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J. Biol. Chem., Vol. 280, Issue 16, 15700-15708, April 22, 2005

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Regulation and Function of the Sonic Hedgehog Signal Transduction Pathway in Isolated Gastric Parietal Cells^*

Vinzenz Stepan, Saravanan Ramamoorthy, Hildegard Nitsche, Yana Zavros, Juanita L. Merchant, and Andrea Todisco

From the Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109

Received for publication, November 18, 2004 , and in revised form, January 27, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Shh (Sonic hedgehog) regulates gastric epithelial cell differentiation. We reported that incubation of purified canine parietal cells with epidermal growth factor (EGF) for 6–16 h, stimulates H⁺/K⁺-ATPase -subunit gene expression through the activation of Akt. We explored if Shh mediates some of the actions of EGF in the parietal cells. EGF induced a 6-fold increase in Shh expression, measured by Western blots, after 5 h of incubation. This effect was inhibited by both the phosphatidylinositol 3-kinase inhibitor LY294002 and by transduction of the cells with an adenoviral vector expressing dominant negative Akt. EGF stimulated the release of Shh-like immunoreactivity from the parietal cells, after 16 h of incubation. Shh induced H⁺/K⁺-ATPase -subunit gene expression, assessed by Northern blots, it stimulated a luciferase reporter plasmid containing the EGF-responsive sequence (ERE) of the canine H⁺/K⁺-ATPase -subunit gene promoter, and it induced parietal cell nuclear protein binding to the ERE. Gli transcription factors mediate the intracellular actions of Shh. Co-transfection of the parietal cells with the H⁺/K⁺-luc plasmid together with one expressing Gli2, induced H⁺/K⁺-luciferase activity 5-fold, whereas co-transfection of the cells with the H⁺/K⁺-luc plasmid together with one expressing dominant negative Gli2, inhibited EGF induction of H⁺/K⁺-luciferase activity. Identical results were observed in the presence of the Shh signal transduction pathway inhibitor, cyclopamine. Transfection of the cells with dominant negative Akt inhibited EGF, but not Shh stimulation of H⁺/K⁺-ATPase-luciferase activity. Thus, EGF but not Shh signals through Akt. Preincubation of the cells for 16 h with either Shh or EGF enhanced histamine-stimulated [¹⁴C]aminopyrine uptake by 50%. In conclusions, some of the actions of EGF in the parietal cells are mediated by the sequential activation of the Akt and the Shh signal transduction pathways. These effects might represent novel mechanisms mediating the actions of growth factors on gastric epithelial cell differentiation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
EGF¹ is a member of a large family of polypeptide growth factors that has been shown to exert numerous physiological actions in the gut such as regulation of growth, differentiation, restitution, and secretion (1–4). Several studies have indicated that stimulation of gastric canine parietal cells in primary culture for 7–16 h with EGF exerts a direct stimulatory action on both the expression and the transcription of the H⁺, K⁺-ATPase -subunit, a gene considered to be a marker of parietal cell differentiation and a key element in the process of gastric acid production (5–12).

The intracellular signal transduction pathways that mediate the multiple, complex actions of growth factors in the stomach have been only partially elucidated. We have recently reported that, in the canine gastric parietal cells, EGF stimulates H⁺, K⁺-ATPase -subunit gene expression and it enhances secretagogue-stimulated gastric acid secretion, through a signal transduction pathway that requires, at least in part, the activation of Akt (9). Akt has been linked to the induction of cellular growth and survival and to the expression and the maintenance of highly differentiated cellular phenotypes (13–16). The specific intracellular targets of the Akt signal transduction pathway in the stomach are currently not known.

Sonic, Indian, and Desert hedgehog (Shh) are members of the family of the Hedgehog proteins, peptides known to exert important regulatory functions in patterning and growth in a large number of tissues during embryogenesis (17–19). In the mammalian stomach, in particular, Shh has been shown to be an important factor for the regulation of gastric epithelial cell maturation and differentiation (20–22). Recent studies have shown that Shh null mice fail to develop a normal gastric epithelium (20) and that inhibition of Shh signaling in the gastric mucosa leads to enhanced cellular proliferation and to diminished expression of factors, such as bone morphogenic protein 4 and hepatocyte nuclear factor 3, which promote gastric epithelial cell differentiation (21).

The intracellular mechanisms that mediate the actions of Shh on target cells have been only partially understood. According to recent investigations, Shh appears to bind to a transmembrane receptor protein, known as patched (Ptc) which, in the absence of Shh, exerts an inhibitory effect on the seven transmembrane receptor smoothened (Smo). Binding of Shh to Ptc blocks the inhibitory effect of Ptc on Smo. Once activated, Smo induces a complex series of intracellular reactions that targets the Gli family of transcription factors (17, 18, 23, 24). At least three members of this family of nuclear proteins have been identified in mammalian tissues (17, 24). Although Gli1 and Gli2 are transcriptional activators, Gli3 seems to function primarily as a transcriptional repressor (17, 24). Gli2 appears to be the principal effector of Shh signaling, because disruption of the Gli2 gene leads to developmental defects involving several Shh target tissues, while Gli1 null mice are born without detectable abnormalities (17, 25–30).

Immunohistochemical studies conducted in the gastric mucosa of both mice and humans have demonstrated that Shh is expressed in the parietal cells (21), highly specialized gastric epithelial cells, known to produce and secrete growth factors, and regulatory peptides in the gastric mucosa (21, 31, 32). Indeed, loss of mature parietal cells, achieved by genetic, pharmacological, and immunological methods, appears to be associated with profound abnormalities in the differentiation and development of multiple cell lineages in the stomach (33–37). Taken together, these observations underscore the importance of the parietal cells in the regulation of complex programs of cellular growth and differentiation in the gastric mucosa.

Accordingly, we took advantage of a well established system based on primary cultures of highly purified canine gastric parietal cells to study the function and regulation of Shh in the stomach. In addition, we investigated if the stimulatory actions of EGF on parietal cell maturation and differentiation are mediated by the activation of the Shh signal transduction pathway.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Adenoviral Vectors and Plasmids—The replication defective adenoviral vector expressing hemagglutinin-tagged, dominant negative Akt 1(with serine 473 and threonine 308 mutated to alanine, Ad.dn-Akt) under the control of the CMV promoter, was a gift of K. Walsh (Tufts University, Boston, MA) (38). The adenoviral vector expressing the -galactosidase enzyme under the control of the CMV promoter (Ad-.CMV--gal) was previously described (39). The plasmid 8X3'Gli-BS-Luc (40) was a gift of H. Sasaki (Osaka University, Osaka, Japan). The vectors expressing Gli2C4p and Gli2p, gifts of Andrzej Dlugosz (University of Michigan, Ann Arbor, MI), were generated by subcloning Gli2C4 (24) and Gli2 (24) into the plasmid pcDNA 3.1. HK(–619 + 35)-Luc and ERE-HK(–54 + 34)-Luc (7) were gifts of T. Yamada (University of Michigan). SRE-Luc was a gift of J. Pessin, (41) (University of Iowa, Iowa City, IA). The plasmid expressing dominant negative Akt/PKB (PKBDN) (42) was a gift of T. Soderling (Portland, OR).

Primary Parietal Cell Preparation and Culture—For preparation of primary parietal cells we utilized a modification of the method of Soll et al. (43–46). The mucosal layer of freshly obtained canine gastric fundus was bluntly separated from the submucosa and rinsed in Hank's balanced salt solution containing 0.1% bovine serum albumin. The cells were dispersed by sequential exposure to collagenase (0.35 mg/ml) and 1 mM EDTA and parietal cells were enriched by centrifugal elutriation using a Beckman JE-6B elutriation rotor. Elutriator fractions 8 and 9, which contain up to 70% parietal cells as determined by hematoxylin and eosin and periodic acid-Shiff reagent staining, were further purified by centrifugation through density gradients generated by 50% Percoll (Amersham Biosciences) at 30,000 x g for 20 min. The cell fraction at = 1.05 consisted of virtually all parietal cells as determined by staining with an anti-H⁺, K⁺-ATPase -subunit monoclonal antibody (MBL, Nagoya, Japan). The isolated parietal cells (2 x 10⁶ cells/well) were cultured according to the method of Chew et al. with some modifications (44, 45). Briefly, the cells were cultured in Ham's F-12/Dulbecco's modified Eagle's medium (1:1) containing 0.1 mg/ml gentamycin, 50 units/ml penicillin G, 0.01 mg/ml ciprofloxacin, and 2% Me₂SO (Sigma) on 6-well culture dishes (Corning Inc., Corning, NY) coated with 150 µl of H₂O-diluted (1:5) growth factor reduced Matrigel (BD Biosciences). For our studies, the parietal cells were incubated, according to the experiments, with EGF (10 nM, BD Biosciences), histamine (100 µM, Sigma), and recombinant Shh amino-terminal peptide (0.1–0.5 µg/ml, R&D Systems, Minneapolis, MN) for various time periods. In some experiments, LY294002 (10 µM, Calbiochem) and cyclopamine (1 µM, Toronto Research Chemicals, North York, Ontario, Canada) were added 30 min prior to the addition of the stimulants. LY294002 and cyclopamine were dissolved in Me₂SO; all other test substances were dissolved in water. Control experiments with untreated cells were performed by incubating the cells in either vehicle (0.1% Me₂SO) or incubation buffer without the test substances. The parietal cells were transduced, when indicated, with a multiplicity of infection of 100 of adenoviral vectors expressing either dominant negative Akt or -galactosidase.

Northern Blot Analysis—The parietal cells were lysed with TRIzol (Invitrogen) according to the manufacturer's instructions. Northern blot hybridization assays were performed as previously described (47). Equal amounts of each RNA sample, with ethidium bromide (10 mg/ml) in a final volume of 20 µl, were electrophoresed on a 1.25% agarose gel containing formaldehyde, and the RNA was transferred from the gel to nitrocellulose filters. The ethidium-stained ribosomal RNA bands in the gel were photographed before and after transfer to ensure that equivalent amounts of RNA were loaded onto each lane and that no residual RNA was left on the gel. The canine H⁺, K⁺-ATPase -subunit cDNA probe was a gift of Il Song (University of Michigan). The human glyceraldehyde-3-phosphate dehydrogenase cDNA probe was obtained from Clontech (Palo Alto, CA). The cDNAs were labeled with [³²P]dCTP by the random priming procedure, and the nitrocellulose filters were hybridized to the ³²P-labeled cDNA probes as previously described (47).

Transfection of Primary Cultured Parietal Cells—The parietal cells were transfected according to previously reported techniques (7, 45, 46). Before transfection the cells were washed once with 1 ml of Opti-MEM I serum-reduced media (Invitrogen) and fed with 400 µl of Opti-MEM I media supplemented with 2% Me₂SO. The cells were transfected with the luciferase reporter plasmids (3 µg of HK(–619 + 35)-Luc, 2 µg of 8X3'Gli-BS-Luc, 5 µg of ERE-HK(–54 + 34)-Luc and 2 µg of SRE-Luc) and, where indicated, with different amounts of the expression vectors (1 µg of dnAkt, 1 µg of Gli2C4, 0.75 µg of Gli2, 0.25 µg of pCMV-Gal). Control experiments were conducted in the presence of the empty vectors. Transfections were carried out using Lipofectin (Invitrogen) as previously described (7, 45, 46). The day after transfection the cells were fed with fresh media and stimulated with the test substances. At the end of the incubation period (16 h), the cells were washed twice with cold calcium-free PBS. After addition of 100 µl of cell lysis buffer (25 mM Tris-HCl, pH 7.8, 2 mM EDTA, 2 mM 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, 1% Triton X-100), the cells were incubated at room temperature for 15 min. The cells were then scraped and transferred to Eppendorf tubes. After quick centrifugation to pellet large debris, the supernatant was transferred to a new tube. An aliquot of cell lysate (20 µl) was mixed with 100 µl of luciferase assay reagent (20 mM Tricine, 1.07 mM (MgCO₃)₄Mg(OH)₂·5H₂O, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM dithiothreitol, 270 µM coenzyme A, 470 µM luciferin, 530 µM ATP, final pH 7.8), and luminescent intensity was measured for 10 s utilizing a Lumat LB9501 luminometer (Berthold, Germany). Luciferase activity was expressed as RLU (relative light units) and normalized for -galactosidase activity. -Galactosidase activity was measured by the luminescent light derived from 10 µl of each sample incubated in 100 µl of Lumi-Gal 530 (Lumigen, Southfield, MI) and used to correct the luciferase assay data for transfection efficiency.

Western Blots—The parietal cells were lysed in 500 µl of lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EDTA, 1.5 mM MgCl₂, 1 mM Na₃VO₄, 10 mM NaF, 10 mM Na₄P₂O₇·10H₂O, 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochlorine (AEBSF, ICN-Biomedicals, Aurora, OH), 1 µg/ml leupeptin, and 1 µg/ml aprotinin). The cell homogenates were spun at 1000 rpm for 5 min at 4 °C. The supernatants were transferred to Eppendorf tubes. Protein concentrations were measured by the Bradford method (48). 80 µg of parietal cell lysates were mixed with 5x electrophoresis buffer (50% glycerol, 25% mercaptoethanol, 10% SDS, 0.3 M Tris (pH 6.8), 0.025% bromphenol blue), boiled for 5 min, and loaded on 10% SDS-polyacrylamide mini-gels, which were run at 200 V for 1 h. The gels were transferred on an Immobilon-P transfer membrane (Millipore, Bedford, MA) in 25 mM Tris, 150 mM glycine, 20% methanol. After transfer the membranes were blocked in 10 ml of TBST (20 mM Tris, 0.15 M NaCl, 0.1% Tween), 5% dry milk for 1 h and then incubated for 16–18 h at 4 °C in 10 ml of TBST, 5% dry milk, containing either specific anti-Shh (sc-1194 and sc-9024, which were raised against a recombinant protein mapping at the amino terminus of Shh (1:200, Santa Cruz Biotechnology, Santa Cruz, CA) or anti-ptc antibodies (sc-9016, which was raised against a recombinant protein mapping at the carboxyl terminus of ptc, 1:250, Santa Cruz Biotechnology). Control blots were performed using anti-actin antibodies (1:500, Santa Cruz Biotechnology). At the end of the incubation periods the membranes were washed in TBST for 30 min at room temperature and then incubated for 1 h in TBST, 5% dry milk, containing protein A directly conjugated to horseradish peroxidase (1:2500, Amersham Biosciences). The membranes were washed in TBST for 30 min at room temperature and then exposed to the Amersham Biosciences ECL detection system according to the manufacturer's instructions.

Electrophoretic Mobility Shift Assays—For gel mobility shift assays, nuclear extracts from parietal cells were prepared using previously described techniques (7, 45). Extracts were stored at –80 °C, and protein concentrations were measured by the Bradford method using bovine serum albumin standards (48). The oligonucleotide probe used to quantify the ERE-binding proteins present in the nuclear extracts was 5'-CTAGCAGACATGGCAGATC-3' (7). Labeling of the probe was performed using [³²P]dATP and the T4 kinase reaction (45). The specificity of nuclear protein binding to the ERE probe was confirmed by competition with an unlabeled ERE probe. Gel shift mobility assays were performed as previously described (7, 45) using 5 µg of parietal cells nuclear extracts and the ³²P-labeled ERE oligonucleotide probe (10,000 cpm/0.5 ng) in a 20-µl solution containing 10 mM Tris-HCl (pH 7.9), 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, and 300 ng of double-stranded poly(dI-dC) as nonspecific competitor. The mixture was preincubated, when indicated, at 25 °C for 10 min with unlabeled competitors, for 10 min with labeled probe and then resolved on a 4% nondenaturing polyacrylamide gel (acrylamide/bisacrylamide ratio, 30:1). After pre-electrophoresis at 10 V/cm for 2 h, gels were run at 10 V/cm in 45 mM Tris base/45 mM boric acid/1 mM EDTA. The gels were dried prior to autoradiography at –80 °C.

Amplification and Purification of Adenoviral Vectors—Briefly, the recombinant adenoviruses were amplified as previously described using HEK293 cells (39). The viruses were subsequently concentrated and purified on a cesium chloride gradient (39). The concentration of the recombinant adenoviruses was assessed on the basis of the absorbance at 260 nm and on a limiting dilution plaque assay (39).

Histochemistry—These studies were carried out according to previously reported methods with minor modifications (49, 50). Briefly, the parietal cells were cultured on glass slides and fixed in 4% paraformaldehyde. The cells were permeabilized in 0.2% Triton X-100 for 15 min at room temperature. After three rinses with PBS the slides were blocked for 1 h in 5% milk, PBS. At the end of the incubation period, the slides were rinsed with PBS and incubated, for 1 h at room temperature, with rabbit polyclonal antibodies directed against either Shh (sc-9024, 1:50) or ptc (sc-9016, 1:50) and with mouse monoclonal antibodies directed against the H⁺, K⁺-ATPase -subunit 1:200, MBL). The primary antibodies were diluted in PBS, 1% milk. Immunostaining for either Shh or ptc preceded staining of the parietal cells with the H⁺, K⁺-ATPase -subunit antibodies. After three rinses with PBS, the slides were incubated with specific donkey secondary antibodies (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA) diluted in PBS, 1% milk, for 1 h at room temperature. The secondary antibodies were labeled with either fluorescein isothiocyanate (FITC) or Cy-3. At the end of the incubation periods with the secondary antibodies, the slides were rinsed with PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA). In control experiments, the parietal cells were incubated with the secondary antibodies without the primary antibodies. Visualization of slides was performed with a Zeiss LSM 510 version 3.2 confocal microscope. The microscope settings, such as lasers, excitation wavelengths, emission filters, and gain settings, were initially defined for proper image acquisition using empirical image quality. The settings were stored in the microscope data base and recalled through the re-use function for analysis of all subsequent experiments. With this system, the microscope settings were strictly controlled and maintained constant for all images generated. Fluorescence excitation was provided by an argon laser at 488 nm (FITC) and a helium-neon laser at 543 nm (Texas Red). The emission filters were a bandpass filter set at 505–550 nm and a longpass filter set at 560 nm. The detector gains were 788 (Zeiss value) and 606 (Zeiss value) for FITC and Texas Red, respectively.

Aminopyrine Uptake—Gastric acid secretion was measured according to previously described methods (7, 9). Briefly, the accumulation of the weak base [¹⁴C]aminopyrine (Amersham Biosciences) was used as an indicator of acid production by parietal cells. The cultured parietal cells were washed once with Earle's balanced salt solution, incubated with 0.1 µCi of [¹⁴C]aminopyrine for 60 min, and then stimulated, when indicated, with histamine for the last 30 min of aminopyrine incubation. In some experiments, the cells were cultured for 16–18 h in the presence of either EGF (10 nM) or Sonic hedgehog (0.5 µg/ml) prior to the addition of histamine. The parietal cells were lysed with 500 µl of 1% Triton X-100, and the radioactivity of lysate was quantified in a liquid scintillation counter as previously reported (7, 9).

Gel Filtration Chromatography—Shh in the parietal cell-conditioned media was identified using previously described methods (51). Briefly, equal volumes of media derived from equal numbers of parietal cells, which were either left untreated or stimulated with 10 nM EGF for 16 h, were collected and centrifuged at 2000 rpm for 15 min. The supernatants were concentrated using Centricon YM-10 (Millipore, Bedford, MA) and loaded on to a Superose 12 (Amersham Biosciences) gel filtration column that had been equilibrated with PBS/0.01% Nonidet P-40. The column was calibrated by denoting the elution fraction of a recombinant Shh, amino-terminal peptide standard. The fractions were collected, concentrated with Centricon YM-10, and subjected to Western blot analysis using specific rabbit anti-Shh antibodies (sc-9024).

Data Analysis—Data are expressed as means ± S.E., wherein n is equal to the number of separate dog preparations from which the parietal cells were obtained. Statistical analysis was performed using Student's t test. p values < 0.05 were considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We first investigated if Shh is expressed in the gastric parietal cells. For these studies we used non-Percoll-purified gastric epithelial cells from elutriator fractions 8 and 9, which are enriched in parietal cells (50). As shown in Fig. 1 (A–C), immunofluorescence staining of the cells, with an anti-H⁺/K⁺-ATPase -subunit primary antibody and a Cy-3-conjugated secondary antibody together with an anti-Shh primary antibody and a FITC-conjugated secondary antibody, demonstrated co-localization of Shh with the H⁺/K⁺-ATPase -subunit in the gastric parietal cells. The specificity of the immunohistochemical staining was underscored by the observation that a few cells did not stain for both the H⁺/K⁺-ATPase -subunit and Shh. Similar results were observed when the cells were stained with the anti-H⁺/K⁺-ATPase -subunit antibody together with an antibody recognizing the Shh transmembrane receptor protein, ptc (Fig. 1, D–F). No staining was detected in control experiments in which the slides were incubated with either the Cy-3- or FITC-conjugated secondary antibodies without the primary antibodies (Fig. 1, G–I). Accordingly, both ptc and Shh appear to be specifically localized in the canine parietal cells.

View larger version (41K): [in this window] [in a new window]   FIG. 1. Expression of Shh and ptc in the parietal cells. Gastric mucosal cells from elutriator fractions 8 and 9 were stained with the anti-H+, K+-ATPase -subunit antibody and a Cy-3-conjugated secondary antibody (A and D). Images of the same cells stained with either anti-Shh or anti-Ptc antibodies and with FITC-conjugated secondary antibodies are shown in B and E, respectively. Corresponding differential interference contrast images (DIC) are shown in C, F, and I. The images are representative 1-µm confocal fluorescence sections. Size bars, 20 µm. The arrows indicate cells that did not stain with both the anti-H+, K+-ATPase -subunit antibody and either the anti-Shh or the anti-Ptc antibodies. Identical results were obtained in at least two other separate parietal cell preparations.

View larger version (24K): [in this window] [in a new window]   FIG. 2. PI3K and Akt regulate Shh expression in the gastric parietal cells. Expression of Shh and actin in lysates from cultured parietal cells stimulated for 16 h with EGF (10 nM), in the presence or absence of 10 µM LY294002 (A and B), or after transduction of the cells with either the adenoviral vector expressing dominant negative Akt (Ad.dom.neg-Akt) or that expressing -galactosidase (Ad.CMV--gal) (C and D), was studied by Western blots using specific anti-Shh and anti-actin antibodies. A and C, representative blots obtained with a single cell preparation. B and D, results obtained from densitometric analysis of Western blots derived from several cell preparations. Data are expressed as -fold induction over control, mean ± S.E., n = 3. O.D., optical density. *, p < 0.05.

View larger version (14K): [in this window] [in a new window]   FIG. 3. EGF induces Shh release from the parietal cells. Media from parietal cells incubated in the presence or absence of EGF (10 nM), for 16 h were collected, concentrated, and subjected to gel filtration chromatography. The peak fractions (fractions 15 and 16) were pooled and subjected to Western blot analysis using specific anti-Shh antibodies. Similar results were observed in experiments with at least two other separate parietal cell preparations.

View larger version (28K): [in this window] [in a new window]   FIG. 4. Shh regulates H+, K+-ATPase -subunit gene expression. Aliquots of total RNA (10 µg) extracted following stimulation of the cultured cells for 16 h with Shh (0.1–0.5 µg/ml), was examined by Northern blot analysis using 32P-labeled cDNA probes for the H+, K+-ATPase -subunit gene. The autoradiograms were controlled for RNA quantity by hybridization of the RNA with a cDNA probe encoding the ubiquitous enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Identical results were observed in experiments with at least two other separate parietal cell preparations.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Shh regulates H+, K+-ATPase -subunit gene transcription. The parietal cells were co-transfected with either HK(–619 + 35)-Luc (A) or ERE-HK(–54 + 34)-Luc (B) together with the pCMV-Gal expression vector and treated with either EGF (10 nM) or Shh (0.1–0.5 µg/ml) for 16 h. Data are expressed as -fold induction over control, mean ± S.E., n = 3in A, n = 4in B. RLU, relative light units. C, mobility shift assays were performed with 5 µg of nuclear extracts from untreated parietal cells or from cells treated with Shh (0.5 µg/ml) for 16 h. Identical results were observed in experiments with at least three other separate parietal cell preparations.

View larger version (21K): [in this window] [in a new window]   FIG. 6. Gli transcription factors regulate HK(–619 + 35)-Luciferase activity. A, co-transfection of the parietal cells with the plasmid 8X3'Gli-BS-Luc together with the pCMV-Gal expression vector, in the presence or absence of 10 nM EGF. B, co-transfection of the parietal cells with the plasmid HK(–619 + 35)-Luc together with vectors expressing -galactosidase (pCMV-Gal) and the transcription factor Gli2 (Gli2). C, co-transfection of the parietal cells with the plasmid HK(–619 + 35)-Luc together with the pCMV-Gal expression vector and a dominant negative Gli2 gene (Gli2C4), in the presence or absence of 10 nM EGF. Control experiments were performed by transfecting the cells with vectors lacking either Gli2 or Gli2C4 coding sequences (PCDNA3). Data are expressed as -fold induction over control, mean ± S.E., n = 3 in A and B, n = 4 in C. *, p < 0.05. RLU, relative light units.

View larger version (20K): [in this window] [in a new window]   FIG. 7. Effect of cyclopamine on EGF-stimulated HK(–619 + 35)-Luc (A) and c-fos-(–357–276)-Luc transcriptional activity. The parietal cells were transfected with either the plasmid HK(–619 + 35)-Luc (A) or with c-fos-(–357–276)-Luc (B), together with the pCMV-Gal expression vector and treated with 10 nM EGF in the presence or absence of 1 µM cyclopamine. Data are expressed as -fold induction over control, mean ± S.E., n = 3. *, p < 0.05. ns, non-significant; RLU, relative light units.

View larger version (22K): [in this window] [in a new window]   FIG. 8. Role of Akt in EGF- and Shh-stimulated HK(–619 + 35)-Luc transcriptional activity. The parietal cells were co-transfected with the plasmid HK(–619 + 35)-Luc together with pCMV-Gal and a plasmid expressing a dominant negative Akt gene (dnAkt) and treated with either 10 nM EGF (A) or 0.5 µg/ml Shh (B). Control experiments were performed by transfecting the cells with a vector lacking dominant negative Akt coding sequences (PCDNA3). Data are expressed as -fold induction over control, mean ± S.E., n = 4in A, n = 3in B.*, p < 0.05. RLU, relative light units. C, phosphorylation and activation of Akt kinase in lysates from cells stimulated for 5 min with either 10 nM EGF or 0.5 µg/ml Shh, was studied by Western blots using a specific anti-phospho-Akt antibody. Identical results were observed in experiments with at least one other separate parietal cell preparation.

View larger version (27K): [in this window] [in a new window]   FIG. 9. Shh enhances secretagogue-stimulated gastric acid secretion. Percoll-purified cultured, canine gastric parietal cells were incubated for 30 min with 100 µM histamine, alone, or after preincubation for 16 h with either 0.5 µg/ml Shh or 10 nM EGF. Data are expressed as -fold induction over control, mean ± S.E., n = 3. *, p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In this study, we demonstrate that the canine parietal cells express both Shh and its transmembrane receptor protein, Ptc. In addition, we report that EGF, a growth factor that has been shown to exert important regulatory actions on the growth and differentiation of the gastric epithelium (1–4), stimulates the expression and the release of Shh from the parietal cells, suggesting that this peptide might be an important mediator of the actions of EGF in the stomach through the activation of both paracrine and autocrine mechanisms.

Shh is known to undergo extensive post-translational modifications to gain biological activity (18, 53). In most cells, the full-length form of Shh migrates with a mobility that corresponds to a relative mass of 46 kDa (53). This protein is then converted to a species of 39 kDa, which is thought to represent the signal-cleaved form of the full-length peptide. Further processing of this molecular form of Shh appears to generate smaller molecules of 19, 25, and 16 kDa, respectively. Of these, the 19-kDa amino-terminal peptide undergoes esterification at its C terminus to a cholesterol molecule prior to being secreted into the extracellular space where it mediates the physiological actions of Shh (18, 53, 54). In agreement with these reports, we observed that, in the parietal cells, EGF stimulates the expression of a protein of 35 kDa, which is likely to represent the signal-cleaved form of full-length Shh. In addition, we noted that EGF induces the release, in the culture medium, of the 19-kDa molecular form of Shh which is thought to mediate the biological activities of Shh on target cells (18, 53, 54). Several mechanisms could be considered to explain these observations. One possibility would be that EGF stimulates the processing of Shh, leading to increased production of the smaller molecular forms of this peptide. Alternatively, EGF might induce either the expression of the Shh gene or the translation of the Shh protein, leading, in the end, to increased production, processing, and release of Shh. It is clear that future studies will be necessary to better define these important biochemical issues.

EGF has been shown to stimulate H⁺, K⁺-ATPase -subunit gene expression. Previous studies conducted in canine parietal cells in primary culture transfected with plasmids containing a series of H⁺, K⁺-ATPase -subunit gene promoter deletion mutants, demonstrated that the sequence comprised between bases –619 to +34 of the promoter, is EGF-responsive (7). In addition, those reports identified and characterized a specific element located between bases –162 and –156 of the promoter, which appears to be sufficient to confer EGF responsiveness (7). In this report, we propose that the regulatory actions of EGF on the H⁺, K⁺-ATPase -subunit gene might be, at least in part, mediated by Shh. In support of this hypothesis, we demonstrate that Shh induces H⁺, K⁺-ATPase -subunit gene expression and that it stimulates the activation of the H⁺, K⁺-ATPase -subunit gene promoter EGF-responsive regulatory element. In addition, we report that both cyclopamine, a specific inhibitor of Smo activation (52), and a dominant negative Gli2 gene are able to inhibit the stimulatory actions of EGF on H⁺, K⁺-ATPase -subunit gene transcription, confirming the notion that the Shh signal transduction pathway is involved in the regulation of some of the actions of EGF in the parietal cells.

Gli transcription factors mediate the intracellular actions of Shh (17, 25–30). Gli2, in particular, appears to be the principal transducer of the physiological effects of Shh in target cells (17, 25–30). Our studies suggest that Gli2 is involved in the regulation of the stimulatory actions of EGF on the H⁺, K⁺-ATPase -subunit gene, because expression of Gli2 potently activates the H⁺, K⁺-ATPase -subunit gene promoter, whereas a dominant negative mutant of this transcription factor blocks EGF stimulation of H⁺, K⁺-ATPase -subunit gene transcription. These findings are further supported by the observation that, in the parietal cells, EGF induces signal transduction pathways that target a luciferase reporter plasmid containing Gli-responsive elements. Although these results strongly support the notion that Gli2 belongs to a cascade of biochemical events that link EGF to the H⁺, K⁺-ATPase -subunit gene promoter, they do not allow us to define if the actions of Gli2 on the H⁺, K⁺-ATPase -subunit gene are direct or indirect. Indeed, it would be conceivable to speculate that Gli2 might induce the sequential transcription of still undetermined, downstream nuclear proteins, which, in turn, could exert regulatory functions on the H⁺, K⁺-ATPase -subunit gene. Alternatively, even if the ERE is not a canonical Gli binding site, we cannot exclude that Gli transcription factors could interact directly with the H⁺, K⁺-ATPase -subunit ERE. Future experiments will be needed to define these intriguing possibilities.

In previous studies we observed that Akt, plays a crucial role in the process of parietal cell maturation and differentiation (9, 50). Here we report that, in the parietal cells, Akt mediates the stimulatory action of EGF on the expression of Shh, underscoring the importance of this kinase in the regulation of gastric epithelial cell differentiation. Recent studies have indicated that Shh induces the activation of Akt in immortalized murine brain capillary endothelial cells (55). In contrast to these findings, our results clearly demonstrate that Shh does not induce Akt in the parietal cells. Thus, in the stomach, Akt appears to regulate the expression of Shh, an important mediator of parietal cell maturation and differentiation, in response to growth factor stimulation. These findings suggest the existence of a novel mechanism linking a growth factor, such as EGF, to Shh, through the induction of the Akt signal transduction pathway.

One important observation of our study is that incubation of the parietal cells with Shh leads to enhancement of secretagogue-stimulated gastric acid secretion, an event that appears to be identical to that observed in the presence of EGF (7, 9). Thus, Shh could be one of the effectors of the stimulatory actions of EGF on H⁺/K⁺-ATPase -subunit gene expression and gastric acid production.

In conclusion, our study demonstrates that Akt is a crucial molecular switch that regulates the expression of Shh. Moreover, the Shh signal transduction pathway appears to mediate some of the actions of EGF in the parietal cells. These findings shed new insight into the complex signal transduction pathways that mediate the actions of growth factors in the stomach, providing new clues for a better understanding of the mechanisms that regulate gastric epithelial cell growth and differentiation.

	FOOTNOTES

 
^* This work was supported in part by NIDDK, National Institutes of Health (NIH) Grants RO1-DK-058312 (to A. T.), PO1-DK-062041 (to A. T. and J. L. M.), and RO1-DK-1410 (to J. L. M.) and by funds from the National Organization for Rare Disorders (NORD) (to A. T.), and the University of Michigan Gastrointestinal Peptide Research Center (NIH Grant P30-DK-34933). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Pilot Feasibility Grant DK-34933 from the Gastrointestinal Peptide Research Center.

To whom correspondence should be addressed: Dept. of Internal Medicine, University of Michigan Medical Center, 6520 MSRB I, Ann Arbor, MI 48109-0682. Tel.: 734-647-2942; Fax: 734-763-2535; E-mail: atodisco{at}umich.edu.

¹ The abbreviations used are: EGF, epidermal growth factor; Shh, Sonic hedgehog; CMV, cytomegalovirus; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; RLU, relative light unit(s); FITC, fluorescein isothiocyanate; PI3K, phosphatidylinositol 3-kinase; ERE, EGF-responsive sequence.

	ACKNOWLEDGMENTS

 
We thank Jung Park for preparing the parietal cells, Daniel Miller, Jace Nielsen, Matthew Brown, and Kristi Brown for technical assistance and Chris Edwards and the University of Michigan Microscopy and Image-analysis Laboratory for assistance with confocal microscopy.

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