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Am J Physiol Gastrointest Liver Physiol 293: G607-G614, 2007. First published June 28, 2007; doi:10.1152/ajpgi.00194.2006
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HORMONES AND SIGNALING

Functional role of bone morphogenetic protein-4 in isolated canine parietal cells

Hildegard Nitsche, Saravanan Ramamoorthy, Mahdi Sareban, Nonthalee Pausawasdi, and Andrea Todisco

Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bone morphogenetic protein (BMP)-4 is an important regulator of cellular growth and differentiation. Expression of BMP-4 has been documented in the gastric mucosa. We reported that incubation of canine parietal cells with EGF for 72 h induced both parietal cell morphological transformation and inhibition of H+/K+-ATPase gene expression through MAPK-dependent mechanisms. We explored the role of BMP-4 in parietal cell maturation and differentiation. Moreover, we investigated if BMP-4 modulates the actions of EGF in parietal cells. H+/K+-ATPase gene expression was examined by Northern blots and quantitative RT-PCR. Acid production was assessed by measuring the uptake of [14C]aminopyrine. Parietal cell apoptosis was quantitated by Western blots with anti-cleaved caspase 3 antibodies and by counting the numbers of fragmented, propidium iodide-stained nuclei. MAPK activation and Smad1 phosphorylation were measured by Western blots with anti-phospho-MAPK and anti-phospho-Smad1 antibodies. Parietal cell morphology was examined by immunohistochemical staining of cells with anti-H+/K+-ATPase {alpha}-subunit antibodies. BMP-4 stimulated Smad1 phosphorylation and induced H+/K+-ATPase gene expression. BMP-4 attenuated EGF-mediated inhibition of H+/K+-ATPase gene expression and blocked EGF induction of both parietal cell morphological transformation and MAPK activation. Incubation of cells with BMP-4 enhanced histamine-stimulated [14C]aminopyrine uptake. BMP-4 had no effect on parietal cell apoptosis, whereas TGF-beta stimulated caspase-3 activation and nuclear fragmentation. In conclusion, BMP-4 promotes the induction and maintenance of a differentiated parietal cell phenotype. These findings may provide new clues for a better understanding of the mechanisms that regulate gastric epithelial cell growth and differentiation.

Smad proteins; mitogen-activated protein kinases/extracellular signal-regulated kinases; cellular differentiation; apoptosis; gastric acid secretion

THE PARIETAL CELLS are highly specialized gastric epithelial cells that are known to secrete gastric acid in response to stimulation with gastric acid secretagogues (7). In addition to this well-established and characterized function, the parietal cells appear to play an important role in the regulation of complex programs of cellular growth and differentiation in the gastric mucosa.

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 (4, 11, 19, 22, 31). These intriguing observations are thought to be secondary to the ability of the parietal cells to produce and secrete growth factors and morphogens, such as TGF-{alpha} and sonic hedgehog (Shh), in the gastric mucosa (2, 5, 34, 39).

Shh, in particular, is a member of the family of hedgehog proteins, which are peptides known to exert important regulatory functions in patterning and growth in a large number of tissues during embryogenesis (10, 27, 28, 30, 34, 39, 40). In the mammalian stomach, Shh has been shown to be an important factor for the regulation of gastric epithelial cell maturation and differentiation (27, 39, 40). Recent studies have shown that Shh-null mice fail to develop a normal gastric epithelium (27). In addition, inhibition of Shh signaling in the gastric mucosa leads to diminished expression of bone morphogenetic protein (BMP)-4 (39), a member of a family of regulatory peptides that have been shown to play an important role in the modulation of embryonic and postnatal vertebrate development (9, 14, 41). Interestingly, this effect of Shh appears to be highly specific, since Shh fails to affect the expression of BMP-2, another member of the BMP family, whose expression has been documented in the gastrointestinal mucosa (13, 39). Thus, BMP-4 but not BMP-2 is a target of the Shh signal transduction pathway in the stomach.

BMPs activate several complex signal transdsuction pathways to exert their biological actions (17, 18, 29, 43). In particular, binding of BMPs to the BMP type I receptor (BMPR-I), leads to the dimerization of BMPR-I with the BMP type II receptor (BMPR-II), a molecule that has serine/threonine kinase activity. This event triggers the phosphorylation of both BMPR-I and of Smad1, -5, and -8, which are proteins known to mediate the intracellular actions of BMPs. Upon phosphorylation, Smad1, -5, and -8 associate with Smad 4 in a heterodimeric complex that transclocates to the nucleus, where it activates gene transcription (17, 18, 29, 43).

We have recently observed that stimulation of highly purified parietal cells in primary culture with EGF for 16 h induces H+/K+-ATPase gene expression, a marker of parietal cell differentiation, through mechanisms that involve the sequential activation of the Akt and Shh signal transduction pathways (21, 33, 34, 36). In particular, we demonstrated that EGF stimulates the production and release of Shh from the parietal cells and that this peptide induces H+/K+-ATPase gene expression (34). Thus, Shh might regulate parietal cell differentiation and maturation through both paracrine and autocrine mechanisms (34, 39). In contrast to these findings, we noted that prolonged exposure (>72 h) of the parietal cells to EGF induces dramatic morphological changes that are associated with inhibition of H+/K+-ATPase gene expression through MAPK-dependent mechanisms (33). On the basis of these observations, we hypothesized that while a short exposure of the parietal cells to growth factors promotes the expression of a highly differentiated cellular phenotype, prolonged activation of the parietal cell EGF receptor appears to activate mechanisms that might induce parietal cell dedifferentiation.

Accordingly, we took advantage of a well-established system based on primary cultures of highly purified canine gastric parietal cells to study the function of BMP-4, a downstream target of the Shh signal transduction pathway (39), in the parietal cells. Moreover, we investigated if BMP-4 modulates some of the actions of EGF in the parietal cells.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Primary parietal cell preparation and culture. For the preparation of primary parietal cells, we used a modification of the method of Soll et al. (6, 24, 32). The mucosal layer of a freshly obtained canine gastric fundus was bluntly separated from the submucosa and rinsed in HBSS containing 0.1% BSA. 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 contained 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 (Pharmacia Biotech, Piscataway, NJ) at 30,000 g for 20 min. The cell fraction at a density = 1.05 consisted of virtually all parietal cells as determined by staining with an anti-H+/K+-ATPase {alpha}-subunit monoclonal antibody (MBL, Nagoya, Japan). Isolated parietal cells (2 x 106 cells/well) were cultured according to the method of Chew et al. with some modifications (6, 24). Briefly, the cells were cultured in Ham's F-12-DMEM (1:1) containing 0.1 mg/ml gentamycin, 50 U/ml penicillin G, 0.01 mg/ml ciprofloxacin, and 2% DMSO (Sigma, St. Louis, MO) in six-well culture dishes (Corning, Corning, NY) coated with 150 µl of H2O-diluted (1:5) growth factor-reduced Matrigel (Becton Dickinson, Bedford, MA). For our experiments, the parietal cells were incubated with BMP-4 (20 ng/ml, R&D Systems, Minneapolis, MN), EGF (10 nM, Becton Dickinson), histamine (100 µM, Sigma), and TGF-beta (3 ng/ml, Oncogene Research Products, Cambridge, MA) for various time periods. EGF and histamine were dissolved in water, and BMP-4 and TGF-beta were dissolved in 4 mM HCl containing 0.1% BSA. Control experiments were performed by incubating the cells with vehicle without the test substances.

Northern blot analysis. The parietal cells were lysed with TRIzol (GIBCO-BRL, Grand Island, NY) according to the manufacturer's instructions. Northern blot hybridization assays were performed as previously described (33, 34). 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 RNA was transferred from the gel to nitrocellulose filters. Ethidium-stained rRNA bands in the gel were photographed before and after the 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 {alpha}-subunit cDNA probe was a gift of Il Song (University of Michigan). The human GAPDH cDNA probe was obtained from Clontech (Palo Alto, CA). cDNAs were labeled with [32P]dCTP by the random priming procedure, and nitrocellulose filters were hybridized to the 32P-labeled cDNA probes as previously described (33, 34).

Quantitative RT-PCR analysis. Quantitative RT-PCR analysis was performed according to previously reported techniques (23). RNA was isolated using TRIzol reagent, purified, and DNase treated using RNeasy (Qiagen, Valencia, CA); 200 ng of RNA were reverse transcribed in a 10-µl volume using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) following the manufacturer's protocol. Quantitative PCR was performed using an Icycler (Bio-Rad) with SYBR green dye (Molecular Probes). Each 20-µl reaction contained 2 µl reverse-transcribed product, PCR buffer (Invitrogen), 5.5 mM MgCl2, 100 nM each primer, 0.1x SYBR green, 10 nM fluorescein, 200 µM dNTPs, and 0.025 UPlatinum Taq polymerase (Invitrogen). Canine H+/K+-ATPase {alpha}-subunit gene primers (forward: 5'-ACCAGACCAGTGCGACAAAGG-3' and reverse: 5'-CCACGACCACGGCGATGAG-3') and canine GAPDH gene primers (forward: 5'-ACCACCGTCCATGCCATCACTG-3' and reverse: 5'-GGATGACCTTGCCCACAGCCTTG-3') for quantitative PCR were obtained from Integrated DNA Technologies (Coralville, IA). Primer pairs were validated by confirming quantitative amplification with a dilution of control cellular RNAs. Cycle parameters for both genes were as follows: 95°C for 3 min; 40 cycles each at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and a final elongation at 72°C for 5 min. After amplification was completed, samples were subjected to melt curve analysis by increasing the temperature to 100°C in 0.5°C intervals every 10 s for 80 steps to assess product purity. No signal was detected with control samples that were not treated with reverse transcriptase (data not shown). The relative abundance of the H+/K+-ATPase {alpha}-subunit gene was calculated using the comparative threshold cycle (CT) method according to previously published techniques (25). Data for the H+/K+-ATPase {alpha}-subunit gene were normalized to the CT of GAPDH, which was measured in the same RNA sample and PCR run. The GAPDH CT was subtracted from the CT of the H+/K+-ATPase {alpha}-subunit, giving the {Delta}CT for each sample. To compare the relative expression of the H+/K+-ATPase {alpha}-subunit gene between untreated and BMP-4-stimulated samples, the {Delta}CT for each control sample was subtracted from the {Delta}CT of each BMP-4-stimulated sample. This value yielded the {Delta}{Delta}CT. The fold change of expression between control and BMP-4-stimulated samples was calculated using the following formula: fold change = 2–{Delta}{Delta}CT.

Western blot analysis. The parietal cells were lysed in 500 µl of lysis buffer [containing 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 Na4P2O7·10 H2O, 1 mM AEBSF (ICN Biomedicals, Aurora, OH), 1 µg/ml leupeptin, and 1 µg/ml aprotinin]. Cell homogenates were spun at 1,000 rpm for 5 min at 4°C. Supernatants were transferred to Eppendorf tubes. Protein concentrations were measured by the Bradford method (3). Parietal cell lysates (80 µg) were mixed with 5x electrophoresis buffer [containing 50% glycerol, 25% mercaptoethanol, 10% SDS, 0.3 M Tris (pH 6.8), and 0.025% bromophenol blue], boiled for 5 min, and loaded on 10% SDS-polyacrylamide minigels, which were run at 200 V for 1 h. Gels were transferred on an Immobilon-P transfer membrane (Millipore, Bedford, MA) in 25 mM Tris, 150 mM glycine, and 20% methanol. After the transfer, membranes were blocked in 10 ml of Tris-buffered saline-Tween (TBST; containing 20 mM Tris, 0.15 M NaCl, and 0.1% Tween) with 5% dry milk for 1 h and then incubated for 16–18 h at 4°C in 10 ml of TBST and 5% dry milk containing specific antibodies recognizing phosphorylated Thr202 and Tyr204 of ERK2 (1:1,000) and phosphorylated Ser463 and Ser465 of Smad1 (1:1,000) (Cell Signaling, Beverly, MA). In additional experiments, membranes were incubated with antibodies recognizing ERK2 and Smad1 independent of their phosphorylation state (1:1,000) and cleaved caspase-3 (1:1,000) (Cell Signaling, Beverly, MA). At the end of the incubation periods, membranes were washed in TBST for 30 min at room temperature and then incubated for 1 h in TBST and 5% dry milk containing protein A directly conjugated to horseradish peroxidase (1:2,500, Amersham Life Science). Membranes were washed in TBST for 30 min at room temperature and then exposed to the Amersham ECL detection system according to the manufacturer's instructions.

Histochemistry. These experiments were carried out according to previously reported methods with minor modifications (33, 34). Briefly, the parietal cells were cultured on glass slides and fixed in 4% paraformaldehyde. For the immunohistochemical experiments, 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 and PBS. At the end of the incubation period, the slides were rinsed with PBS and incubated for 1 h at room temperature with mouse monoclonal antibodies directed against the H+/K+-ATPase {alpha}-subunit (1:200, MBL). Primary antibodies were diluted in PBS and 1% milk. After three rinses with PBS, the slides were incubated with specific donkey Cy3-conjugated secondary antibodies (1:100, Jackson Immunoresearch Laboratories, West Grove, PA) diluted in PBS and 1% milk for 1 h at room temperature. 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. The slides were analyzed using 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. Settings were stored in the microscope database and recalled through the reuse function for analysis of all subsequent experiments. With this system, microscope settings were strictly controlled and maintained constant for all images generated. Fluorescence excitation was provided by a HeNe laser at 543 nm (Texas red). Emission filters were a bandpass filter set at 505–550 nm and a longpass filter set at 560 nm.

Measurement of nuclear fragmentation. Parietal cell nuclei were stained with propidium iodide (PI) according to previously reported methods (37). Briefly, cells were washed with PBS and incubated in 0.1% sodium citrate, 0.1% Triton X-100, 100 µg/ml RNAase, and 50 µg/ml PI for 20 min at room temperature. Nuclear fragmentation was measured by counting the numbers of PI-stained fragmented parietal cell nuclei and analyzing, in a blinded fashion, 200 nuclei/field of 5 different fields of each slide. Visualization of the slides was performed with a Nikon Eclipse E 800 fluorescence microscope.

Aminopyrine uptake. Gastric acid secretion was measured according to previously described methods (26). Briefly, the accumulation of the weak base [14C]aminopyrine (Amersham Life Science) was used as an indicator of acid production by parietal cells. Cultured parietal cells were washed once with Earle's balanced salt solution, incubated with 0.1 µCi [14C]aminopyrine for 60 min, and then stimulated with histamine for the last 30 min of aminopyrine incubation. In some experiments, the cells were cultured for 72 h in the presence of BMP-4 (20 ng/ml) prior to the addition of histamine (100 µM). The parietal cells were lysed with 500 µl of 1% Triton X-100, and the radioactivity of the lysate was quantified in a liquid scintillation counter as previously reported (26).

Data analysis. Data are expressed as means ± SE, where n is the number of separate dog preparations from which the parietal cells were obtained. Statistical analysis was performed using a two-tailed paired Student's t-test. P values of <0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We first examined if BMP-4 regulates H+/K+-ATP-ase {alpha}-subunit gene expression, a marker of a differentiated parietal cell phenotype (33). As shown in Fig. 1A, which depicts data generated using quantitative RT-PCR, BMP-4 stimulated H+/K+-ATPase {alpha}-subunit gene expression in a time-dependent manner, with a maximal effect observed after 72 h of incubation. To confirm the validity of these observations, we performed Northern blots using a specific canine H+/K+-ATPase {alpha}-subunit cDNA probe. In agreement with the quantitative RT-PCR data, BMP-4 stimulated H+/K+-ATPase {alpha}-subunit gene expression after 72 h of stimulation (Fig. 1B).


Figure 1
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  		Fig. 1. Bone morphogenetic protein (BMP)-4 regulates H+/K+-ATPase {alpha}-subunit gene expression. A: aliquots of total RNA extracted following exposure of parietal cells for 24, 48, and 72 h to 20 ng/ml BMP-4 were examined by quantitative (Q)RT-PCR. Data are expressed as means ± SE of relative H+/K+-ATPase {alpha}-subunit expression and reflect RNA samples isolated from 3 separate dog preparations. B: aliquots of total RNA extracted following exposure of parietal cells for 72 h to BMP-4 were examined by Northern blot analysis using a 32P-labeled canine cDNA probe for the H+/K+-ATPase {alpha}-subunit gene, as shown in representative blots (left) obtained with a single parietal cell preparation. Results obtained from densitometric analysis of blots derived from 5 parietal cell preparations are shown in the bar graphs (right). Data are expressed as means ± SE of fold induction over control. O.D., optic density. Autoradiograms were controlled for RNA quantity by hybridization of the RNA with a cDNA probe encoding the ubiquitous enzyme GAPDH. *P < 0.05.

 
One of the best-characterized signal transduction pathways that mediates the actions of BMP-4 is that leading to the phosphorylation of Smad1, 5, and 8 (17, 18, 29, 43). Accordingly, we examined the effect of BMP-4 on phosphorylation of Smad1, 5, and 8 using Western blots with specific anti-phospho-Smad1-5-8 antibodies. As shown in Fig. 2A, BMP-4 stimulated the phosphorylation of Smad1, 5, and 8 after 24, 48, and 72 h of incubation.


Figure 2
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  		Fig. 2. Effect of BMP-4 on Smad1-5-8 in parietal cells. The expression of phosphorylated (P-)Smad1-5-8 and GAPDH in lysates from parietal cells stimulated for 24, 48, and 72 h with BMP-4 was studied by Western blots using specific anti-phospho-Smad 1-5-8 and GAPDH antibodies (A). Total Smad1 levels were monitored by Western blot analysis with an antibody recognizing Smad1 independent of its phosphorylation state (B). Similar results were observed in experiments performed with cells derived from at least 2 other separate dog preparations.

 
The specificity of this effect was underscored by the observation that BMP-4 failed to stimulate the expression of Smad1 (Fig. 2B).

Since treatment of the parietal cells with EGF for 72 h potently inhibits H+/K+-ATPase {alpha}-subunit gene expression (33), we investigated if BMP-4 could modulate this effect. As shown in the Northern blot depicted in Fig. 3, EGF inhibited H+/K+-ATPase {alpha}-subunit gene expression. In contrast, treatment of the cells with EGF in combination with BMP-4 prevented the occurrence of the inhibitory effects of EGF, supporting the notion that BMP-4 might play an important role in the induction and maintenance of a differentiated parietal cell phenotype.


Figure 3
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  		Fig. 3. BMP-4 reverses the inhibitory effect of EGF on H+/K+-ATPase {alpha}-subunit gene expression. Aliquots of total RNA extracted following exposure of parietal cells for 72 h to EGF (10 nM), either alone or in combination with BMP-4 (20 ng/ml), were examined by Northern blot analysis using a 32P-labeled canine cDNA probe for the H+/K+-ATPase {alpha}-subunit gene, as shown in the representative blots obtained with a single parietal cell preparation (A). Bar graphs represent results obtained from densitometric analysis of blots from 6 parietal cell preparations (B). Data are expressed as means ± SE of fold induction over control. Autoradiograms were controlled for RNA quantity by hybridization of the RNA with a cDNA probe encoding the ubiquitous enzyme GAPDH. *P < 0.05.

 
We investigated if BMP-4 could affect EGF-stimulated parietal cell morphological transformation. As indicated in the confocal images shown in Fig. 4, which depict cells stained with an anti-H+/K+-ATPase {alpha}-subunit antibody and Cy3-conjugated secondary antibody, incubation of the cells with EGF induced significant morphological changes in the parietal cells. These changes were characterized by the loss of the typical round parietal cell morphology and by the appearance of fusiform cells. In contrast, as shown in Fig. 4, bottom right, cells incubated with EGF in combination with BMP-4 failed to exhibit any morphological transformation, suggesting that BMP-4 preserves the normal shape and appearance of the parietal cells. No staining was observed when the slides were incubated with Cy3-conjugated secondary antibodies without primary antibodies (data not shown).


Figure 4
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  		Fig. 4. BMP-4 inhibits EGF-induced parietal cell morphological transformation. Images of histochemical staining with anti-H+/K+-ATPase {alpha}-subunit antibody and Cy3-conjugated secondary antibody of untreated, Percoll-purified, cultured gastric parietal cells (control) or of cells incubated for 72 h with either 10 nM EGF or 20 ng/ml BMP-4 alone or in combination with EGF are shown. Images are representative 1-µm confocal fluorescence sections. Similar results were observed in experiments with at least 2 other separate parietal cell preparations. Objective magnification: x63. Bar = 20 µm.

 
MAPK mediates both EGF-stimulated parietal cell morphological transformation and EGF inhibition of H+/K+-ATPase {alpha}-subunit gene expression (33). Accordingly, we investigated the effect of BMP-4 on EGF induction of MAPK in the parietal cells after 72 h of incubation. As shown in Fig. 5, which depicts Western blots with specific anti-phospho-ERK2 antibodies, BMP-4 inhibited EGF-stimulated ERK2 activation.


Figure 5
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  		Fig. 5. BMP-4 inhibits EGF-stimulated ERK2 activation. Phosphorylation and activation of ERK2 in lysates from cells stimulated for 72 h with 10 nM EGF, alone or in combination with 20 ng/ml BMP-4, were studied by Western blots using a specific anti-phospho-ERK2 antibody. Total ERK2 levels were monitored by Western blots with an antibody recognizing ERK2 independent of its phosphorylation state (A). Bar graphs represent results obtained from densitometric analysis of blots from 4 parietal cell preparations (B). NT, no treatment. *P < 0.05.

 
To examine the functional significance of BMP-4 in gastric acid production, we tested the effect of BMP-4 on [14C]aminopyrine uptake. Exposure of the parietal cells for 30 min to histamine led to a sevenfold increase in aminopyrine uptake (7.15 ± 2.1-fold induction over control, mean ± SE, n = 5), whereas incubation of cells with BMP-4 had no significant effect (1.27 ± 0.15-fold induction over control, mean ± SE, n = 5). In contrast, preincubation of the cells for 72 h with BMP-4, prior to the addition of histamine, led to a 50% enhancement of histamine-stimulated aminopyrine uptake (Fig. 6). The difference between the effect of histamine and that of histamine in association with BMP-4, assessed using a paired Student's t-test, was found to be statistically significant (P < 0.05). Thus, BMP-4 appears to be important for the enhancement of secretagogue-stimulated gastric acid production.


Figure 6
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  		Fig. 6. BMP-4 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 72 h with 20 ng/ml BMP-4. Data are expressed as means ± SE of percentages of histamine-stimulated aminopyrine uptake in the absence of BMP-4; n = 5.

 
Since, in some systems, BMP-4 regulates cellular apoptosis (13, 38), we investigated the effect of BMP-4 and TGF-beta, another member of the same family of regulatory peptides (18, 43), on parietal cell apoptosis. Apoptosis requires the coordinate activation of a family of cellular proteases known as caspases (8, 35). Since caspase-3 is responsible for the execution of one of the final steps of the apoptotic cascade (8, 35), we examined the effect of BMP-4 and TGF-beta on caspase-3 activation by Western blot analysis using a specific anti-cleaved caspase-3 antibody. As shown in Fig. 7, TGF-beta but not BMP-4 stimulated caspase-3 activation.


Figure 7
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  		Fig. 7. Effect of BMP-4 and TGF-beta on caspase-3 activation. Activation of caspase-3 in lysates from parietal cells stimulated for 24 h with either 20 ng/ml BMP-4 or 3 ng/ml TGF-beta was studied by Western blots using a specific anti-cleaved caspase-3 antibody. Identical results were observed in experiments with at least 2 other separate parietal cell preparations.

 
One of the best-characterized features of cellular apoptosis is nuclear fragmentation (8, 35). To confirm the validity of the observations acquired with the caspase-3 Western blots, we investigated the effect of BMP-4 and TGF-beta on parietal cell nuclear fragmentation. Nuclear fragmentation was measured by counting the number of PI-stained fragmented parietal cell nuclei. As indicated in the images shown in Fig. 8, TGF-beta but not BMP-4 significantly stimulated parietal cell nuclear fragmentation.


Figure 8
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  		Fig. 8. Effect of BMP-4 and TGF-beta on parietal cell nuclear fragmentation. A: representative images of propidium iodide-stained parietal cell nuclei. Arrows pointing at fragmented parietal cell nuclei are shown in the bottom right. B: mean values ± SE of percentages of fragmented nuclei; n = 4. *P < 0.05.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Numerous studies have underscored the importance of the parietal cells in the biology and pathobiology of the gastric mucosa (2, 4, 5, 7, 11, 19, 22, 31). In this article, we report a series of novel observations regarding the actions of BMP-4 in the parietal cells. The crucial role of the BMPs in the regulation of growth and differentiation of the gastrointestinal epithelium has been the focus of numerous investigations. In the gut, the BMPs appear to inhibit cellular proliferation and to induce cellular apoptosis. Indeed, transgenic expression of the BMP inhibitor Noggin or conditional deletion of the BMPR-I gene in the intestinal epithelium lead to the formation of new crypts and to an increased number of proliferative cells in normally differentiated areas of the villi (12, 15). Interestingly, similar abnormalities have been detected in patients suffering from juvenile polyposis syndrome, a disease characterized by the formation of hamartomatous polyps throughout the gastrointestinal tract. Genetic analysis of these subjects has revealed that these abnormalities are due to the presence of germline mutations in genes involved in BMP signal transduction (16, 44). Additional studies performed in mice carrying a targeted mutation of the transcription factor Fkh6 have indicated that these animals exhibit decreased expression of BMP-4 in the gastric mucosa with increased proliferation of the gastric epithelium and decreased number of parietal cells (20).

We reported that BMP-4 is a potent inducer of H+/K+-ATPase {alpha}-subunit gene expression, a marker of parietal cell differentiation, confirming the notion that BMP signaling in the gastric epithelium might be an important regulator of the complex series of events that lead to the differentiation and maturation of the gastric parietal cells. The physiological significance of this observation was underscored by the finding that incubation of the parietal cells with BMP-4 led to enhancement of secretagogue-stimulated gastric acid secretion.

The expression of BMP-4 has been documented in myofibroblast-like cells located in submucosal layers of the stomach (38). Thus, in contrast to Shh, which is expressed in the parietal cells (34, 39), BMP-4 cannot be detected in gastric epithelial cells. Accordingly, although we previously reported that in isolated parietal cells Shh stimulates H+/K+-ATPase {alpha}-subunit gene expression (34), it is possible that some of the actions of Shh, in vivo, could be mediated by BMP-4, a well-established target of the Shh signal transduction pathway in the stomach (39). According to this model, Shh might exert both direct and indirect effects on parietal cells, with the latter being mediated by its ability to induce the expression and release of BMP-4 from gastric mesenchymal cells.

In this report, we demonstrated that BMP-4 induces the sustained phosphorylation of Smad1, a well-established transducer of BMP-4-activated signals (17, 18, 30, 44). Although it is currently unknown if Smad transcription factors bind to the H+/K+-ATPase {alpha}-subunit gene promoter, it is conceivable to speculate that Smads might interact with parietal cell-specific nuclear proteins to direct the transcription of the H+/K+-ATPase {alpha}-subunit gene.

In a previous study (33), we presented evidence that prolonged exposure of parietal cells to EGF leads to both inhibition of H+/K+-ATPase {alpha}-subunit gene expression and to parietal cell morphological transformation through MAPK-dependent mechanisms. In this report, we demonstrated that BMP-4 blocks these actions of EGF in the parietal cells. In particular, we report that BMP-4 potently inhibits EGF-stimulated MAPK activation. Thus, the ability of BMP-4 to inhibit MAPK in the parietal cells might represent an important biological mechanism responsible for the modulation of the actions of growth factors in the gastric mucosa. Several possible mechanisms might mediate the inhibitory actions of BMP-4 on MAPK activation. BMP-4 might induce phosphatases, such as the MAPK phosphatases, that specifically dephosphorylate and inactivate MAPK (42), or it might inhibit other elements of the signal transduction cascade that links the EGF receptor to MAPK activation (42), such as MEK, Raf, Ras, or the EGF receptor itself.

The complex interplay that might occur between MAPK and the signals generated by the interaction of BMPs with their specific cellular receptors has been further underscored by a series of recent investigations (1). According to this report (1), MAPK phosphorylates a region of the Smad1 molecule known as the linker region. Phosphorylation of the linker prevents the nuclear localization of Smad1, leading to inhibition of Smad1-dependent gene transcription. Indeed, mice carrying mutations of the linker region that prevents MAPK phosphorylation have significant alterations in the normal architecture of the gastric mucosa (1). One of the most intriguing abnormalities displayed by the stomach of these animals is represented by a significant increase in the number of parietal cells (1). Thus, MAPK signaling can negatively influence parietal cell maturation and BMP-4 signal transduction in the stomach.

Since BMPs are known to stimulate cellular apoptosis in the gastrointestinal tract (13, 38), we examined if BMP-4 could activate caspase-3 and induce parietal cell nuclear fragmentation. Our data indicated that BMP-4 does not induce apoptosis, suggesting that activation of the BMP-4 signal transduction pathway specifically activates programs of parietal cell maturation and differentiation, but not of programmed cell death.

In conclusion, our study demonstrates that BMP-4 plays a crucial role in the activation of programs of parietal cell differentiation. 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.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant PO1-DK-06204, by the University of Michigan Gastrointestinal Peptide Research Center (NIDDK Grant P30-DK-34933), and by funds from the National Organization for Rare Disorders.


   ACKNOWLEDGMENTS
 
The authors thank Jung Park for preparing the parietal cells; Maria Mao, Yinghua Xiao, 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.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Todisco, 6520 MSRB I, Ann Arbor, MI 48109-0682 (e-mail: atodisco{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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