Molecular mechanisms for the growth factor action of gastrin (original) (raw)
Related papers
Gut, 1987
Trophic changes of the exocrine pancreas after in vivo gastrin (G)/CCK treatment are well documented but up to now the study of the mechanisms involved is restricted by the lack of a suitable in vitro model. Nevertheless the in vivo trophic effect induced by gastrin/CCK peptides has been associated with an increase of ornithine decarboxylase (ODC) activity. In the present work, using the AR42J cell line in which CCK receptors and stimulation of amylase release by CCK peptides has already been demonstrated, we investigated the presence of gastrin binding sites and the possible modulation of proliferation by an inhibitor of ODC activity. '25I-BH-Gl7ns binding is saturable, reversible and specific. Potencies of the different analogues tested are G17ns > CCK8 > CCK8ns > G6s > G/CCK4. Furthermore dBt cGMP, a non-peptide antagonist for CCK recep-
Cell type-specific requirement of the MAPK pathway for the growth factor action of gastrin
American Journal of Physiology-gastrointestinal and Liver Physiology, 1999
has a CCK B 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 GH 3. Both AR42J and GH 3 cells displayed equal levels of CCK B receptor expression and similar binding kinetics of 125 I-labeled G17. G17 stimulation of cell proliferation was identical in both cell lines. G17 stimulation of GH 3 cell proliferation was completely blocked by the CCK B 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 GH 3 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 GH 3 cells, demonstrating the integrity of this signaling system. G17 induced Ca 2ϩ mobilization in both the GH 3 and AR42J cells. The calmodulin inhibitor N-(6aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited AR42J cell proliferation by 20%, whereas it completely blocked G17 induction of GH 3 cell growth. The Ca 2ϩ ionophore ionomycin stimulated GH 3 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 GH 3 cells, G17 fails to activate MAPK, and it induces cell proliferation through Ca 2ϩ-dependent signaling pathways. Furthermore, induction of Ca 2ϩ mobilization in the AR42J cells appears not to be sufficient to sustain cell proliferation. cellular proliferation; early response genes; protein kinases; transcriptional regulation; c-fos; mitogen-activated protein kinases; extracellular signal-regulated protein kinases THE PEPTIDE HORMONE GASTRIN is a potent growth factor for both normal and malignant gastrointestinal tissues (23-26, 28, 29, 32, 35). Gastrin is known to interact 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.
Characterization of gastrin-cholecystokinin 2 receptor interaction in relation to c-fos induction
Endocrine Related Cancer, 2008
The interaction of gastrin with the cholecystokinin 2 (CCK2)/gastrin receptor has been studied extensively in relation to gastric acid secretion. However, not much is known about the contribution of individual amino acids of gastrin interacting with the CCK2 receptor, when gastrin is acting as a tumor growth factor. The purpose of the present study was to determine the significance of each individual amino acid residue of human gastrin-17 with respect to CCK2 receptor-mediated cell proliferation. Activation of this receptor was assessed using an in vitro bioassay based on gastrininduced expression of a c-fos-luciferase reporter, transfected in AR42JB13 and Colo 320 cells, a rat pancreatic and human colorectal cell line respectively. Gastrin-17 dose dependently increased c-fos induction in both cancer cell lines. L365,260, a known CCK2 receptor antagonist, completely blocked the gastrin signal, demonstrating the specificity of this assay. We demonstrated for the first time that four carboxy-terminal amino acids of gastrin-17 are essential for activation of the CCK2 receptor with respect to c-fos induction. Also other residues of gastrin-17, notably glycine-2 for the rat CCK2 receptor and glutamic acid 8-10 and tyrosine-12 for the human receptor, were found to be important, although to a lesser extent. Alanine-substitution variants of each of the four carboxy-terminal amino acids of gastrin-17 showed strongly reduced receptor activation but did not act as competitive inhibitors of gastrin-17. Identification of the essential role of the carboxyterminal tetrapeptide of gastrin-17 in CCK2 receptor-mediated c-fos induction indicates that gastrin inhibitory therapeutic strategies should mainly be targeted toward this region of gastrin.
Gastrin induces c-fos gene transcription via multiple signaling pathways
Gastroenterology, 1998
observed that the trophic actions of gastrin (G17) on the AR42J rat acinar cell line are mediated by mitogen-activated protein kinase (MAPK)-induced c-fos gene transcription via protein kinase C (PKC)-dependent and-independent pathways. In this study, we further investigated the signaling pathways that target c-fos in response to G17. G17 led to a sixfold induction in luciferase activity in cells transfected with plasmids containing the Ϫ356ϩ109 sequence of the murine c-fos promoter, which includes the Sis-inducible element (SIE), serum response element (SRE), and the Ca 2ϩ / cAMP response element (CRE) regulatory elements. Addition of either the selective PKC inhibitor GF-109203X or the MAPK/extracellular signal-regulated kinase inhibitor PD-98059 resulted in an 80% reduction in luciferase activity. G17 induced the transcriptional activity of both Elk-1 and Sap-1a, transcription factors that bind to the E26 transformation specific (Ets) DNA sequence of the SRE, and this effect was inhibited by both GF-109203X and PD-98059. Point mutations in the Ets sequence led to a 4-fold induction of c-fos transcription stimulated by G17 and to a 1.3-fold induction in response to epidermal growth factor (EGF). In contrast, mutations in the CA rich G (CArG) sequence of the SRE prevented transcriptional activation by both G17 and EGF. G17 induction of the Ets mutant construct was unaffected by either GF-109203X or PD-98059. Because activation of the SRE involves the small GTP-binding protein Rho A, we examined the role of Rho A in G17 induction of c-fos transcription. Inactivation of Rho A by either the specific inhibitor C3 or by expression of a dominant negative Rho A gene inhibited G17 induction of both the wild-type and the Ets mutant constructs by 60%. C3 also inhibited G17-stimulated AR42J cell proliferation. Thus G17 targets the c-fos promoter CArG sequence via Rho A-dependent pathways, and Rho A appears to play an important role in the regulation of the trophic action of G17. cellular proliferation; early response genes; protein kinases; transcriptional regulation; mitogen-activated protein kinase; extracellular signal-regulated kinases ALTHOUGH CHARACTERIZED as a stimulant of gastric acid secretion (14), the peptide hormone gastrin also exerts growth-promoting effects on normal and malignant gastrointestinal tissues (37, 43, 45, 51). Gastrin is an important growth factor for the fetal pancreas
Hepatology, 2000
We studied the role of gastrin in regulating cholangiocyte proliferation induced by bile duct ligation (BDL). In purified cholangiocytes, we evaluated (1) for the presence of cholecystokinin-B (CCK-B)/gastrin receptors, (2) the effect of gastrin on D-myo-Inositol 1,4,5-triphosphate (IP 3 ) levels, and (3) the effect of gastrin on DNA synthesis and adenosine 3Ј, 5Ј-monophosphate (cAMP) levels in the absence or presence of CCK-A (L-364,718) and CCK-B/gastrin (L-365,260) receptor inhibitors, 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid tetrakis(acetxymethyl ester) (BAPTA/AM; an intracellular Ca 2؉ chelator), and 2 protein kinase C (PKC) inhibitors, 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7) and staurosporin. To evaluate if gastrin effects on cholangiocyte proliferation are mediated by the isoform PKC␣, we evaluated (1) for the presence of PKC␣ in cholangiocytes and (2) the effect of gastrin on the PKC␣ protein expression in a triton-soluble (containing cytoplasm ؉ membrane) and a triton-insoluble (containing cytoskeleton) fraction. To evaluate the effects of gastrin in vivo, immediately following BDL, gastrin or bovine serum albumin (BSA) was infused by minipumps for 7 days to rats and we measured cholangiocyte growth and cAMP levels. We found CCK-B/gastrin receptors on cholangiocytes. Gastrin increased IP 3 levels. Gastrin inhibited DNA synthesis and cAMP synthesis in cholangiocytes. Gastrin effects on cholangiocyte functions were blocked by L-365,260, BAPTA/AM, H7, and staurosporin but not by L-364,718. Gastrin induced translocation of PKC␣ from cholangiocyte cytoskeleton to membrane. In vivo, gastrin decreased cholangiocyte growth and cAMP synthesis compared with controls. We concluded that gastrin inhibits cholangiocyte growth in BDL rats by interacting with CCK-B/gastrin receptors through a signal transduction pathway involving IP 3 , Ca 2؉ , and PKC␣. (HEPATOLOGY
Pharmacology & Toxicology, 2001
Gastrin has a growth-promoting effect on the oxyntic mucosa of the stomach but has been claimed also to affect other parts of the gastrointestinal tract and pancreas. This report describes the effects of the cholecystokinin 2 (CCK 2 ) receptor antagonists YM022 and YF476 on various growth parameters in the gastrointestinal tract and pancreas of the rat. YM022 and YF476 were given subcutaneously in doses known to produce maximum and sustained CCK 2 receptor blockade. The body weight was not affected. However, the oxyntic mucosal weight, thickness and protein and DNA contents were reduced by 15-20% already within 1-2 days and by about 30% after 4-8 weeks of CCK 2 receptor blockade. Hence, the response of the oxyntic mucosa to CCK 2 receptor blockade was in the form of hypotrophy (reduced protein content) and hypoplasia (reduced DNA content). There were no obvious effects of CCK 2 receptor blockade on the intestine or pancreas (nor on liver, kidney or thyroid). The proton pump inhibitor omeprazole was used to induce hypergastrinaemia and was given with or without YM022. Omeprazole treatment for 4 weeks increased the oxyntic mucosal weight and thickness by 15-20%. YM022 prevented these effects. We conclude that while elevated circulating gastrin levels, acting on CCK 2 receptors, exert a growth-promoting effect on the oxyntic mucosa (but not elsewhere), normal serum gastrin levels exert a mucosa-preserving effect.
Gut, 2002
Background: Activation of the gastrin-cholecystokinin B (CCK B ) receptor stimulates cell proliferation and increases production of ligands for the epidermal growth factor receptor (EGF-R). Aims: To determine the role of gastrin-CCK B activation in stimulation of cell proliferation via paracrine activation of EGF-R. Methods: AGS cells were transfected with the gastrin-CCK B receptor (AGS-G R cells) or with green fluorescent protein (AGS-GFP cells). Proliferation was determined by [ 3 H] thymidine incorporation, flow cytometry, and cell counting. Results: Gastrin inhibited proliferation of AGS-G R cells by delaying entry into S phase. However, when AGS-G R cells were cocultured with AGS-GFP cells, gastrin stimulated proliferation of the latter. Immunoneutralisation and pharmacological studies using metalloproteinase and kinase inhibitors indicated that the proliferative response was mediated by paracrine stimulation of EGF-R and activation of the mitogen activated protein kinase pathway through release of heparin binding EGF. Conclusions: Gastrin can directly inhibit, and indirectly stimulate, proliferation of gastric AGS cells.
AJP: Gastrointestinal and Liver Physiology, 2006
Inducible cAMP early repressor suppresses gastrin-mediated activation of cyclin D1 and c-fos gene expression. The gastric hormone gastrin and its precursors promote proliferation in several gastrointestinal cell types. Here we show that gastrin induces transcription of cell cycle gene cyclin D1 and protooncogene c-fos in the neuroendocrine pancreatic cell line AR42J and that this gastrin response is inhibited by endogenous inducible cAMP early repressor (ICER). The transcriptional repressor ICER is known to downregulate both its own expression and the expression of other genes containing cAMPresponsive elements (CREs). Using siRNA, we also show that CRE promoter elements are the targets of endogenous ICER in AR42J cells as well as in the neuroendocrine cell line RIN5F. Our results suggest that ICER plays an important role in molecular mechanisms governing gastrin-mediated growth by modulating gastrin's transcriptional activation of growth-related genes. Our finding that ICER modulates pituitary adenylate cyclase-activating polypeptide-activated gene expression also indicates a regulatory effect of ICER in the responses of neuroendocrine cells to peptides other than gastrin. neuroendocrine gene regulation; gastrointestinal proliferation; gastrin THE PEPTIDE HORMONE GASTRIN is well characterized as a stimulant of gastric acid secretion by stimulation of enterochromaffin-like (ECL) cells to produce histamine, which in turn stimulates the parietal cells in the oxyntic mucosa to release HCl (36, 48). Gastrin is an important growth factor for the fetal pancreas (50) and a potent stimulant for the growth of gastric mucosa (4, 5, 46). Transgenic mice overexpressing gastrin exhibit increased proliferation of the oxyntic mucosa (51), whereas gastrin-deficient mice develop abnormal gastrointestinal (GI) mucosa with immature cells (7, 12). In addition, there is abundant evidence to suggest that gastrin may play an important role in tumor biology, as gastrin is shown to regulate tumor cell growth (4, 5) and stimulate tumor cell invasion (4, 5, 13). Hypergastrinemia is associated with the occurrence of gastric ECL cell carcinoid tumors (4, 5, 28, 46) and with an increased risk of gastric and colorectal carcinoma (5), indicating a role in carcinogenesis. The biological actions of gastrin are exerted through binding to the gastrin/cholecystokinin (CCK2) receptor. This receptor has been shown to be coexpressed with gastrin in several GI tumor cell lines (53) and in human gastric carcinoids (38), indicating the existence of an autocrine growth stimulatory loop. The malignant potential of carcinoids is associated with hypergastrinemia (24). These studies suggest that gastrin plays an important role in neuroendocrine tumor biology.
Biochemical and Biophysical Research Communications, 1997
cause of the non selectivity of the compounds used in Glycine-extended gastrin precursors (G-Gly) were these studies, the notion of autocrine loop for gastrin considered as processing intermediates devoid of bioremains controversial. logical activity. However, we have recently identified Like many other regulatory peptides, gastrin is charselective receptors for G-Gly which mediate the prolifacterized by the presence of a carboxyl-terminal amide erative effects of this precursor. Little is known about group. Post-translational processing of progastrin, that the signaling pathways activated by G-Gly. In the presleads to amidated gastrin, involves the formation of ent study, we demonstrate that PI-3-kinase is rapidly glycine-extended processing intermediates (G-Gly) and transiently activated by G-Gly. We also observed which serve as substrates for alpha amidation. Until a rapid increase in the tyrosine phosphorylation of recently, the alpha-amide group was presumed to be IRS-1 and an activation of the PI-3-kinase in anti-IRSessential for full biological activity. 1 immunoprecipitates, suggesting that PI-3-kinase In peptide-producing tumors, post-translational promay be activated by association with tyrosine phosphorylated IRS-1. We also demonstrated that gastrin cessing is often incomplete. Thus non-amidated progasprecursors activate the serine/threonine kinase, p70 trin-derived peptides have been shown to be present in kDa S6 kinase (p70 S6K), through a wortmannin sensilung, pancreas and colon tumors at concentrations tive pathway. ᭧ 1997 Academic Press higher than in normal tissues (10-12). In addition, levels of processing intermediates of gastrin are elevated in the circulation of patients with colorectal carcinomas (13). We have recently reported that glycine-extended pro-Gastrin, a peptide hormone produced from G cells in gastrin processing intermediates exert trophic effects the gastric antrum was first characterized as a regulathrough selective membrane receptors distinct from tor of gastric acid secretion (1). Gastrin also functions the G protein-coupled gastrin receptor (G/CCK B) that as a growth-promoting factor for normal gut mucosal mediates the effects of amidated gastrin (14). Similar cells (2). This peptide has also been reported to have results were obtained by another laboratory on colon trophic effects on a number of gastrointestinal cancers. cancer cells, normal fetal intestinal cells as well as fi-Many studies have demonstrated the stimulatory efbroblasts (15, 16). fects of exogenous gastrin on the growth in vitro of cell Little is known about the mitogenic signal transduclines established from pancreatic, gastric and colonic tion pathways activated by the binding of G-Gly to its carcinomas (3, 4). In addition, gastrin enhances the specific membrane receptors. Recent studies indicate growth in vivo of tumor cells transplanted in animals that activation of c-jun amino-terminal kinase (JNK) (5, 6). A potential autocrine effect of gastrin on colon may contribute to the intracellular events that are recancers has been proposed by different laboratories sponsible for the growth promoting effects of G-Gly based on the inhibitory effects of gastrin receptor an-(17). This enzyme has been shown to increase the phostagonists and gastrin antibodies (7-9). However, bephorylation and the transcriptional activity of the cjun proteins which control the transcription of early 1 Address correspondence to Catherine Seva, INSERM U.151, response genes including c-jun itself. Groupe de Recherche de Biologie et Pathologie digestive Institut PI-3-kinase is a lipid kinase that phosphorylates Louis Bugnard,
British Journal of Pharmacology, 1997
1In the pig, the secretory response of the pancreas is not inhibited by the antagonist MK329 suggesting that cholecystokininA (CCKA) receptors are not involved.2Membranes were isolated from the pancreas of 6 Large White pigs to characterize their CCK receptors.3The binding of [M125I]-BH-[Thr, Nle]CCK-9 was dependent on pH, maximal after a 90 min incubation period, saturable and reversible. Saturation analysis of the binding demonstrated a single class of high affinity sites (Kd = 0.22 ± 0.02 nM) and a binding capacity, Bmax= 110.64±12.50 fmol mg−1 protein.4Competition binding by agonists and antagonists of CCKA and CCKB/gastrin receptors demonstrated the presence of two distinct binding components, sites presenting a high affinity for [Thr, Nle]CCK-9, gastrin, PD 135158, L-365,260 and a low affinity for MK329, SR 27897, and sites presenting a high affinity for [Thr, Nle]CCK-9, MK329, SR 27897 and a low affinity for gastrin, PD 135158, L-365,260.5These pharmacological data demonstrate the presence of both CCKA and CCKB/gastrin receptors in the pig pancreas, the latter being predominant.6Two distinct membrane proteins (50 and 85–100 kDa, respectively) display pharmacological features of CCKB/gastrin and CCKA receptors.7In pigs, as in calves and humans, CCKB/gastrin receptors are predominant in the pancreas.In the pig, the secretory response of the pancreas is not inhibited by the antagonist MK329 suggesting that cholecystokininA (CCKA) receptors are not involved.Membranes were isolated from the pancreas of 6 Large White pigs to characterize their CCK receptors.The binding of [M125I]-BH-[Thr, Nle]CCK-9 was dependent on pH, maximal after a 90 min incubation period, saturable and reversible. Saturation analysis of the binding demonstrated a single class of high affinity sites (Kd = 0.22 ± 0.02 nM) and a binding capacity, Bmax= 110.64±12.50 fmol mg−1 protein.Competition binding by agonists and antagonists of CCKA and CCKB/gastrin receptors demonstrated the presence of two distinct binding components, sites presenting a high affinity for [Thr, Nle]CCK-9, gastrin, PD 135158, L-365,260 and a low affinity for MK329, SR 27897, and sites presenting a high affinity for [Thr, Nle]CCK-9, MK329, SR 27897 and a low affinity for gastrin, PD 135158, L-365,260.These pharmacological data demonstrate the presence of both CCKA and CCKB/gastrin receptors in the pig pancreas, the latter being predominant.Two distinct membrane proteins (50 and 85–100 kDa, respectively) display pharmacological features of CCKB/gastrin and CCKA receptors.In pigs, as in calves and humans, CCKB/gastrin receptors are predominant in the pancreas.