RESUMEN
Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated ß-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.
Asunto(s)
Adenocarcinoma , Receptores de Apelina/metabolismo , Apelina/metabolismo , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/genética , Ciclina D1/metabolismo , Glucosa , Humanos , Ratones , Oncogenes , Neoplasias Pancreáticas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , beta Catenina/metabolismo , Neoplasias PancreáticasRESUMEN
Apelin signaling plays an important role during embryo development and regulates angiogenesis, cardiovascular activity, and energy metabolism in adulthood. Overexpression and hyperactivity of this signaling pathway is observed in various pathologic states, such as cardiovascular diseases and cancer, which highlights the importance of inhibiting apelin receptor (APJ); therefore, we developed a cell-based screening assay that uses fluorescence microscopy to identify APJ antagonists. This approach led us to identify the U.S. Food and Drug Administration-approved compound protamine-already used clinically after cardiac surgery-as an agent to bind to heparin and thereby reverse its anticlotting activity. Protamine displays a 390-nM affinity for APJ and behaves as a full antagonist with regard to G protein and ß-arrestin-dependent intracellular signaling. Ex vivo and in vivo, protamine abolishes well-known apelin effects, such as angiogenesis, glucose tolerance, and vasodilatation. Remarkably, protamine antagonist activity is fully reversed by heparin treatment both in vitro and in vivo Thus, our results demonstrate a new pharmacologic property of protamine-blockade of APJ-that could explain some adverse effects observed in protamine-treated patients. Moreover, our data reveal that the established antiangiogenic activity of protamine would rely on APJ antagonism.-Le Gonidec, S., Chaves-Almagro, C., Bai, Y., Kang, H. J., Smith, A., Wanecq, E., Huang, X.-P., Prats, H., Knibiehler, B., Roth, B. L., Barak, L. S., Caron, M. G., Valet, P., Audigier, Y., Masri, B. Protamine is an antagonist of apelin receptor, and its activity is reversed by heparin.
Asunto(s)
Heparina/farmacología , Protaminas/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Receptores de Apelina , Línea Celular Tumoral , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
While many factors that modulate the morphogenesis and patterning of the embryonic heart have been identified, relatively little is known about the molecular events that regulate the differentiation of progenitor cells fated to form the myocardium. Here, we show that zebrafish grinch (grn) mutants form a reduced number of myocardial progenitor cells, which results in a profound deficit in cardiomyocyte numbers in the most severe cases. We show that grn encodes the G protein-coupled receptor (GPCR) Agtrl1b, a known regulator of adult cardiovascular physiology. Ectopic expression of Apelin, an Agtrl1b ligand, results in the complete absence of cardiomyocytes. Data from transplantation and transgenic approaches indicate that Agtrl1 signaling plays a cell-autonomous role in myocardial specification, with activity being required coincident with the onset of gastrulation movements. These results support a model in which agtrl1b regulates the migration of cells fated to form myocardial progenitors.
Asunto(s)
Corazón/embriología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mioblastos Cardíacos/metabolismo , Organogénesis/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Xenopus/metabolismo , Proteínas de Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/genética , Receptores de Apelina , Diferenciación Celular/genética , Movimiento Celular/genética , Gástrula/citología , Gástrula/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Mesodermo/citología , Mesodermo/metabolismo , Mutación/genética , Mioblastos Cardíacos/citología , Miocardio/citología , Miocardio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Proteínas de Xenopus/genética , Pez Cebra , Proteínas de Pez Cebra/genéticaRESUMEN
The apelin receptor is a G protein-coupled receptor activated by several apelin fragments. Its tissue distribution suggests that apelin signalling is involved in a broad range of physiological functions. Endothelial cells, which express high levels of apelin receptors, respond to apelin through the phosphorylation of key intracellular effectors associated with cell proliferation and migration. In addition, apelin is a mitogen for endothelial cells and exhibits angiogenic properties in matrigel experiments. This review focuses on the therapeutic potential of apelin signalling, which is associated with pathologies that result from decreased vascularisation (ischemias) or neovascularisation (retinopathies and solid tumors).
Asunto(s)
Receptores Acoplados a Proteínas G/fisiología , Transducción de Señal/fisiología , Receptores de Apelina , Diseño de Fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Humanos , Neovascularización Patológica/fisiopatología , Neovascularización Patológica/prevención & control , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
The results presented herein demonstrate that apelin is expressed and secreted by both human and mouse adipocytes. Apelin mRNA levels in isolated adipocytes are close to other cell types present in white adipose tissue or other organs known to express apelin such as kidney, heart, and to a lesser extent brown adipose tissue. Apelin expression is increased during adipocyte differentiation stage. A comparison of four different models of obesity in mice showed a large increase in both apelin expression in fat cells and apelin plasma levels in all the hyperinsulinemia-associated obesities and clearly demonstrated that obesity or high-fat feeding are not the main determinants of the rise of apelin expression. The lack of insulin in streptozotocin-treated mice is associated with a decreased expression of apelin in adipocytes. Furthermore, apelin expression in fat cells is strongly inhibited by fasting and recovered after refeeding, in a similar way to insulin. A direct regulation of apelin expression by insulin is observed in both human and mouse adipocytes and clearly associated with the stimulation of phosphatidylinositol 3-kinase, protein kinase C, and MAPK. These data provide evidence that insulin exerts a direct control on apelin gene expression in adipocytes. In obese patients, both plasma apelin and insulin levels were significantly higher, suggesting that the regulation of apelin by insulin could influence blood concentrations of apelin. The present work identifies apelin as a novel adipocyte endocrine secretion and focuses on its potential link with obesity-associated variations of insulin sensitivity status.
Asunto(s)
Proteínas Portadoras/genética , Insulina/farmacología , Obesidad/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Adipoquinas , Tejido Adiposo/metabolismo , Animales , Apelina , Proteínas Portadoras/fisiología , Diferenciación Celular , Femenino , Regulación de la Expresión Génica , Humanos , Hiperinsulinismo/metabolismo , Insulina/sangre , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Regulación hacia ArribaRESUMEN
We have recently identified a new G protein-coupled receptor, msr/apj, whose transcripts are detected in the endothelium of the primary blood vessels and the newly forming heart (Mech. Dev. 1999;84:199). Its expression during formation of the embryonic vasculature incited us to investigate whether expression of the receptor also increased during the formation of retinal vessels, which occurs postnatally in the mouse. Interestingly, msr/apj transcripts are indeed associated with the forming vessels and trace the centrifugal extension of the superficial vasculature. We also show that expression of the endogenous ligand apelin is upregulated at the leading edge of vessel formation.
Asunto(s)
Proteínas Portadoras/genética , Receptores de Superficie Celular/genética , Receptores de Dopamina D2/genética , Receptores Acoplados a Proteínas G , Vasos Retinianos/crecimiento & desarrollo , Adipoquinas , Animales , Apelina , Receptores de Apelina , Regulación del Desarrollo de la Expresión Génica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Ratones , Ratones Endogámicos C57BL , Vasos Retinianos/metabolismo , Regulación hacia ArribaRESUMEN
We report here that apelin (65-77) activates p70 S6 kinase (p70S6K), not only in CHO cells that have been stably transfected with the apelin receptor, but also in umbilical endothelial cells (HUVEC), which express it endogenously. Apelin (65-77) induces a time-dependent phosphorylation of p70S6K at residues T421/S424 and T389. This dual phosphorylation is associated with two transduction cascades, involving a PI3K pathway and an ERK pathway, respectively. The PI3K pathway, which can be blocked by wortmannin, leads to phosphorylation of Akt at residues T308 or S473, which then promotes the phosphorylation of p70S6K at T421/S424 and T389. The ERK pathway is blocked by PD 098059, a MEK inhibitor, and results in the phosphorylation of p70S6K at T421/S424. Phosphorylation both of Akt and p70S6K is abrogated by pretreatment with pertussis toxin (PTX) and an inhibitor of atypical PKCs. In addition, we demonstrate that apelin (65-77) also increases the enzymatic activity of p70S6K and that the effects of the previously mentioned inhibitors on the level of T389 phosphorylation correlate with their action on enzyme activity. Interestingly, the main findings were reproduced in umbilical endothelial cells and apelin (65-77) promoted thymidine incorporation into DNA of these cells, revealing that apelin is a new mitogenic peptide for the endothelial cell.
Asunto(s)
Proteínas Portadoras/farmacología , Endotelio Vascular/enzimología , Mitógenos/farmacología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Flavonoides/farmacología , Humanos , Toxina del Pertussis/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteína Quinasa C/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Sirolimus/farmacología , Cordón Umbilical/citología , Familia-src Quinasas/metabolismoRESUMEN
Expression of the new G protein-coupled msr/apj receptor in the mouse embryo is restricted to the endothelial layer of the primary blood vessels and the newly forming heart (Mech. Dev. 84 (1999) 199). During development of the retinal vasculature, the msr/apj gene is not expressed throughout the vascular network, indicating a possible relationship between the localization of expression and the acquisition of arterial or venous identity (Mech. Dev. 110 (2002) 183). Here we first established that retinal expression of ephrin-B2 and its putative receptor EphB4 correlates with arterial and venous phenotype, respectively. Then we analyzed the expression pattern of msr/apj in the retinal vessels at various stages of postnatal development by in situ hybridization. In contrast to the expression of ephrin-B2 or EphB4, msr/apj transcripts can be detected as early as postnatal day P3. From P3 to P12, msr/apj expression in the vascular network is restricted to the venules and the associated capillaries. The msr/apj gene is thus an early and specific marker of the venous phenotype in the retinal vasculature.
Asunto(s)
Biomarcadores/análisis , Efrina-B2/metabolismo , Receptor EphB4/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Vena Retiniana/crecimiento & desarrollo , Animales , Receptores de Apelina , Arteriolas/metabolismo , Hibridación in Situ , Ligandos , Ratones , Ratones Endogámicos C57BL , Fenotipo , Sondas ARN , Receptores Acoplados a Proteínas G/genética , Vena Retiniana/metabolismoRESUMEN
Using a cancer profiling array, our laboratory has shown that apelin gene is up-regulated in half of colon adenocarcinomas. We have therefore postulated that apelin signalling might play a prominent role in the growth of colon tumours. We first confirmed by immunohistochemistry that apelin peptide is overexpressed in human colon adenomas and adenocarcinomas. We also observed a significant overexpression of apelin receptor (APJ) in adjacent sections. We then demonstrated that several colorectal cancer cell lines also expressed apelin and its receptor, the highest gene and peptide expression being detected in LoVo cells. In this cell line, the expression and functionality of apelin receptor were revealed by apelin-induced adenylyl cyclase inhibition and Akt phosphorylation. In addition, apelin clearly protected LoVo cells from apoptosis by inactivating a caspase-dependent pathway and decreasing the degradation of poly ADP ribose polymerase protein (PARP). Finally, treatment of these tumour cells by the (F13A)apelin13 receptor antagonist significantly reduced their proliferation rate. Altogether, these data suggest the existence of an autocrine loop by which constitutive activation of apelin signalling should participate in the growth of colon adenocarcinomas. Accordingly, apelin signalling is a promising pharmacological target for the treatment of human colon adenomas and adenocarcinomas.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Receptores Acoplados a Proteínas G/biosíntesis , Adenocarcinoma/genética , Apelina , Receptores de Apelina , Apoptosis/fisiología , Comunicación Autocrina , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Neoplasias del Colon/genética , Perfilación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Fosforilación , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de SeñalRESUMEN
G Protein-Coupled Receptors (GPCRs) share the same topology made of seven-transmembrane segments and represent the largest family of membrane receptors. Initially associated with signal transduction in differentiated cells, GPCRs and heterotrimeric G proteins were shown to behave as proto-oncogenes whose overexpression or activating mutations confer transforming properties. The first part of this review focuses on the link between biochemical interactions of a GPCR with other receptors, such as dimerization or multiprotein complexes, and their oncogenic properties. Alteration of these interactions or deregulation of transduction cascades can promote uncontrolled cell proliferation or cell transformation that leads to tumorigenicity and malignancy. The second part concerns the design of drugs specifically targeting these complex interactions and their promise in cancer therapy.
Asunto(s)
Antineoplásicos/uso terapéutico , Diseño de Fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antineoplásicos/farmacología , Humanos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The formation of the vascular system is an early step in organogenesis that involves the participation of various signalling pathways. Integration of the extracellular signals decoded by their cognate membrane receptors orchestrate the cell events, which act at different stages, from the primitive network formed by vasculogenesis to the arborescent network remodeled by angiogenesis. Our laboratory showed the participation of a new signalling pathway in physiological angiogenesis and tumour neovascularisation. This signalling pathway named apelin comprises a G protein-coupled receptor and a peptide ligand. Expression of apelin receptors is observed during the embryonic formation of blood vessels where it is localized in the endothelium. In HUVECs, which endogenously express apelin receptors, apelin promotes the phosphorylation of ERKs, Akt and p70 S6 Kinase. In addition, apelin increases in vitro the proliferation of these endothelial cells. Finally, injection of apelin in the vitreous induces in vivo the sprouting and the proliferation of endothelial cells from the retinal vascular network. Accordingly, all these results led us to study the role of apelin signalling in tumour neovascularisation. In two tumoral cell lines, we showed that hypoxia induces the expression of apelin gene. In addition, the overexpression of apelin gene resulting from stable transfection of these cell lines clearly accelerates in vivo tumour growth, as a consequence of an increased number of vessels irrigating these tumours. The pathological relevance of these data has been validated by the characterization of an overexpression of apelin gene in one third of human tumours. Taken together, apelin signalling is both involved in physiological angiogenesis and pathological neoangiogenesis, and therefore represents an interesting pharmacological target for anti-angiogenic therapies.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Neoplasias/irrigación sanguínea , Neovascularización Patológica/fisiopatología , Neovascularización Fisiológica/fisiología , Receptores Acoplados a Proteínas G/fisiología , Adipoquinas , Animales , Apelina , Receptores de Apelina , Proteínas Portadoras/fisiología , Hipoxia de la Célula/fisiología , Células Cultivadas , Embrión no Mamífero/irrigación sanguínea , Células Endoteliales/citología , Células Endoteliales/metabolismo , Gástrula/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/toxicidad , Ratones , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neovascularización Fisiológica/genética , Fosforilación , Procesamiento Proteico-Postraduccional , Vasos Retinianos/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriologíaRESUMEN
The apelin receptor is a G protein-coupled receptor to which two ligand fragments, apelin-(65-77) and apelin-(42-77), can bind. To address the physiological significance of the existence of dual ligands for a single receptor, we first compared the ability of the apelin fragments to regulate intracellular effectors, to promote G protein coupling, and to desensitize the response in Chinese hamster ovary cells expressing the murine apelin receptor. We found that both apelin fragments inhibited adenylyl cyclase and increased the phosphorylation of ERK or Akt. Using stably transfected cells expressing a pertussis toxin-insensitive alpha(i) subunit, we demonstrated that each apelin fragment promoted coupling of the apelin receptor to either Galpha(i1) or Galpha(i2) but not to Galpha(i3). Although preincubation with each apelin fragment induced a desensitization at the level of the three effectors, preincubation with apelin-(42-77) also increased basal effector activity. In addition, a C-terminal deletion of the apelin receptor decreased the desensitization induced by apelin-(65-77) but did not alter the desensitization pattern induced by apelin-(42-77). Finally, in umbilical endothelial cells, which we have recently shown to express the apelin receptor, the Galpha(i1) and Galpha(i2) subunits are also expressed, ERK and Akt phosphorylation is desensitized after preincubation with apelin-(65-77), and basal levels of Akt phosphorylation are increased after preincubation with apelin-(42-77). In summary, apelin fragments regulate the same effectors, via the preferential coupling of the apelin receptor to G(i1) or G(i2), but they promote a differential desensitization pattern that may be central to their respective physiological roles.
Asunto(s)
Proteínas Portadoras/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Adipoquinas , Animales , Apelina , Receptores de Apelina , Células CHO , Proteínas Portadoras/química , Cricetinae , Cricetulus , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Ligandos , Ratones , Fragmentos de Péptidos/metabolismoRESUMEN
Phosphoinositide 3-kinase (PI3K) mediates essential functions of vascular endothelial growth factor (VEGF), including the stimulation of endothelial cell proliferation and migration. Nevertheless, the mechanisms coupling the receptor VEGFR-2 to PI3K remain obscure. We observed that the Grb2-bound adapter Gab1 is tyrosine-phosphorylated and relocated to membrane fractions upon VEGF stimulation of endothelial cells. We could detect the PI3K regulatory subunit p85 in immunoprecipitates of endogenous Gab1, and vice versa, and measure a Gab1-associated lipid kinase activity upon VEGF stimulation. Furthermore, transfection of the Gab1-YF3 mutant lacking all p85-binding sites strongly repressed PI3K activation measured in vitro. Moreover, Gab1-YF3 severely decreased the cellular amount of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generated in response to VEGF. Furthermore, adenoviral expression of Gab1-YF3 suppressed both Akt phosphorylation and recovery of wounded human umbilical vein endothelial cell monolayers, a VEGF-dependent process involving cell migration and proliferation under PI3K control. Transfection of other Gab1 mutants, lacking Grb2-binding sites or the pleckstrin homology (PH) domain, also prevented Akt activation, further demonstrating Gab1 involvement in PI3K activation. These mutants were also used to show that interactions with both Grb2 and PtdIns(3,4,5)P3 mediate Gab1 recruitment by VEGFR-2. Importantly, Gab1 mobilization was impaired by (i) PI3K inhibitors, (ii) deletion of Gab1 PH domain, (iii) PTEN (phosphatase and tensin homolog deleted on chromosome 10) overexpression to repress PtdIns(3,4,5)P3 production, and (iv) overexpression of a competitor PH domain for PtdIns(3,4,5)P3 binding, which altogether demonstrated that PI3K is also an upstream regulator of Gab1. Gab1 thus appears as a primary actor in coupling VEGFR-2 to PI3K/Akt, recruited through an amplification loop involving PtdIns(3,4,5)P3 and its PH domain.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenoviridae/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Endotelio Vascular/citología , Activación Enzimática , Eliminación de Gen , Humanos , Mutación , Transfección , Venas Umbilicales/citología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de HeridasRESUMEN
We report here that apelin (65-77) induces activation of extracellular-regulated kinases (ERKs) in Chinese hamster ovary (CHO) cells expressing the msr/apj receptor. This concentration-dependent activation was transient, peaking at 5 min. Pretreatment of CHO cells with pertussis toxin fully abrogated ERK phosphorylation, whereas overexpression of the beta-adrenergic receptor kinase-1 C-terminal fragment did not alter ERK activation. Transfection with a dominant-negative mutant of Ras was without effect on ERK activation, whereas an inhibitor of many protein kinase C isoforms, GF109203X, strongly decreased it. These results demonstrate that stimulation of the murine msr/apj receptor promotes ERK activation via the alpha subunit of a pertussis toxin-sensitive protein in a Ras-independent pathway.