RESUMEN
Angiogenesis is essential in tumor progression and metastatic process, and increased angiogenesis has been associated with poor prognosis and relapse of colorectal cancer (CRC). VEGF has become the main target of anti-angiogenic therapy. However, most patients relapse after an initial response or present a resistance to the treatment. Identification of new pro-angiogenic factors may help to improve anti-angiogenic therapy. In this study, we demonstrated that the pro-hormone progastrin (PG), over-expressed in CRC, recognized as a growth factor, is a potent pro-angiogenic factor. In transgenic mice and human colorectal HPs producing high levels of PG, we correlated PG overexpression with an increased vascularization. In vitro, exogenous PG and conditioned media (CM) from CRC cells producing PG increased endothelial cell proliferation and migration. We also showed that treatment with exogenous PG can increase the ability of endothelial cells to form capillary-like structures. Moreover, we demonstrated that PG enhanced endothelial permeability. The finding that PG stimulated the phosphorylation of vascular endothelial (VE)-cadherin, p125-FAK, paxillin and induced actin remodelling was consistent with a role of these components in PG-stimulated endothelial cell migration and permeability. The pro-angiogenic effects observed with CM were significantly inhibited when CRC cells expressed a PG shRNA. In vivo, we found an important decrease in tumor growth and neovascularization when the CRC cells expressing the PG shRNA were xenografted in mice or in the chick chorioallantoic membrane model. We also observed an increase in the coverage of blood vessels by pericytes and a decrease in endothelial permeability when PG expression was blocked. Our results demonstrate that PG is a new pro-angiogenic factor in CRC and an attractive therapeutic target.
Asunto(s)
Neoplasias Colorrectales/irrigación sanguínea , Gastrinas/fisiología , Neovascularización Patológica/genética , Precursores de Proteínas/fisiología , Animales , Células Cultivadas , Embrión de Pollo , Neoplasias Colorrectales/patología , Gastrinas/genética , Gastrinas/farmacología , Células HCT116 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones SCID , Ratones Transgénicos , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , ARN Interferente Pequeño/farmacologíaRESUMEN
We previously demonstrated that expression of the gastrin receptor, CCK2R, in pancreatic acini of transgenic ElasCCK2 mice induced alteration of acinar morphology and differentiation, increased sensitivity to a carcinogen and development of preneoplastic lesions and tumours. Reg proteins are suggested to be involved in pancreatic cancer and in regeneration of endocrine pancreas. Reg I gene is a known target of gastrin. We examined whether an expression of CCK2R in the pancreatic acini of ElasCCK2 mice is linked to induction of Reg proteins expression. We analyzed Reg expression by Western-blot and immunohistochemistry in pancreas from ElasCCK2 and control mice. Islet neogenesis, glucose homeostasis, insulin secretion and content were also evaluated. Reg I is exclusively produced in acini in ElasCCK2 and control mice. In tumoral pancreas, Reg I and Reg III proteins are expressed in duct-like cells in preneoplastic lesions or in the periphery of tumours and in adjacent acini. The expression of Reg III proteins is increased in ElasCCK2 pancreas before the development of preneoplastic lesions in a subpopulation of islet cells and in small islet-like cell clusters dispersed within the acinar tissue. Several criteria of an enhanced neogenesis are fulfilled in ElasCCK2 pancreas. Moreover, ElasCCK2 mice have an improved response to glucose load, an increased insulin secretion and a doubling of insulin content compared to control mice. We show that Reg proteins are targets of CCK2R activation and are induced during early steps of carcinogenesis in ElasCCK2 mice pancreas. Alterations of exocrine tissue homeostasis in ElasCCK2 pancreas concomitantly activate regenerative responses of the endocrine pancreas possibly linked to paracrine actions of Reg III proteins.
Asunto(s)
Páncreas/metabolismo , Proteínas/genética , Receptor de Colecistoquinina B/metabolismo , Animales , Antígenos de Neoplasias , Biomarcadores de Tumor , Regulación de la Expresión Génica , Prueba de Tolerancia a la Glucosa , Inmunohistoquímica , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Lectinas Tipo C , Ratones , Ratones Transgénicos , Tamaño de los Órganos , Proteínas Asociadas a Pancreatitis , Análisis por Matrices de Proteínas , Proteínas/metabolismo , Receptor de Colecistoquinina B/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Several lines of evidence suggest that gastrin and the CCK-2 receptor (CCK2R) could contribute to pancreatic carcinogenesis by modulating processes such as proliferation, cell adhesion or migration. In the current study, we used a 'cancer gene array' and identified beta1-integrin subunit as a new gastrin-regulated gene in human pancreatic cancer cells. We also demonstrated that Src family kinases and the phosphatidylinositol-3-kinase (PI-3-kinase) pathway play a crucial role in the expression of beta1-integrin induced by gastrin. Our results also showed that gastrin modulates cell-substrate adhesion via beta1-integrin. Indeed, using blocking anti-beta1-integrin monoclonal antibodies, we completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we observed that in response to gastrin, beta1-integrin is tyrosine phosphorylated by Src family kinases and associates with paxillin, a scaffold protein involved in focal adhesion and integrin signalling. This mechanism might be involved in gastrin-induced cell adhesion. Moreover, we showed in vivo that targeted CCK2R expression in the pancreas of Elas-CCK2 mice leads to the overexpression of beta1-integrin. This process may contribute to pancreatic tumour development observed in these transgenic animals.
Asunto(s)
Integrina beta1/fisiología , Neoplasias Pancreáticas/metabolismo , Receptor de Colecistoquinina B/fisiología , Animales , Adhesión Celular/genética , Adhesión Celular/fisiología , Línea Celular Tumoral , Gastrinas/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias Pancreáticas/genética , Receptor de Colecistoquinina B/genéticaRESUMEN
Long-term amino acid starvation represents a form of metabolic stress which stimulates gene expression. Here we report that depriving HeLa cells for any one of a series of amino acids activates c-Jun N-terminal kinase-1 (JNK-1). In contrast, the other mitogen-activated protein kinases (MAPKs) ERK-1 and, to a lesser extent, p38 activities decreased under such conditions. In methionine- or leucine-deprived cells, JNK-1 activation occurred after 4 or 6 h, respectively, and reached a steady maximum of 5- to 7-fold over control cells afterwards. This activation was dependent on the amino acid concentration and it could be reversed by resupplying the complete medium. Limitation for all amino acids also augmented JNK-1 activity, whereas increased amino acid concentrations had an opposite effect. The free radical scavenging thiol antioxidant N-acetylcysteine (NAC) alleviated partially JNK-1 activation in amino acid-deprived cells. The data indicate that activation of JNK-1 by long-term amino acid deprivation may be mediated in part by oxidative stress.
Asunto(s)
Aminoácidos/deficiencia , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Acetilcisteína/farmacología , Activación Enzimática , Depuradores de Radicales Libres/farmacología , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Estrés Oxidativo , Pruebas de Precipitina , Especificidad por Sustrato , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
We present evidence that gastrin, binding to a G protein-coupled receptor, activates the p38-mitogen-activated protein kinase (MAPK) pathway. Blockage of protein kinase C (PKC) by GF109203X, depletion of intracellular calcium by thapsigargin or inhibition of Src family kinases by PP2 prevented p38-MAPK activation and the Src kinase activity stimulated by gastrin. Inhibition of the PI 3-kinase by wortmannin or LY294002 did not affect these responses. In addition, the p38-MAPK inhibitor, SB203580, repressed gastrin-induced [(3)H]thymidine incorporation, indicating a major role of p38-MAPK in the growth-promoting effect of gastrin. Our results demonstrate that gastrin-induced DNA synthesis requires p38-MAPK activation through mechanisms that involve calcium mobilization, PKC and Src family kinases.
Asunto(s)
ADN/biosíntesis , Gastrinas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Quinasa C/metabolismo , Familia-src Quinasas/metabolismo , Androstadienos/farmacología , Animales , Células CHO , Calcio/metabolismo , Cromonas/farmacología , Cricetinae , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Gastrinas/farmacología , Humanos , Líquido Intracelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/metabolismo , Transducción de Señal/efectos de los fármacos , Tapsigargina , Transfección , Wortmanina , Proteínas Quinasas p38 Activadas por Mitógenos , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Among the most conserved regions in the G-protein-coupled receptors is the (N/D)PX(2-3)Y motif of the seventh transmembrane domain (X represents any amino acid). The mutation of the Asn/Asp residue of this motif in different G-protein-coupled receptors was shown to affect the activation of either adenylyl cyclase or phospholipase C. We have mutated the Asn residue (Asn-391) of the NPXXY motif in the CCKBR to Ala and determined the effects of the mutation on binding, signaling, and G-proteins coupling after expression of the mutated receptor in COS cells. The mutated receptor displayed similar expression levels and high affinity CCK binding compared with the wild type CCKBR. However, unlike the wild type CCKBR, the mutated receptor was completely unable to mediate activation of either phospholipase C and protein kinase C-dependent and -independent mitogen-activated protein kinase pathways, indicating an essential role of Asn-391 in CCKBR signaling. Coimmunoprecipitation experiments allowed us to show that the inactive mutant retains an intact capacity to form stable complexes with G(q)alpha subunits in response to CCK. These results indicate that the formation of high affinity CCK-receptor-G(q) protein complexes is not sufficient to activate G(q) and suggest that Asn-391 is specifically involved in G(q) proteins activation.
Asunto(s)
Asparagina , Proteínas de Unión al GTP/metabolismo , Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ácido Aspártico , Sitios de Unión , Células COS , Colecistoquinina/farmacología , Secuencia Conservada , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Fosfatos de Inositol/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/farmacología , Proteína Quinasa C/metabolismo , Ratas , Receptor de Colecistoquinina B , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc.Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src. IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de Colecistoquinina/metabolismo , Familia-src Quinasas/metabolismo , Animales , Células CHO , Cricetinae , Replicación del ADN , Activación Enzimática , Proteína Adaptadora GRB2 , Gastrinas/metabolismo , Humanos , Proteínas Sustrato del Receptor de Insulina , Proteína Quinasa 3 Activada por Mitógenos , Fosfoproteínas/metabolismo , Fosforilación , Proteínas/metabolismo , Receptor de Colecistoquinina B , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Tirosina/metabolismoRESUMEN
The molecular events whereby gastrin occupancy of G/CCK(B) receptors leads to phosphatidylinositol (PI) 3-kinase activation have been examined. We report here that this peptide promotes the association between two non-receptor tyrosine kinases, p60Src and p125FAK, and elicits a parallel increase in tyrosine phosphorylation and activity of both kinases. Gastrin-induced PI 3-kinase activity was coprecipitated with p60Src and p125FAK and was inhibited by herbimycin A, the selective Src inhibitor PP-2 or cytochalasin D, which disrupts the actin cytoskeleton and prevents p125FAK activity. These results indicate, for the first time, that a p60Src/p125FAK complex acts upstream of the gastrin-stimulated PI 3-kinase pathway.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Gastrinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Gastrinas/farmacología , Fosforilación , Pruebas de Precipitina , Ratas , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Gastrin via its G-protein coupled specific receptor induces transcription of c-fos and c-jun genes through a ras-MAPK pathway. Ornithine Decarboxylase (ODC), a growth regulated proto-oncogene, was chosen to investigate gastrin effects on translation initiation of mRNAs exhibiting a 5'UnTranslated Region (5'UTR) responsible for translation repression in quiescent cells. In AR4-2J tumoral cells, we first demonstrated that gastrin increases ODC mRNA translation. Transient transfections with various CAT chimeric constructs suggested a direct involvement of the 5'UTR in this observation. Translation of this group of mRNAs is enhanced by the availability of the cap-binding protein (eIF4E) that is increased after phosphorylation of its specific binding protein eIF4E-BP1. We found that AR4-2J cells over-expressed eIF4E protein which was not modulated by gastrin treatment. Rapamycin which inhibits 4E-BP1 phosphorylation, completely prevents gastrin-mediated increase of ODC translation indicating that 4E-BP1 could be involved in regulating ODC translation. Implication of 4E-BP1 in mediating gastrin effects is corroborated by the capacity of the ligand to affect 4E-BP1 phosphorylation. These results indicate that gastrin enhances ornithine decarboxylase mRNA translation through a rapamycin sensitive pathway and provide the first evidence in the control of 4E-BP1 phosphorylation after occupancy of a G protein-coupled receptor.
Asunto(s)
Proteínas Portadoras , Gastrinas/farmacología , Ornitina Descarboxilasa/genética , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , ARN Mensajero/genética , Animales , Células COS , Inducción Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Ornitina Descarboxilasa/biosíntesis , Inhibidores de la Ornitina Descarboxilasa , Fosforilación/efectos de los fármacos , Polienos/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Ratas , Proteínas Represoras/farmacología , Sirolimus , Células Tumorales CultivadasRESUMEN
The gastrin/CCKB (G/CCKB) G protein-coupled receptor has been shown to mediate the proliferative effects of gastrin on normal and neoplastic gastro-intestinal tissues. In the present study, we examined the signal transduction mechanisms coupled to this receptor. We report here that phosphorylation and activity of the p70S6K, whose major substrate is the ribosomal S6 protein, are enhanced in response to gastrin. These effects were completely reversed by a commonly used PI-3-kinase inhibitor, wortmannin, suggesting that p70S6K may be a downstream target of PI-3-kinase in a signaling cascade induced by gastrin. In addition, blocking PI-3-kinase activity by wortmannin partially decreased gastrin-induced MAPK activation (42% +/- 3) as well as the tyrosine phosphorylation of She (50% +/- 6), an upstream regulator of the Ras-dependent MAPK pathway. These results indicate that at least two signaling pathways lead to MAPK activation by gastrin, only one of which is sensitive to PI-3-kinase inhibitors.
Asunto(s)
Androstadienos/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Unión al GTP/metabolismo , Gastrinas/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Colecistoquinina/fisiología , Proteínas Ribosómicas/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Gastrinas/metabolismo , Neoplasias Pancreáticas , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Polienos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Receptor de Colecistoquinina B , Proteínas Quinasas S6 Ribosómicas , Sirolimus , Células Tumorales Cultivadas , Wortmanina , Dominios Homologos src/efectos de los fármacosRESUMEN
The proliferative effects of gastrin on normal and neoplastic gastro-intestinal tissues have been shown to be mediated by the gastrin/CCKB (G/CCKB) G-protein-coupled receptors. We have recently reported that gastrin stimulates the tyrosine phosphorylation of Shc proteins and their subsequent association with the Grb2/Sos complex, leading to mitogen-activated protein kinase (MAPK) activation, a pathway known to play an important role in cell proliferation. We undertook the present study to characterize the signalling pathways used by this receptor to mediate the activation of the Shc/Grb2 complex. Since G/CCKB receptor occupancy leads to the activation of the phospholipase C (PLC)/protein kinase C (PKC) pathway, we examined whether PKC stimulation and Ca2+ mobilization contribute to the phosphorylation of Shc proteins and their association with Grb2 in response to gastrin. Our results indicate that Shc proteins are tyrosine phosphorylated and associate with Grb2 in response to phorbol esters, suggesting that activation of PKC is a potential signalling pathway leading to activation of the Shc/Grb2 complex. Inhibition of PKC by GF109203X completely blocked the effect of PMA on Shc tyrosine phosphorylation and its subsequent association with Grb2, but had a partial inhibitory effect on the response to gastrin. Depletion of the intracellular Ca2+ pools by treatment with thapsigargin blocked the increase in intracellular free calcium concentration induced by gastrin and diminished the ability of the peptide to stimulate Shc phosphorylation and recruitment of Grb2. In addition, removal of extracellular Ca2+ partially inhibited the effect of gastrin on Shc phosphorylation as well as its association with Grb2, indicating that the effects of gastrin are also mediated by Ca2+-dependent mechanisms. Furthermore, we show that blockage of the two major early signals generated by activation of PLC, which induced the activation of the Shc/Grb2 complex, also blocked gastrin-induced MAPK activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Gastrinas/farmacología , Proteínas Quinasas Activadas por Mitógenos , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Animales , Western Blotting , Activación Enzimática , Proteína Adaptadora GRB2 , Humanos , Proteína Quinasa 3 Activada por Mitógenos , Neoplasias Experimentales , Fosforilación , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal/fisiología , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/farmacología , Células Tumorales Cultivadas , Tirosina/metabolismoRESUMEN
Glycine-extended gastrin precursors (G-Gly) were considered as processing intermediates devoid of biological activity. However, we have recently identified selective receptors for G-Gly which mediate the proliferative effects of this precursor. Little is known about the signaling pathways activated by G-Gly. In the present study, we demonstrate that PI-3-kinase is rapidly and transiently activated by G-Gly. We also observed a rapid increase in the tyrosine phosphorylation of IRS-1 and an activation of the PI-3-kinase in anti-IRS-1 immunoprecipitates, suggesting that PI-3-kinase may be activated by association with tyrosine phosphorylated IRS-1. We also demonstrated that gastrin precursors activate the serine/threonine kinase, p70 kDa S6 kinase (p70S6K), through a wortmannin sensitive pathway.
Asunto(s)
Gastrinas/farmacología , Glicina/farmacología , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotirosina/metabolismo , Precursores de Proteínas/farmacología , Androstadienos/farmacología , Animales , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Proteínas Sustrato del Receptor de Insulina , Cinética , Neoplasias Pancreáticas , Fosfatidilinositol 3-Quinasas , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Células Tumorales Cultivadas , WortmaninaRESUMEN
The growth-promoting effects of gastrin on normal and neoplastic gastrointestinal tissues have been shown to be mediated by the gastrin/CCKB receptor, which belongs to the family of G protein-coupled receptors. However, the downstream signaling pathways activated by gastrin are not well characterized. In the present study, we demonstrate that gastrin stimulates tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1), the major cytoplasmic substrate of the insulin receptor. The gastrin-induced phosphorylation of IRS-1 was rapid and transient, occurring within 30 s of treatment and diminishing thereafter. IRS-1 binds several proteins containing Src homology 2 domains through its multiple tyrosine phosphorylation sites. Following gastrin stimulation, we observed a time- and dose-dependent association of IRS-1 with the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). In addition, activation of PI 3-kinase was detected in anti-IRS-1 immunoprecipitates from gastrin-treated cells, suggesting that tyrosine phosphorylation of IRS-1, which leads to the rapid recruitment of p85, might be one mechanism used by gastrin to activate PI 3-kinase. We have previously reported that tyrosine phosphorylation of Shc and its association with the Grb2-Sos complex may contribute to the activation of the mitogen-activated protein kinase pathway by gastrin. We report here that Grb2 also interacts with tyrosine-phosphorylated IRS-1 in response to gastrin. Taken together, our results suggest that IRS-1 may serve as a converging target in the signaling pathways stimulated by receptors that belong to different families, such as the gastrin/CCKB G protein-coupled receptor and the insulin receptor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Receptores ErbB/metabolismo , Gastrinas/farmacología , Fosfoproteínas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Animales , Activación Enzimática , Proteína Adaptadora GRB2 , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Páncreas/efectos de los fármacos , Páncreas/enzimología , Fosfatidilinositol 3-Quinasas , Fosforilación , Ratas , Células Tumorales CultivadasRESUMEN
Gastrin/CCKB G protein-coupled receptors have been shown to mediate proliferative effects of their endogenous ligands. In the present study, we examined the signal transduction mechanisms linked to the G/CCKB receptor occupancy. We report here that gastrin stimulates MAP kinase activation in a dose- and time-dependent manner, a pathway known to play a key role in cell proliferation. We also characterized the molecular events, upstream of p21-Ras, that may link the MAP kinase pathway to G/CCKB receptors. Gastrin induced a rapid and transient increase in tyrosine phosphorylation of several proteins including the 2 isoforms (46 and 52 kDa) of the adaptor protein Shc. Phosphorylated Shc subsequently associated with a complex that includes Grb2 and the p21-Ras activator, Sos. Our results also indicate that Sos becomes phosphorylated in response to gastrin as shown by a reduction in electrophoretic mobility of the protein. Tyrosine phosphorylation of Shc and subsequent complex formation with Grb2 and Sos appear to be a common mechanism by which tyrosine kinase receptors and the G/CCKB G protein-coupled receptor stimulate the Ras-dependent MAP kinase pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Gastrinas/farmacología , Proteínas de la Membrana/metabolismo , Fosfotirosina/metabolismo , Proteínas/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Activación Enzimática/efectos de los fármacos , Proteína Adaptadora GRB2 , Cinética , Neoplasias Pancreáticas , Fosforilación , Ratas , Receptores de Colecistoquinina/fisiología , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Proteínas Son Of Sevenless , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Células Tumorales CultivadasRESUMEN
We recently reported that gastrin and glycine-extended progastrin processing intermediates (G-Gly) exert growth-promoting effects on AR4-2J cells (derived from rat pancreas) via interaction with distinct receptors. In this study we sought to investigate the mechanisms by which gastrin and G-Gly stimulate cell proliferation. While gastrin increased [Ca2+]i in AR4-2J cells, G-Gly had no effect. Similarly, G-Gly had no effect either on basal and 10(-7) M vasoactive intestinal polypeptide-stimulated cAMP generation, although gastrin is known to inhibit cAMP generation. Gastrin dose dependently stimulated AR4-2J cell mRNA content of both c-fos and c-jun, two genes known to function in regulating cell proliferation, but G-Gly had no effect. Gastrin also induced the expression of luciferase in AR4-2J cells transfected with a construct consisting of a luciferase reporter gene coupled to the serum response element of the c-fos gene promoter. In similar fashion, gastrin stimulated the activity of mitogen-activated protein kinase, an enzyme known to mediate the induction of the c-fos serum response element in response to growth factor stimulation. Although G-Gly had none of these effects of gastrin in AR4-2J cells, it stimulated activity of c-Jun amino-terminal kinase, an enzyme known to phosphorylate and transcriptionally activate c-Jun. These data support the notion that gastrin stimulates cell proliferation by inducing c-fos and c-jun gene expression, while G-Gly acts by post-translationally regulating early gene transcriptional activation. Our studies represent a novel model in which both the precursor and the product of a key processing reaction, peptide alpha-amidation, act cooperatively to stimulate cell proliferation via distinct receptors linked to different signal transduction pathways.
Asunto(s)
Gastrinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Animales , Calcio/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , AMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Genes fos , Genes myc , Luciferasas/biosíntesis , Páncreas , Fragmentos de Péptidos/farmacología , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Transducción de Señal , Activación Transcripcional , TransfecciónRESUMEN
We have reported previously that the widespread inhibitory actions of somatostatin might be mediated by its ability to inhibit the expression of the immediate early genes c-fos and c-jun. The products of these genes form a heterodimeric transcription factor complex [activator protein 1 (AP-1)], which is known to be induced by treatment with phorbol esters. In the present study, we sought to investigate the mechanisms by which somatostatin inhibits immediate early gene expression. For our experiments, we used a rat pituitary adenoma cell line (GH3), which is known to express multiple subclasses of somatostatin receptors. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated both AP-1 binding and transcriptional activity in GH3 cells and the somatostatin analogue octreotide inhibited this response by 40-70%. In the presence of two different phosphatase inhibitors, sodium orthovanadate or okadaic acid, the ability of somatostatin to inhibit AP-1 binding and transcriptional activity was abolished. This effect of octreotide, which appears to be mediated by the SSTR2 and SSTR5 subtypes of somatostatin receptors, was paralleled by its ability to inhibit TPA-stimulated GH3 cell proliferation. Pretreatment of the GH3 cells with pertussis toxin (200 ng/ml) reversed the inhibitory effect of octreotide on both AP-1 function and cellular proliferation. Our observations lead us to conclude that somatostatin not only inhibits immediate early gene expression but also inhibits AP-1 binding and transcriptional activity via the action of several classes of protein phosphatases. This effect, which is pertussis toxin sensitive, might be one mechanism by which somatostatin inhibits cellular proliferation.
Asunto(s)
Fosfoproteínas Fosfatasas/fisiología , Somatostatina/farmacología , Factor de Transcripción AP-1/antagonistas & inhibidores , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Éteres Cíclicos/farmacología , Sondas Moleculares/genética , Datos de Secuencia Molecular , Octreótido/farmacología , Ácido Ocadaico , Toxina del Pertussis , Ratas , Receptores de Somatostatina/fisiología , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Vanadatos/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Biologically active amidated gastrin is synthesized by carboxyl-terminal alpha-amidation of a glycine-extended progastrin post-translational processing intermediate (G-Gly). Although plasma levels of G-Gly are equivalent to those of gastrin, G-Gly has essentially no acute effect on gastric acid secretion. However, we have observed that inhibition of gastrin amidation leads to increased plasma concentrations of G-Gly and enhanced gastric acid secretion. We hypothesized, therefore, that G-Gly might have a chronic effect to increase H+,K(+)-ATPase expression in gastric parietal cells. In the present studies, we observed that a 2-day preincubation with G-Gly significantly enhanced histamine-stimulated [14C]aminopyrine uptake by isolated canine gastric parietal cells but acutely administered G-Gly had no effect. On Northern blot analysis, both G-Gly and gastrin dose-dependently increased H+,K(+)-ATPase alpha-subunit gene expression with maximal induction (225 +/- 35 and 170 +/- 29% of basal, mean +/- S.E.) achieved at concentrations of 10(-9) M G-Gly and 10(-8) M gastrin, respectively. Using an H+,K(+)-ATPase alpha-subunit gene-luciferase chimeric reporter construct transfected into primary cultured parietal cells, we observed that both G-Gly and gastrin increased luciferase activity in a manner similar to that obtained by Northern blot analysis. L365,260, a specific gastrin/CCKB receptor antagonist, completely reversed the stimulation of luciferase activity induced by gastrin but had no effect on G-Gly-stimulated activity. Gastrin increased [Ca2+]i, although G-Gly did not, however, genistein (a tyrosine kinase inhibitor) significantly reduced induction of luciferase activity by both G-Gly and gastrin. Specific binding of 125I-Leu15-G2-17-Gly to gastric parietal cells was dose-dependently displaced by G2-17-Gly but not by gastrin nor L365,260. Gastrin peptides truncated at the carboxyl- (G1-13) and amino terminus (G5-17-Gly) both induced H+,K(+)-ATPase alpha-subunit gene expression and inhibited 125I-Leu15-G2-17-Gly binding, but were less potent than G2-17-Gly. These data indicate that G-Gly may have a functional role in potentiating gastric acid secretagogue action via enhanced expression of the gene responsible for H+ generation through action at a novel receptor that can be distinguished from the gastrin/CCKB receptor. Thus, both the substrate and product of the terminal progastrin processing reaction appear to have complementary functions in regulation of gastric acid secretion.
Asunto(s)
Gastrinas/metabolismo , Gastrinas/farmacología , Expresión Génica/efectos de los fármacos , Glicina/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/biosíntesis , Células Parietales Gástricas/metabolismo , Compuestos de Fenilurea , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacología , Procesamiento Proteico-Postraduccional , Aminopirina/metabolismo , Animales , Secuencia de Bases , Benzodiazepinonas/farmacología , Transporte Biológico , Northern Blotting , Calcio/metabolismo , Células Cultivadas , Perros , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Histamina/farmacología , Humanos , Cinética , Luciferasas/biosíntesis , Sustancias Macromoleculares , Datos de Secuencia Molecular , Células Parietales Gástricas/efectos de los fármacos , Células Parietales Gástricas/enzimología , Regiones Promotoras Genéticas , Receptores de Colecistoquinina/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , beta-Galactosidasa/biosíntesisRESUMEN
Peptide alpha amidation is required to produce some hormones, such as gastrin, from their glycine-extended precursors. This terminal posttranslational processing reaction is thought to be essential for the biological activation of many peptide hormones; only amidated gastrin exerts a physiological effect that results in gastric acid secretion. However, both amidated gastrin and glycine-extended gastrin stimulate proliferation of exocrine pancreatic cell line AR4-2J through selective receptors for the substrate and the product, respectively, of peptide alpha amidation. Thus, the amidation reaction may function as a determinant of the specific biological actions of products derived from prohormones.
Asunto(s)
División Celular/efectos de los fármacos , Gastrinas/farmacología , Compuestos de Fenilurea , Benzodiazepinonas/farmacología , Sitios de Unión , Gastrinas/metabolismo , Humanos , Indoles/farmacología , Meglumina/análogos & derivados , Meglumina/farmacología , Octreótido/farmacología , Ornitina Descarboxilasa/metabolismo , Receptores de Colecistoquinina/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
Gastrin and cholecystokinin (CCK) have proven trophic effects on the gut. We have previously demonstrated that these peptides stimulate an early event in cellular proliferation, namely ornithine decarboxylase activity (ODC), in a rat exocrine pancreatic cell line AR4-2J. Furthermore, this effect is mediated through a G/CCKB receptor. Thus, in the present study we sought to examine the signal transduction mechanisms linked to the G/CCKB receptor occupancy. Both gastrin and CCK induced a rapid (maximum at 40 s) increase in inositol triphosphates (InsP3) and diacylglycerol (DAG) formation in a dose-dependent manner (EC50 = 5.6 nM) that quickly returned to baseline. Although InsP3 levels remained at baseline, DAG levels demonstrated a second gradual increase that was maximal at 15 min. CCK/gastrin efficiency to stimulate DAG and InsP3 formation (EC50 = 5.6 nM) could be correlated to the G/CCKB receptor occupancy, suggesting a coupling of this receptor to phospholipase C. To examine the involvement of protein kinase C (PKC) activation in the increase in ODC activity, we stimulated the AR4-2J cells with the phorbol ester TPA and observed an increase in ODC activity with a maximal effect at 100 nM. TPA stimulation of ODC activity was completely abolished by the PKC inhibitor staurosporine (50 nM). However, 50 nM staurosporine inhibited only 65% of the gastrin and CCK induced increase in ODC activity suggesting that a portion of the G/CCKB receptor-mediated increase in ODC activity is PKC independent.
Asunto(s)
Proteína Quinasa C/metabolismo , Receptores de Colecistoquinina/metabolismo , Fosfolipasas de Tipo C/metabolismo , Alcaloides/farmacología , Animales , Sitios de Unión , Colecistoquinina/farmacología , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Gastrinas/farmacología , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Ornitina Descarboxilasa/metabolismo , Páncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Transducción de Señal/fisiología , Estaurosporina , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
In order to characterize the biological functions coupled to cholecystokinin (CCK) A and B receptors, the effects of gastrin(2-17 ds) and caerulein were compared. An isolated cell model, the pancreatic acinar cell line AR4-2J, was used and the experiments were carried out in serum-free media. Caerulein was found to evoke no mitogenic effects either alone or in the presence of the CCK antagonists L364,718 and CR1409. Gastrin(2-17 ds) increased cell proliferation by 2-fold with an IC50 of 150 pM, corresponding to the occupancy of the CCK B receptors. CR1409, at concentrations that fully occupied CCK B receptors, inhibited the gastrin(2-17 ds) effects. Caerulein enhanced chymotrypsinogen biosynthesis by 100% and the corresponding mRNA level by 75%; amylase biosynthesis and mRNA level were enhanced by 40% only. Half-maximal increases in chymotrypsin activity and mRNA level were recorded in response to caerulein at concentrations of 100 pM and 50 pM respectively. Gastrin(2-17 ds) at 100 nM enhanced chymotrypsinogen biosynthesis by 26% and its mRNA level by 35%; these responses were lower than those evoked by 0.1 nM caerulein. Furthermore, CR1409 completely inhibited caerulein- and gastrin(2-17 ds)-stimulated chymotrypsinogen synthesis, with similar IC50 (4 microM). These results suggest that both peptides induced the synthesis of the secretory enzyme after occupancy of CCK A receptors.