Andrea Todisco

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

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 α-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 α-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 α-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.

Treatment of Helicobacter gastritis with IL-4 requires somatostatin.

Fifty percent of the worlds population is infected with Helicobacter pylori; however, treatment has been insufficient to eradicate the organisms due to rising antibiotic resistance. Helicobacter infection is characterized by induction of a T helper 1 lymphocyte (Th1) immune response, hypergastrinemia, and suppressed tissue somatostatin (SOM) levels. However, the mechanism by which the immune response regulates acid secretion is not known. We show here that treatment with IFN-γ, a Th1 cytokine, was sufficient to induce gastritis, increase gastrin, and decrease SOM levels within 7 days. In contrast, the T helper 2 lymphocyte cytokine IL-4 increased SOM levels and effectively suppressed gastrin expression and secretion. This result demonstrated reciprocal regulation of acid regulatory peptides by immune modulators. IL-4 pretreatment prevented gastritis in infected wild-type but not in SOM null mice. Thus, the ability of IL-4 to oppose a Th1-mediated infection required SOM. Immunofluorescence was used to document the presence of IL-4 receptors on the gastric SOM-secreting cell (D cell). Moreover, IL-4 stimulated SOM release from primary D cell cultures. Treatment of mice chronically infected with Helicobacter felis for 2 mo with the SOM analogue octreotide resolved the inflammation. Thus, a mechanism by which IL-4 resolves inflammation in the stomach is by stimulating the release of SOM from gastric D cells.

Interleukin-1β Promotes Gastric Atrophy Through Suppression of Sonic Hedgehog

BACKGROUND & AIMS In both human subjects and rodent models, Helicobacter infection leads to a decrease in Shh expression in the stomach. Sonic Hedgehog (Shh) is highly expressed in the gastric corpus and its loss correlates with gastric atrophy. Therefore, we tested the hypothesis that proinflammatory cytokines induce gastric atrophy by inhibiting Shh expression. METHODS Shh-LacZ reporter mice were infected with Helicobacter felis for 3 and 8 weeks. Changes in Shh expression were monitored using beta-galactosidase staining and immunohistochemistry. Gastric acidity was measured after infection, and interleukin (IL)-1beta was quantified by quantitative reverse-transcription polymerase chain reaction. Mice were injected with either IL-1beta or omeprazole before measuring Shh mRNA expression and acid secretion. Organ cultures of gastric glands from wild-type or IL-1R1 null mice were treated with IL-1beta then Shh expression was measured. Primary canine parietal or mucous cells were treated with IL-1beta. Shh protein was determined by immunoblot analysis. Changes in intracellular calcium were measured by Fura-2. RESULTS All major cell lineages of the corpus including surface pit, mucous neck, zymogenic, and parietal cells expressed Shh. Helicobacter infection reduced gastric acidity and inhibited Shh expression in parietal cells by 3 weeks. IL-1beta produced during Helicobacter infection inhibited gastric acid, intracellular calcium, and Shh expression through the IL-1 receptor. Suppression of parietal cell Shh expression by IL-1beta and omeprazole was additive. IL-1beta did not suppress Shh expression in primary gastric mucous cells. CONCLUSIONS IL-1beta suppresses Shh gene expression in parietal cells by inhibiting acid secretion and subsequently the release of intracellular calcium.

Molecular mechanisms for the growth factor action of gastrin

We have previously observed that gastrin has a cholecystokinin B (CCK-B) receptor-mediated growth-promoting effect on the AR42J rat pancreatic acinar cell line and that this effect is paralleled by induction of expression of the early response gene c- fos. We undertook these experiments to elucidate the mechanism for induction of c- fos and the linkage of this action to the trophic effects of gastrin. Gastrin (0.1-10 nM) dose dependently induced luciferase activity in AR42J cells transfected with a construct consisting of a luciferase reporter gene coupled to the serum response element (SRE) of the c- fos promoter. This effect was blocked by the specific CCK-B receptor antagonist D2 but not by the specific CCK-A receptor antagonist L-364,718 or by pertussis toxin, indicating that gastrin targets the SRE via specific CCK-B receptors through a mechanism independent of Gi. Inhibition of protein kinase C (PKC) either by prolonged (24 h) exposure of the cells to the phorbol ester 12- O-tetradecanoylphorbol 13-acetate (100 nM) or by incubation with the selective inhibitor GF-109203X (3.5 μM) resulted in an 80% reduction in luciferase activity. Similar results were observed in the presence of the specific extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD-98059 (50 μM). We measured ERK2 activity in AR42J cells via in-gel kinase assays and observed that gastrin (1 pM-100 nM) induced ERK2 enzyme activity in a dose-dependent manner. Addition of GF-109203X and PD-98059, either alone or in combination, produced, respectively, partial and total inhibition of gastrin-induced ERK2 activity. Gastrin induction of ERK2 activity also resulted in a threefold increase in the transcriptional activity of Elk-1, a factor known to bind to the c- fos SRE and to be phosphorylated and activated by ERK2. PD-98059 blocked the growth-promoting effect of gastrin on the AR42J cells, demonstrating that this effect depends on activation of MEK. Our data lead us to conclude that the trophic actions of gastrin are mediated by ERK2-induced c- fos gene expression via PKC-dependent and -independent pathways.

Reduced Pepsin A Processing of Sonic Hedgehog in Parietal Cells Precedes Gastric Atrophy and Transformation

Sonic hedgehog (Shh) is not only essential to the development of the gastrointestinal tract, but is also necessary to maintain the characteristic acid-secreting phenotype of the adult stomach. Gastrin is the only hormone capable of stimulating gastric acid and is thus required to maintain functional parietal cells. We have shown previously that gastrin-null mice display gastric atrophy and metaplasia prior to progression to distal, intestinal-type gastric cancer. Because reduced levels of Shh peptide correlate with gastric atrophy, we examined whether gastrin regulates Shh expression in parietal cells. We show here that gastrin stimulates Shh gene expression and acid-dependent processing of the 45-kDa Shh precursor to the 19-kDa secreted peptide in primary parietal cell cultures. This cleavage was blocked by the proton pump inhibitor omeprazole and mediated by the acid-activated protease pepsin A. Pepsin A was also the protease responsible for processing Shh in tissue extracts from human stomach. By contrast, extracts prepared from neoplastic gastric mucosa had reduced levels of pepsin A and did not process Shh. Therefore processing of Shh in the normal stomach is hormonally regulated, acid-dependent, and mediated by the aspartic protease pepsin A. Moreover parietal cell atrophy, a known pre-neoplastic lesion, correlates with loss of Shh processing.

Bone Morphogenetic Protein Signaling Regulates Gastric Epithelial Cell Development and Proliferation in Mice

BACKGROUND & AIMS We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach. METHODS The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses. RESULTS In the stomachs of the transgenic mice, phosphorylation of Smad 1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and Griffonia (Bandeiraea) simplicifolia lectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2. CONCLUSIONS Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis.

Cell type-specific requirement of the MAPK pathway for the growth factor action of gastrin

Gastrin (G17) has a CCKB receptor-mediated growth-promoting effect on the AR42J rat acinar cell line that is linked to induction of both mitogen-activated protein kinase (MAPK) and c- fos gene expression. We investigated the mechanisms that regulate the growth factor action of G17 on the rat pituitary adenoma cell line GH3. Both AR42J and GH3 cells displayed equal levels of CCKB receptor expression and similar binding kinetics of125I-labeled G17. G17 stimulation of cell proliferation was identical in both cell lines. G17 stimulation of GH3 cell proliferation was completely blocked by the CCKBreceptor antagonist D2 but not by the MEK inhibitor PD-98059 or the protein kinase C inhibitor GF-109203X, which completely inhibited G17 induction of AR42J cell proliferation. G17 induced a c- fos SRE-luciferase reporter gene plasmid more than fourfold in the AR42J cells, whereas it had no effect in the GH3 cells. In contrast to what we observed in the AR42J cells, G17 failed to stimulate MAPK activation and Shc tyrosyl phosphorylation and association with the adapter protein Grb2. Epidermal growth factor induced the MAPK pathway in the GH3 cells, demonstrating the integrity of this signaling system. G17 induced Ca2+ mobilization in both the GH3 and AR42J cells. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited AR42J cell proliferation by 20%, whereas it completely blocked G17 induction of GH3 cell growth. The Ca2+ ionophore ionomycin stimulated GH3 cell proliferation to a level similar to that observed in response to G17, but it had no effect on AR42J cell proliferation. Thus there are cell type specific differences in the requirement of the MAPK pathway for the growth factor action of G17. Whereas in the AR42J cells G17 stimulates cell growth through activation of MAPK and c- fos gene expression, in the GH3 cells, G17 fails to activate MAPK, and it induces cell proliferation through Ca2+-dependent signaling pathways. Furthermore, induction of Ca2+mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation.Gastrin (G17) has a CCKB receptor-mediated growth-promoting effect on the AR42J rat acinar cell line that is linked to induction of both mitogen-activated protein kinase (MAPK) and c-fos gene expression. We investigated the mechanisms that regulate the growth factor action of G17 on the rat pituitary adenoma cell line GH3. Both AR42J and GH3 cells displayed equal levels of CCKB receptor expression and similar binding kinetics of 125I-labeled G17. G17 stimulation of cell proliferation was identical in both cell lines. G17 stimulation of GH3 cell proliferation was completely blocked by the CCKB receptor antagonist D2 but not by the MEK inhibitor PD-98059 or the protein kinase C inhibitor GF-109203X, which completely inhibited G17 induction of AR42J cell proliferation. G17 induced a c-fos SRE-luciferase reporter gene plasmid more than fourfold in the AR42J cells, whereas it had no effect in the GH3 cells. In contrast to what we observed in the AR42J cells, G17 failed to stimulate MAPK activation and Shc tyrosyl phosphorylation and association with the adapter protein Grb2. Epidermal growth factor induced the MAPK pathway in the GH3 cells, demonstrating the integrity of this signaling system. G17 induced Ca2+ mobilization in both the GH3 and AR42J cells. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited AR42J cell proliferation by 20%, whereas it completely blocked G17 induction of GH3 cell growth. The Ca2+ ionophore ionomycin stimulated GH3 cell proliferation to a level similar to that observed in response to G17, but it had no effect on AR42J cell proliferation. Thus there are cell type specific differences in the requirement of the MAPK pathway for the growth factor action of G17. Whereas in the AR42J cells G17 stimulates cell growth through activation of MAPK and c-fos gene expression, in the GH3 cells, G17 fails to activate MAPK, and it induces cell proliferation through Ca2+-dependent signaling pathways. Furthermore, induction of Ca2+ mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation.

Glycine-extended gastrin regulates HEK cell growth

Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH2) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH2, glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH2 and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH2 can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH2 and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for 125I-labeled Leu15-G17-NH2and125I-Leu15-G2-17-Gly. Both G-NH2 and G-Gly induced a dose-dependent increase in [3H]thymidine incorporation, and both peptides together significantly increased [3H]thymidine incorporation above the level of either peptide alone. G-NH2 and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH2 and G-Gly lead to a significant reduction in [3H]thymidine incorporation. G-NH2 but not G-Gly increased intracellular Ca2+concentration. We conclude that G-NH2 and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.Posttranslational processing of progastrin to a carboxy terminally amidated form (G-NH(2)) is essential for its effect on gastric acid secretion and other biological effects mediated by gastrin/CCK-B receptors. The immediate biosynthetic precursor of G-NH(2), glycine-extended gastrin (G-Gly), does not stimulate gastric acid secretion at physiological concentrations but is found in high concentrations during development. G-NH(2) and G-Gly have potent growth stimulatory effects on gastrointestinal tissues, and G-NH(2) can stimulate proliferation of human kidney cells. Thus we sought to explore the actions of G-NH(2) and G-Gly on the human embryonic kidney cell line HEK 293. HEK 293 cells showed specific binding sites for (125)I-labeled Leu(15)-G17-NH(2) and (125)I-Leu(15)-G(2-17)-Gly. Both G-NH(2) and G-Gly induced a dose-dependent increase in [(3)H]thymidine incorporation, and both peptides together significantly increased [(3)H]thymidine incorporation above the level of either peptide alone. G-NH(2) and G-Gly were detected by radioimmunoassay in serum-free conditioned media. Antibodies directed against G-NH(2) and G-Gly lead to a significant reduction in [(3)H]thymidine incorporation. G-NH(2) but not G-Gly increased intracellular Ca(2+) concentration. We conclude that G-NH(2) and G-Gly act cooperatively via distinct receptors to stimulate the growth of a nongastrointestinal cell line (HEK 293) in an autocrine fashion.

Activation of the human histamine H2 receptor is linked to cell proliferation and c-fos gene transcription

We examined whether histamine could regulate cell proliferation and expression of the early response gene c- fos in HEK-293 cells stably transfected with the human H2receptor (HEK-H2). Histamine stimulated [3H]thymidine incorporation [50% effective concentration (EC50) = 3.6 × 10-6 M] in HEK-H2 cells in a cimetidine-sensitive manner and increased c- fos mRNA in a time-dependent fashion, reaching maximal induction after 30 min. Histamine induced luciferase activity in HEK-H2cells transiently transfected with a construct containing the luciferase reporter gene ( Luc) coupled to the serum response element (SRE) of the c- fos gene promoter (EC50 = 1.5 × 10-6 M) or a plasmid containing the SRE core fragment (bases -320 to -298). The protein kinase C (PKC) inhibitor staurosporine and long-term pretreatment of HEK cells with phorbol ester inhibited the effect of histamine on PKC activation, SRE- Lucactivity, and [3H]thymidine incorporation. We have demonstrated that activation of the human H2 receptor can lead to induction of c- fos gene transcription and cell proliferation through a PKC-dependent mechanism.

Functional role of extracellular signal-regulated protein kinases in gastric acid secretion

Epidermal growth factor (EGF) has acute inhibitory and chronic stimulatory effects on gastric acid secretion. Because a cascade of intracellular events culminating in the activation of a family of serine-threonine protein kinases called extracellular signal-regulated protein kinases (ERKs) is known to mediate the actions of EGF, we undertook studies to explore the functional role of the ERKs in gastric acid secretion. ERK2 was immunoprecipitated from cell lysates of highly purified (> 95%) gastric canine parietal cells, and its activity was quantified using in-gel kinase assays. Of the primary gastric secretagogues, carbachol was the most potent inducer of ERK2 activity. Gastrin and EGF had weaker stimulatory effects, whereas no induction was noted in response to histamine. The effect of carbachol appeared to be independent of Ca2+ signaling. PD-98059, a selective inhibitor of the upstream ERK activator mitogen-activated protein kinase/ERK kinase, dose-dependently inhibited both carbachol- and EGF-stimulated ERK2 activity, with a maximal effect observed between 50 and 100 microM. ERKs activation is required for induction of the early gene c-fos via phosphorylation of the transcription factor Elk-1 which binds to the c-fos serum response element (SRE). Carbachol stimulated a two- to threefold induction of luciferase activity in cultured parietal cells transfected with either a SRE-luciferase reporter plasmid or with a chimeric GAL4-ElkC expression vector and the 5 x GAL-luciferase reporter plasmid. To examine the significance of ERK activation in gastric acid secretion, we tested the effect of PD-98059 on carbachol-stimulated uptake of 14C-labeled aminopyrine (AP). Acute inhibition of the ERKs by PD-98059 led to a small increase in AP uptake and a complete reversal of the acute inhibitory effect of EGF on AP uptake induced by either carbachol or histamine. In contrast, exposure of the cells to PD-98059 for 16 h led to a reversal of the chronic stimulatory effect of EGF on AP uptake induced by carbachol. Our data led us to conclude that carbachol induces a cascade of events in parietal cells that results in ERK activation. Although the acute effect of the ERKs on gastric acid secretion appears to be inhibitory, the activation of transcription factors and of early gene expression could be responsible for its chronic stimulatory effects.Epidermal growth factor (EGF) has acute inhibitory and chronic stimulatory effects on gastric acid secretion. Because a cascade of intracellular events culminating in the activation of a family of serine-threonine protein kinases called extracellular signal-regulated protein kinases (ERKs) is known to mediate the actions of EGF, we undertook studies to explore the functional role of the ERKs in gastric acid secretion. ERK2 was immunoprecipitated from cell lysates of highly purified (>95%) gastric canine parietal cells, and its activity was quantified using in-gel kinase assays. Of the primary gastric secretagogues, carbachol was the most potent inducer of ERK2 activity. Gastrin and EGF had weaker stimulatory effects, whereas no induction was noted in response to histamine. The effect of carbachol appeared to be independent of Ca2+ signaling. PD-98059, a selective inhibitor of the upstream ERK activator mitogen-activated protein kinase/ERK kinase, dose-dependently inhibited both carbachol- and EGF-stimulated ERK2 activity, with a maximal effect observed between 50 and 100 μM. ERKs activation is required for induction of the early gene c- fos via phosphorylation of the transcription factor Elk-1 which binds to the c- fos serum response element (SRE). Carbachol stimulated a two- to threefold induction of luciferase activity in cultured parietal cells transfected with either a SRE-luciferase reporter plasmid or with a chimeric GAL4-ElkC expression vector and the 5×GAL-luciferase reporter plasmid. To examine the significance of ERK activation in gastric acid secretion, we tested the effect of PD-98059 on carbachol-stimulated uptake of14C-labeled aminopyrine (AP). Acute inhibition of the ERKs by PD-98059 led to a small increase in AP uptake and a complete reversal of the acute inhibitory effect of EGF on AP uptake induced by either carbachol or histamine. In contrast, exposure of the cells to PD-98059 for 16 h led to a reversal of the chronic stimulatory effect of EGF on AP uptake induced by carbachol. Our data led us to conclude that carbachol induces a cascade of events in parietal cells that results in ERK activation. Although the acute effect of the ERKs on gastric acid secretion appears to be inhibitory, the activation of transcription factors and of early gene expression could be responsible for its chronic stimulatory effects.