that mechanisms other
than induction of H
,K
-ATPase also
might be involved in the stimulatory effects of EGF on gastric acid
secretion.
,K
-ATPase
-subunit gene
transcription in gastric parietal cells through a cis-regulatory ERE. Unlike many response elements, the ERE
does not activate basal transcription of the
H,K
-ATPase
-subunit gene (data
not shown). Electrophoretic mobility shift assays indicate that the ERE
forms a sequence-specific DNA-protein complex with a parietal cell
nuclear protein, and this complex appears to mediate the EGF
responsiveness of the H,K
-ATPase
-subunit gene. The results obtained with the ERE linked to the
minimal H,K
-ATPase promoter or the
thymidine kinase promoter indicate that it confers EGF responsiveness
to both homologous and heterologous promoters in gastric parietal
cells. The observation that the ERE is also active in MDCK cells but
not in L cells indicates the high specificity of the DNA-protein
interaction required to mediate EGF inducibility.
,K
-ATPase
-subunit gene.
Cloning of the gene encoding the parietal cell ERE-binding protein will
permit further characterization of the process by which EGF exerts a
physiologically important regulatory effect on gastric acid secretion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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