©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Epidermal Growth Factor Induces H,K-ATPase -Subunit Gene Expression through an Element Homologous to the 3` Half-site of the c-fos Serum Response Element (*)

(Received for publication, March 14, 1995; and in revised form, June 7, 1995)

Mitsuru Kaise (1) Akira Muraoka (1) Junko Yamada (1) Tadataka Yamada (1) (2)(§)

From the  (1)Departments of Internal Medicine and (2)Physiology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0368

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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,K-ATPase, the principle enzyme responsible for acid secretion. EGF in concentrations equivalent to those in plasma increased H,K-ATPase alpha-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 alpha-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.


INTRODUCTION

Epidermal growth factor (EGF) (^1)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) .

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,K-ATPase alpha-subunit, which is the principle enzyme responsible for gastric acid secretion(20) .


EXPERIMENTAL PROCEDURES

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(2), 5% CO(2). 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 alpha-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 alpha-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).

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,K-ATPase alpha-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 alpha-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 alpha-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 alpha-subunit gene was linked to the H,K-ATPase alpha-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 ^14C-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 ^14C-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(2), 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(2), 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(2), 50 mM NaCl, 0.5 mM EDTA, 4% glycerol, 250 ng of poly(dI-dC), 10 fmol (approx30,000 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(2) followed by sequential cooling to 25 °C. Annealed oligonucleotides (10 pmol) were radiolabeled with 40 µCi of [alphaP]dCTP and 10 units of Escherichia coli DNA polymerase 1 Klenow fragment (Boehringer Mannheim) for 30 min at 25 °C. Free [alpha-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).


RESULTS

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.


Figure 1: Effects of EGF on ^14C-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 ^14C-aminopyrine uptake when compared with cells cultured without EGF (means ± S.E., n = 6).



Effects of EGF on Parietal Cell H,K-ATPase alpha-subunit Gene Expression

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 alpha-subunit, the principle enzyme responsible for gastric acid secretion. By Northern blot analysis (Fig. 2A), we observed that EGF induced H,K-ATPase alpha-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 alpha-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 10M, 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.


Figure 2: Effect of EGF on parietal cell H,K-ATPase alpha-subunit mRNA levels. A, EGF increased H,K-ATPase alpha-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).



Employing a luciferase reporter construct (HK-619+34) containing the first exon and 619 bp of the 5`-flanking region of the H,K-ATPase alpha-subunit gene, we examined the transcriptional effects of EGF. EGF significantly stimulated H,K-ATPase alpha-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 10M. These data indicate that physiological concentrations of EGF are capable of inducing H,K-ATPase alpha-subunit gene expression at the transcriptional level. Preincubation with genistein, a tyrosine kinase inhibitor, significantly reduced H,K-ATPase alpha-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 alpha-subunit gene expression through its well characterized receptor tyrosine kinase activity.


Figure 3: Effect of EGF on H,K-ATPase alpha-subunit gene transcription. Primary cultured parietal cells transiently transfected with an H,K-ATPase alpha-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).




Figure 4: Effect of genistein on H,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 10M) for 30 min and stimulated with or without 10M EGF and 10M 8-Br-cAMP (8BcAMP) for 3 h (means ± S.E., n = 6).



Characterization of the EGF Response Element of the H,K-ATPase alpha-Subunit Gene

To identify a sequence motif that mediates EGF responsiveness of the H,K-ATPase alpha-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 alpha-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).


Figure 5: Effects of EGF on expression of luciferase following transfection of parietal cells with vectors constructed with deletion mutants of the H,K-ATPase alpha-subunit gene. Deletion mutants of the H,K-ATPase alpha-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 alpha-subunit gene promoter was expressed as percentage of basal (means ± S.E., n = 6).




Figure 6: Effects of EGF on the H,K-ATPase EGF response element linked to H,K-ATPase alpha-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 alpha-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 alpha-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).



To examine whether a sequence-specific DNA-binding protein can bind to the ERE, we carried out electrophoretic mobility shift assays using a 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.


Figure 7: Electrophoretic mobility shift assays with an ERE probe and parietal cell nuclear extracts. The wild type 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.



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,K-ATPase alpha-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.


Figure 8: Structure of the c-fos serum response element and its DNA-binding sites. The ERE of the H,K-ATPase alpha-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.




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 P-labeled ERE-WT probe and parietal cell nuclear extracts.




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 P-labeled ERE-WT probe and parietal cell nuclear extracts.



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(4)C(1) 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 alpha-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).


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 P-labeled ERE-WT probe with or without a 100-fold excess of unlabeled ERE-WT probe.




Figure 12: Effects of EGF on the H,K-ATPase EGF response element linked to the H,K-ATPase alpha-subunit gene minimal promoter- and thymidine kinase promoter-luciferase vectors in L cells. L cells were transiently transfected with H,K-ATPase alpha-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 alpha-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).




DISCUSSION

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,K-ATPase alpha-subunit gene since the concentrations of EGF required for both effects are similar and in the range observed in the circulation under physiological conditions.

Gastric H,K-ATPase, a member of the phosphorylating ion-motive ATPase family, is expressed in parietal cells as a heterodimer of the catalytic alpha- and beta-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 alpha-subunit mRNA levels in gastric parietal cells(22) . In the present studies we have demonstrated that EGF induces H,K-ATPase alpha-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.

We have observed in the present studies that EGF induces H,K-ATPase alpha-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 alpha-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 alpha-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.

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,K-ATPase alpha-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.


FOOTNOTES

*
These studies were supported by National Institutes of Health Grants R30-DK33500 and R01-DK34306 as well as funds from the University of Michigan Gastrointestinal Peptide Research Center (National Institutes of Health Grant P30-DK34933). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Internal Medicine, University of Michigan Medical Center, 3101 Taubman Center, Ann Arbor, MI 48109-0368. Tel.: 313-936-4770; Fax: 313-936-7024.

^1
The abbreviations used are: EGF, epidermal growth factor; UBCP, ubiquitin carboxyl-terminal precursor; bp, base pair(s); ERE, EGF response element; SRE, serum response element; MDCK, Madin-Darby canine kidney; SRE-BP, SRE binding protein.


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