RESUMEN
BACKGROUND: Adropin, a unique peptide hormone, has been associated with the regulation of several physiological processes, including glucose homeostasis, fatty acid metabolism, and neovascularization. However, its possible role in ovarian function is not understood. Our objective was to examine the expression of adropin and its putative receptor, GPR19, in the ovaries of mice at various phases of the estrous cycle. METHODS: Immunohistochemistry and western blot analysis were performed to explore the localization and changes in expression of adropin and GPR19 in the ovaries during different phases of the estrous cycle in mice. Hormonal assays were performed with ELISA. An in vitro study was performed to examine the direct effect of adropin (10, 100 ng/ml) on ovarian function. RESULTS: A western blot study showed that adropin and GPR19 proteins were maximum during the estrus phase of the estrous cycle. Interestingly, adropin and GPR19 displayed intense immunoreactivity in granulosa cells of large antral follicles and corpus luteum. This suggested the possible involvement of adropin in corpus luteum formation. Adropin treatment stimulated progesterone synthesis by increasing GPR19, StAR, CYP11A1, and 3ß-HSD expressions, while it decreased estrogen synthesis by inhibiting 17ß-HSD and aromatase protein expressions. Moreover, adropin treatment upregulated the cell cycle arrest-CDK inhibitor 1B (p27kip1), pERK1/2, and angiogenic protein (EG VEGF) that are involved in the process of luteinization. CONCLUSIONS: Adropin GPR19 signaling promotes the synthesis of progesterone and upregulates the expression of p27kip1, EG VEGF, and erk1/2, resulting in cell cycle arrest and neovascularization, which ultimately leads to corpus luteum formation.
Asunto(s)
Ovario , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina , Femenino , Ratones , Animales , Ovario/metabolismo , Progesterona/farmacología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Cuerpo Lúteo/metabolismo , Ciclo EstralRESUMEN
Trophoblast invasion is an important event in embryo implantation and placental development. During these processes, endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is the key regulator mediating the crosstalk at the feto-maternal interface. The primary cilium is a cellular antenna receiving environmental signals and is crucial for proper development. However, little is known regarding the role of the primary cilium in early human pregnancy. Here, we demonstrate that EG-VEGF regulates trophoblast cell invasion via primary cilia. We found that EG-VEGF activated ERK1/2 signaling and subsequent upregulation of MMP2 and MMP9, thereby facilitating cell invasion in human trophoblast HTR-8/SVneo cells. Inhibition of ERK1/2 alleviated the expression of MMPs and trophoblast cell invasion after EG-VEGF treatment. In addition, primary cilia were observed in all the trophoblast cell lines tested and, more importantly, in human first-trimester placental tissue. The receptor of EG-VEGF, PROKR1, was detected in primary cilia. Depletion of IFT88, the intraflagellar transporter required for ciliogenesis, inhibited primary cilium growth, thereby ameliorating ERK1/2 activation, MMP upregulation, and trophoblast cell invasion promoted by EG-VEGF. These findings demonstrate a novel function of primary cilia in controlling EG-VEGF-regulated trophoblast invasion and reveal the underlying molecular mechanism. J. Cell. Physiol. 232: 1467-1477, 2017. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Movimiento Celular , Cilios/metabolismo , Transducción de Señal , Trofoblastos/citología , Trofoblastos/enzimología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Línea Celular , Movimiento Celular/efectos de los fármacos , Cilios/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Fosforilación/efectos de los fármacos , Embarazo , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Trofoblastos/efectos de los fármacos , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
Pregnancy-induced hypertension diseases are classified as gestational hypertension, preeclampsia, or eclampsia. The mechanisms of their development and prediction are still to be discovered. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic factor secreted by the placenta during the first trimester of human pregnancy that was shown to control trophoblast invasion, to be upregulated by hypoxia, and to be abnormally elevated in pathological pregnancies complicated with preeclampsia and intrauterine growth restriction. These findings suggested that sustaining EG-VEGF levels beyond the first trimester of pregnancy may contribute to pregnancy-induced hypertension. To test this hypothesis, osmotic minipumps delivering EG-VEGF were implanted subcutaneously into gravid OF1 (Oncins France 1) mice on day 11.5 post coitus, which is equivalent to the end of the first trimester of human pregnancy. Mice were euthanized at 15.5 and 18.5 days post coitus to assess (1) litter size, placental, and fetal weights; (2) placental histology and function; (3) maternal blood pressure; (4) renal histology and function; and (5) circulating soluble fms-like tyrosine kinase 1 and soluble endoglin. Increased EG-VEGF levels caused significant defects in placental organization and function. Both increased hypoxia and decreased trophoblast invasion were observed. Treated mice had elevated circulating soluble fms-like tyrosine kinase 1 and soluble endoglin and developed gestational hypertension with dysregulated maternal kidney function. EG-VEGF effect on the kidney function was secondary to its effects on the placenta as similarly treated male mice had normal kidney functions. Altogether, these data provide a strong evidence to confirm that sustained EG-VEGF beyond the first trimester of pregnancy contributes to the development of pregnancy-induced hypertension.
Asunto(s)
Hipertensión Inducida en el Embarazo/fisiopatología , Placenta/efectos de los fármacos , Preñez , Receptores Acoplados a Proteínas G/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/sangre , Animales , Biopsia con Aguja , Western Blotting , Modelos Animales de Enfermedad , Femenino , Hipertensión Inducida en el Embarazo/genética , Inmunohistoquímica , Ratones , Ratones Endogámicos , Fenotipo , Placenta/patología , Embarazo , Primer Trimestre del Embarazo , Distribución Aleatoria , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
CONTEXT: Fetal ovarian development and primordial follicle formation underpin future female fertility. Prokineticin (PROK) ligands regulate cell survival, proliferation, and angiogenesis in adult reproductive tissues including the ovary. However, their expression and function during fetal ovarian development remains unclear. OBJECTIVE: This study aimed to investigate expression and localization of the PROK ligands, receptors, and their downstream transcriptional targets in the human fetal ovary. SETTING: This study was conducted at the University of Edinburgh. PARTICIPANTS: Ovaries were collected from 37 morphologically normal human fetuses. DESIGN AND MAIN OUTCOME MEASURES: mRNA and protein expression of PROK ligands and receptors was determined in human fetal ovaries using qRT-PCR, immunoblotting, and immunohistochemistry. Functional studies were performed using a human germ cell tumor line (TCam-2) stably transfected with Prokineticin receptor 1 (PROKR1). RESULTS: Expression of PROK1 and PROKR1 was significantly higher in mid-gestation ovaries (17-20 wk) than at earlier gestations (8-11 and 14-16 wk). PROK2 significantly increased across the gestations examined. PROKR2 expression remained unchanged. PROK ligand and receptor proteins were predominantly localized to germ cells (including oocytes within primordial follicles) and endothelial cells, indicating these cell types to be the targets of PROK signaling in the human fetal ovary. PROK1 treatment of a germ cell line stably expressing PROKR1 resulted in ERK phosphorylation and elevated COX2 expression. CONCLUSIONS: Developmental changes in expression and regulation of COX2 and phosphorylated ERK (pERK) by PROK1 suggest that PROK ligands may be novel regulators of germ cell development in the human fetal ovary, interacting within a network of growth and survival factors prior to primordial follicle formation.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Hormonas Gastrointestinales/metabolismo , Células Germinativas/metabolismo , Neuropéptidos/metabolismo , Ovario/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Ciclooxigenasa 2/genética , Femenino , Desarrollo Fetal , Hormonas Gastrointestinales/genética , Hormonas Gastrointestinales/farmacología , Células Germinativas/efectos de los fármacos , Humanos , Neuropéptidos/genética , Ovario/embriología , Fosforilación/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
Endocrine glandderived vascular endothelial growth factor (EGVEGF) is a newly cloned factor that selectively acts on the endothelium of endocrine gland cells. EGVEGF was previously identified as an important cytokine, involved in the modulation of apoptosis in pancreatic cancer cell lines. The present study examined the effects of EGVEGF proliferation and migration, in pancreatic cancer cells. To determine the potential for EGVEGF as a therapeutic target for pancreatic cancer, the expression of EGVEGF were measured in pancreatic cancer tissue, and the association between its expression and the clinicopathological characteristics of the pancreatic cancer patients was determined. The results of the present study suggest that EGVEGF may act as a novel tumor gene in pancreatic cancer. EGVEGF was rarely expressed in the normal pancreatic tissue, but was highly expressed in the pancreatic cancer tissue. These data suggest that EGVEGF may be a cancerspecific, and possibly tissuespecific, survival factor in the pancreas. In the Mia PaCa2 pancreatic cancer cell line, EGVEGF was shown to promote proliferation and cellular invasion, and modulate the phosphorylation of mitogenactivated protein kinase, a modulator for the malignant phenotype.
Asunto(s)
Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Neoplasias Pancreáticas/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Adulto , Anciano , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismoRESUMEN
The mechanisms that regulate the induction of term or preterm delivery (PTD) are not fully understood. Infection is known to play a role in the induction of pro-inflammatory cascades in uteroplacental tissues associated with preterm pathological parturition. Similar but not identical cascades are evident in term labour. In the current study, we used a mouse model to evaluate the role of prokineticins in term and preterm parturition. Prokineticins are multi-functioning secreted proteins that signal through G-protein-coupled receptors to induce gene expression, including genes important in inflammatory responses. Expression of prokineticins (Prok1 and Prok2) was quantified in murine uteroplacental tissues by QPCR in the days preceding labour (days 16-19). Prok1 mRNA expression increased significantly on D18 in fetal membranes (compared with D16) but not in uterus or placenta. Intrauterine injection of PROK1 on D17 induced fetal membrane mRNA expression of the pro-inflammatory mediators Il6, Il1b, Tnf, Cxcl2 and Cxcl5, which are not normally up-regulated until D19 of pregnancy. However, intrauterine injection of PROK1 did not result in PTD. As expected, injection of lipopolysaccharide (LPS) induced PTD, but this was not associated with changes in expression of Prok1 or its receptor (Prokr1) in fetal membranes. These results suggest that although Prok1 exhibits dynamic mRNA regulation in fetal membranes preceding labour and induces a pro-inflammatory response when injected into the uterus on D17, it is insufficient to induce PTD. Additionally, prokineticin up-regulation appears not to be part of the LPS-induced inflammatory response in mouse fetal membranes.
Asunto(s)
Membranas Extraembrionarias/efectos de los fármacos , Inflamación/inducido químicamente , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Animales , Membranas Extraembrionarias/inmunología , Membranas Extraembrionarias/metabolismo , Femenino , Inflamación/genética , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Inyecciones , Lipopolisacáridos/farmacología , Masculino , Ratones , Embarazo , Nacimiento Prematuro , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Útero , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismoRESUMEN
Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has recently been identified as one of the vascular endothelial growth factors, and it is considered that the overexpression of EG-VEGF in colon cancer is related to hepatic metastasis. In this study, we report our recent novel findings of the involvement of EG-VEGF in cell invasion of colon cancer cells. Colon cancer cell lines (DLD-1 and HCT116) with high expression of prokineticin receptor (PK-R) 1 and 2 were stimulated with the EG-VEGF protein. Furthermore, Matrigel cell invasion assay was performed to examine the changes in cancer cell invasion. In addition, we investigated the mRNA expression of matrix metalloproteinase (MMP)-2, -7 and -9 in cancer cells. Finally, the EG-VEGF receptor on the colon cancer cell membrane was blocked by anti-PK-R1 and -PK-R2 antibodies to study whether cell invasion ability would be altered. In colon cancer cell lines where the expression of PK-R1 and 2 was confirmed, stimulation with EG-VEGF increased cell invasion a maximum of ~3-5 times. Furthermore, an increase in the mRNA and protein expression of MMP-2, -7 and -9 was observed. We also observed that the cell invasion rate decreased only after exposure to the anti-PK-R2 antibody. The study showed that the EG-VEGF protein may act on MMP-2, -7 and -9 via PK-R2 to strengthen cell invasion ability in colon cancer cell lines.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Hormonas Gastrointestinales/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Anticuerpos/farmacología , Neoplasias del Colon/genética , Hormonas Gastrointestinales/inmunología , Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Invasividad Neoplásica , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/inmunología , Receptores de Péptidos/inmunología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/inmunología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismoRESUMEN
Prokineticin-1 (PK1) has been identified as a mitogen-specific protein for the endothelium of steroidogenic glands. Here we report a novel function of PK1 in the regulation of multiple myeloma (MM) cells. PK1 activates multiple signals including mitogen-activated protein kinase (MAPK), PI3K-AKT, and Jak-STAT3, sphingosine kinase-1 (SPK1) in MM cells. Treatment of MM cells with PK1 causes a time- and dose-dependent phosphorylation of MAPK, AKT and STAT3 and upregulation of SPK1 expression and cellular activity. We also show that PK1 upregulates Mcl-1 expression in a time- and dose-dependent manner in human MM cell lines and in the cells of patients with MM. Pertussis toxin, a pan-PK1 receptor inhibitor, can block PK1-induced upregulation of Mcl-1, indicating it relates to a G-protein-coupled receptor. We also show that PK1 protects MM cells against apoptosis induced by starvation for fetal calf serum (FBS), or for FBS and IL-6. Taken together, PK1 activates multiple signaling pathways and, upregulates Mcl-1 expression, leading to proliferation and survival of MM cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero/farmacología , Expresión Génica , Humanos , Inmunohistoquímica , Interleucina-6/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Fosforilación/efectos de los fármacos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/metabolismo , Células Tumorales Cultivadas , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismoRESUMEN
Endocrine gland derived vascular endothelial growth factor (EG-VEGF) also called prokineticin (PK1), has been identified and linked to several biological processes including angiogenesis. EG-VEGF is abundantly expressed in the highest vascularized organ, the human placenta. Here we characterized its angiogenic effect using different experimental procedures. Immunohistochemistry was used to localize EG-VEGF receptors (PROKR1 and PROKR2) in placental and umbilical cord tissue. Primary microvascular placental endothelial cell (HPEC) and umbilical vein-derived macrovascular EC (HUVEC) were used to assess its effects on proliferation, migration, cell survival, pseudovascular organization, spheroid sprouting, permeability and paracellular transport. siRNA and neutralizing antibody strategies were used to differentiate PROKR1- from PROKR2-mediated effects. Our results show that 1) HPEC and HUVEC express both types of receptors 2) EG-VEGF stimulates HPEC's proliferation, migration and survival, but increases only survival in HUVECs. and 3) EG-VEGF was more potent than VEGF in stimulating HPEC sprout formation, pseudovascular organization, and it significantly increases HPEC permeability and paracellular transport. More importantly, we demonstrated that PROKR1 mediates EG-VEGF angiogenic effects, whereas PROKR2 mediates cellular permeability. Altogether, these data characterized angiogenic processes mediated by EG-VEGF, depicted a new angiogenic factor in the placenta, and suggest a novel view of the regulation of angiogenesis in placental pathologies.
Asunto(s)
Células Endoteliales/efectos de los fármacos , Neovascularización Fisiológica/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Permeabilidad de la Membrana Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Placenta/irrigación sanguínea , Placenta/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Interferencia de ARN , Receptores Acoplados a Proteínas G/genética , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cordón Umbilical/irrigación sanguínea , Cordón Umbilical/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Prokineticin-1 (PROK1) is a multifunctional secreted protein which signals via the G-protein coupled receptor, PROKR1. Previous data from our laboratory using a human genome survey microarray showed that PROK1-prokineticin receptor 1 (PROKR1) signalling regulates numerous genes important for establishment of early pregnancy, including the cytokine interleukin (IL)-11. Here, we have shown that PROK1-PROKR1 induces the expression of IL-11 in PROKR1 Ishikawa cells and first trimester decidua via the calcium-calcineurin signalling pathway in a guanine nucleotide-binding protein (G(q/11)), extracellular signal-regulated kinases, Ca(2+) and calcineurin-nuclear factor of activated T cells dependent manner. Conversely, treatment of human decidua with a lentiviral miRNA to abolish endogenous PROK1 expression results in a significant reduction in IL-11 expression and secretion. Importantly, we have also shown a regulatory role for the regulator of calcineurin 1 isoform 4 (RCAN1-4). Overexpression of RCAN1-4 in PROKR1 Ishikawa cells using an adenovirus leads to a reduction in PROK1 induced IL-11 indicating that RCAN1-4 is a negative regulator in the calcineurin-mediated signalling to IL-11. Finally, we have shown the potential for both autocrine and paracrine signalling in the human endometrium by co-localizing IL-11, IL-11Ralpha and PROKR1 within the stromal and glandular epithelial cells of non-pregnant endometrium and first trimester decidua. Overall we have identified and characterized the signalling components of a novel PROK1-PROKR1 signalling pathway regulating IL-11.
Asunto(s)
Calcineurina/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-11/metabolismo , Factores de Transcripción NFATC/fisiología , Receptores Acoplados a Proteínas G/fisiología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/fisiología , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Línea Celular Tumoral , Ciclosporina/farmacología , Decidua/metabolismo , Ácido Egtácico/farmacología , Endometrio/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Flavonoides/farmacología , Humanos , Inmunohistoquímica , Interleucina-11/genética , Factores de Transcripción NFATC/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Embarazo , Primer Trimestre del Embarazo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
Endocrine glands-derived vascular endothelial growth factor (EG-VEGF, also termed as Prok1)--a novel cytokine that selectively acts on the endothelial cells of endocrine glands--was recently reported to be involved in the regulation of tumor cell growth and survival. However, its roles in the regulation of pancreatic cancer progression remain unclear. In this report, we investigated the suppressive effects of EG-VEGF on pancreatic cancer cell apoptosis and the relevant mechanisms. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we found that the Mia PaCa II cells of the pancreatic cancer cell line express the mRNAs of both EG-VEGF (Prok1) and its receptors. EG-VEGF protects pancreatic cancer cells from apoptosis through upregulation of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein of the bcl-2 family. Treatment of pancreatic cancer cells with EG-VEGF results in the rapid phosphorylation of mitogen-activated protein kinase (MAPK), STAT3, and AKT, which are involved in the upregulation of Mcl-1 expression. EG-VEGF (Prok1) protects Mia PaCa II cells from apoptosis through G protein-coupled receptor (GPR)-induced activation of multiple signal pathways, and hence can be a novel target for pancreatic cancer therapy.
Asunto(s)
Apoptosis , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Línea Celular Tumoral , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Factor de Transcripción STAT3/metabolismo , Regulación hacia Arriba , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
OBJECTIVE: To study the angiogenic functions of endocrine gland-derived vascular endothelial growth factor (EG-VEGF) on a normal myometrial uterine microvascular endothelial cell line (UtMVEC-Myo) and the signaling pathways elicited by EG-VEGF in UtMVEC-Myo. DESIGN: Experimental laboratory study. SETTING: University gynecology unit. PATIENT(S): Infertile women undergoing diagnostic laparoscopy for assessment of tubal patency. INTERVENTION(S): Real-time polymerase chain reaction (PCR) analysis of mRNA of EG-VEGF and its receptors, PKR1 and PKR2, in UtMVEC-Myo and endometrial samples. The effects of EG-VEGF on the cell proliferation, tube formation, and cell signaling pathways of UtMVEC-Myo were studied. MAIN OUTCOME MEASURE(S): Cell proliferation, tube formation, and molecules of cell-signaling pathways in the treated UtMVEC-Myo. RESULT(S): UtMVEC-Myo cells had PKR1 and PKR2 but not EG-VEGF mRNA. EG-VEGF significantly stimulated cell proliferation and tube formation in UtMVEC-Myo cells. EG-VEGF activated p44/42 mitogen-activated protein kinase (MAPK) but not Akt signaling pathway. The effects of EG-VEGF on p44/42 MAPK phosphorylation and cell proliferation were nullified by the specific MAPK inhibitor, PD98059. CONCLUSION(S): EG-VEGF has a direct angiogenic effect on UtMVEC-Myo that expresses EG-VEGF receptors (PKR1 and PKR2) and modulates cell proliferation and sprouting of the endothelial cells. It is suggested that EG-VEGF enhanced cell proliferation through the activation of MAPK pathway but not through the Akt pathway.
Asunto(s)
División Celular/efectos de los fármacos , Endotelio Vascular/citología , Microcirculación/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/fisiología , Útero/irrigación sanguínea , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/fisiología , Técnicas de Cultivo de Célula , Glándulas Endocrinas/fisiología , Endotelio Vascular/efectos de los fármacos , Femenino , Humanos , Microcirculación/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
Prokineticin 1 and 2 (PROK1 and PROK2) are two small proteins largely expressed in inflammatory tissues and involved in monocyte activation and differentiation. The focus of this study was to evaluate whether PROK1 was able to induce chemokine secretion in human monocytes, in monocyte-derived macrophages and in monocyte-derived dendritic cells, an aspect not addressed thus far. Here, we show for the first time, using flow cytometry, that PROK receptors 1 and 2 are present on the surface of human monocytes. Subsequently, monocytes were selected to investigate the chemokine response after stimulation by PROK1. Our results show that only three chemokines (CCL4, CXCL1 and CXCL8) were significantly induced at both the transcript and protein level, and that PROK1 induces most potently CXCL8, in a dose-dependent manner. From a mechanistic point of view, by blocking independently Galphai protein or intracellular calcium, monocytes lose the ability to secrete CXCL8 in response to PROK1. Finally, we observed that CCL4, CXCL1 and CXCL8 secretion, following PROK1 induction, is only observed in monocytes and not in monocyte-derived macrophages and dendritic cells. Our results demonstrate that, in vitro, the differentiation status of monocytes influences chemokine production after stimulation by PROK1, and that this chemokine production is geared toward a pro-inflammatory response. This could represent a novel amplification loop of leukocyte recruitment, extravasation and tissue invasion.
Asunto(s)
Quimiocina CCL4/metabolismo , Quimiocina CXCL1/metabolismo , Interleucina-8/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Animales , Células Cultivadas , Quimiocina CCL4/genética , Quimiocina CXCL1/genética , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Humanos , Interleucina-8/genética , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Transcripción Genética/genéticaRESUMEN
BACKGROUND: Bv8, prokineticin-1, or endocrine gland-vascular endothelial growth factor, and prokineticin-2 are recently isolated peptide agonists of two G protein-coupled receptors, prokineticin receptor-1 (PROKR 1) and PROKR 2, and have been described as affecting a number of myeloid cell functions. We evaluated the impact of Bv8 on lymphoid cells by investigating its ability to modulate T cell cytokine balance in mouse. RESULTS: The production of T-helper1 cytokines (IL-2, IFN-gamma and IL-1beta), the T-helper 2 cytokine IL-4, and the anti-inflammatory cytokine IL-10 by mouse splenocytes was evaluated after polyclonal stimulation or immunisation with the keyhole limpet hemocyanin protein antigen by measuring cytokine levels. When added in vitro to Con-A-stimulated splenocytes, Bv8 significantly increased IL-1beta and decreased IL-4 and IL-10; IL-2 and IFN-gamma were not affected. Similar results were obtained when Bv8 was administered in vivo. In KLH-immunised mice, splenocytes restimulated in vitro with KLH and Bv8 produced significantly smaller amounts of IL-4 and IL-10. KLH-induced IL-10 and IL-4 production was also significantly blunted in animals administered Bv8 in vivo at the time of KLH immunisation or two weeks later. The Bv8-induced effects were lost in mice lacking the PROKR 1 gene, thus indicating that PROKR 1 is the receptor involved in the modulation of cytokines. CONCLUSION: These findings indicate that Bv8/prokineticin-1 is a novel modulator of lymphoid functions, and may be a suitable target for new immunopharmacological strategies.
Asunto(s)
Interleucina-10/antagonistas & inhibidores , Interleucina-10/biosíntesis , Interleucina-4/antagonistas & inhibidores , Interleucina-4/biosíntesis , Receptores Acoplados a Proteínas G/fisiología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Animales , Concanavalina A/farmacología , Hemocianinas/farmacología , Interferón gamma/biosíntesis , Interleucina-1/biosíntesis , Ratones , Ratones Endogámicos BALB C , Receptores Acoplados a Proteínas G/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismoRESUMEN
Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) and its homolog Bombina variegata (Bv8), also termed prokineticin-1 and -2 (PK1 and PK2) respectively, are newly identified peptides with specific mitogenic activity on endocrine gland-derived endothelial cells. In the present study, we analyzed the sites of expression of EG-VEGF/PK1, Bv8/PK2, and their receptors (PKR1 and PKR2) in the adrenal cortex and checked for new biological functions of these factors on the endocrine cell compartment. RT-PCR and immunostaining analyses revealed that glomerulosa and fasciculata cells express both factors and both receptors. EG-VEGF/PK1 had no effect on the steroidogenic activity of both bovine glomerulosa and fasciculata cells but appeared to be mitogenic for both cell types. Binding of EG-VEGF/PK1 to fasciculata cells stimulated the phosphorylation of ERK1/2. Pretreatment with pertussis toxin suppressed this effect, indicating that it was Gi mediated. EG-VEGF/PK1 also increased the phosphorylation of Akt in endocrine cells of the adrenal cortex. EG-VEGF/PK1 and Bv8/PK2 thus represent new regulatory peptides acting as autocrine mitogens for endocrine cells.
Asunto(s)
Corteza Suprarrenal/metabolismo , Hormonas Gastrointestinales/metabolismo , Sustancias de Crecimiento/metabolismo , Neuropéptidos/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Corteza Suprarrenal/citología , Hormona Adrenocorticotrópica/farmacología , Angiotensina II/farmacología , Animales , Bovinos , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Hormonas Gastrointestinales/genética , Hormonas Gastrointestinales/farmacología , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/farmacología , Inmunohistoquímica , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuropéptidos/genética , Neuropéptidos/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropéptido/genética , Receptores de Neuropéptido/metabolismo , Proteínas Recombinantes/farmacología , Esteroides/metabolismo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
Prokineticin-1 (PK1) is a recently described protein with a wide range of functions, including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hemopoiesis. The aim of this study was to investigate the localization and expression of PK1 and PK receptor-1 (PKR1), their signaling pathways, and the effect of PK1 on expression of the inflammatory mediators cyclooxygenase (COX)-2 and IL-8 in third-trimester placenta. PK1 and PKR1 were highly expressed in term placenta and immunolocalized to syncytiotrophoblasts, cytotrophoblasts, fetal endothelium, and macrophages. PK1 induced a time-dependent increase in expression of IL-8 and COX-2, which was significantly reduced by inhibitors of Gq, cSrc, epidermal growth factor receptor (EGFR), and MAPK kinase. Treatment of third-trimester placenta with 40 nm PK1 induced a rapid phosphorylation of cSrc, EGFR, and ERK1/2. Phosphorylation of ERK1/2 in response to PK1 was dependent on sequential phosphorylation of cSrc and EGFR. Using double-immunofluorescent immunohistochemistry, PKR1 colocalized with IL-8 and COX-2 in placenta. These data suggest that PK1 may have a novel role as a mediator of the inflammatory response in placenta.
Asunto(s)
Placenta/metabolismo , Tercer Trimestre del Embarazo , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Western Blotting , Ciclooxigenasa 2/metabolismo , Receptores ErbB/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Técnicas In Vitro , Interleucina-8/metabolismo , Queratinas/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Miometrio/metabolismo , Fosforilación/efectos de los fármacos , Placenta/efectos de los fármacos , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Embarazo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
Effects of prokineticins (PKs), a novel family of bioactive peptides with a mitogenic action to endothelial cells of the endocrine gland and testis, on astrocytic functions were examined. Mouse cultured astrocytes expressed PK-R1 type PK receptors, while there was little expression of the PK-R2 type. PKs caused increases in astrocytic cytosolic Ca2+ levels and BrdU incorporation. Increases in Ca2+ levels by PK-2 were diminished by U73122 (a phospholipase C inhibitor). PK-induced BrdU incorporation was inhibited by U73122, GF109203 (a protein kinase C inhibitor) and PD98059 (a MEK/ERK inhibitor). These results indicate that PK receptors are expressed in astrocytes and regulate astrocytic proliferation.
Asunto(s)
Astrocitos/metabolismo , Proliferación Celular , Expresión Génica/fisiología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Bromodesoxiuridina/metabolismo , Calcio/metabolismo , Células Cultivadas , Cerebelo/citología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Flavonoides/farmacología , Hormonas Gastrointestinales/farmacología , Ratones , Neuropéptidos/farmacología , Pirrolidinonas/farmacología , Receptores Acoplados a Proteínas G/genética , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
This project identified a novel family of six 66-68 residue peptides from the venom of two Australian funnel-web spiders, Hadronyche sp. 20 and H. infensa: Orchid Beach (Hexathelidae: Atracinae), that appear to undergo N- and/or C-terminal post-translational modifications and conform to an ancestral protein fold. These peptides all show significant amino acid sequence homology to atracotoxin-Hvf17 (ACTX-Hvf17), a non-toxic peptide isolated from the venom of H. versuta, and a variety of AVIT family proteins including mamba intestinal toxin 1 (MIT1) and its mammalian and piscine orthologs prokineticin 1 (PK1) and prokineticin 2 (PK2). These AVIT family proteins target prokineticin receptors involved in the sensitization of nociceptors and gastrointestinal smooth muscle activation. Given their sequence homology to MIT1, we have named these spider venom peptides the MIT-like atracotoxin (ACTX) family. Using isolated rat stomach fundus or guinea-pig ileum organ bath preparations we have shown that the prototypical ACTX-Hvf17, at concentrations up to 1muM, did not stimulate smooth muscle contractility, nor did it inhibit contractions induced by human PK1 (hPK1). The peptide also lacked activity on other isolated smooth muscle preparations including rat aorta. Furthermore, a FLIPR Ca2+ flux assay using HEK293 cells expressing prokineticin receptors showed that ACTX-Hvf17 fails to activate or block hPK1 or hPK2 receptors. Therefore, while the MIT-like ACTX family appears to adopt the ancestral disulfide-directed beta-hairpin protein fold of MIT1, a motif believed to be shared by other AVIT family peptides, variations in the amino acid sequence and surface charge result in a loss of activity on prokineticin receptors.
Asunto(s)
Procesamiento Proteico-Postraduccional , Venenos de Araña/genética , Arañas/genética , Secuencia de Aminoácidos , Animales , Señalización del Calcio/efectos de los fármacos , Línea Celular , Hormonas Gastrointestinales/farmacología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/metabolismo , Péptidos/genética , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Homología de Secuencia de Aminoácido , Venenos de Araña/farmacología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacologíaRESUMEN
Macrophages exist as sentinels in innate immune response and react by expressing proinflammatory cytokines and up-regulating antigen-presenting and costimulatory molecules. We report a novel function for prokineticin-1 (PK1)/endocrine gland-derived vascular endothelial growth factor. Screening of murine tissue sections and cells for specific binding site leads to the identification of macrophages as an in vivo cellular target for PK1. We demonstrate PK1 induces differentiation of murine and human bone marrow cells into the monocyte/macrophage lineage. Human peripheral blood monocytes respond to PK1 by morphological changes and down-regulation of B7-1, CD14, CC chemokine receptor 5, and CXC chemokine receptor 4. Monocytes treated with PK1 have elevated interleukin (IL)-12 and tumor necrosis factor alpha and down-regulated IL-10 production in response to lipopolysaccharide. PK1 induces a distinct monocyte-derived cell population, which is primed for release of proinflammatory cytokines that favor a T helper cell type 1 response.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Macrófagos/fisiología , Células Progenitoras Mieloides/fisiología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/farmacología , Animales , Antígeno B7-1/biosíntesis , Células CHO , Diferenciación Celular/fisiología , Cricetinae , Cricetulus , Citocinas/biosíntesis , Humanos , Receptores de Lipopolisacáridos/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/citología , Ratones , Receptores CCR5/biosíntesis , Receptores CXCR4/biosíntesis , Células TH1/fisiología , Factor de Crecimiento Endotelial Vascular Derivado de Glándula Endocrina/metabolismoRESUMEN
A novel family of angiogenic mitogens have been recently characterized. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF), and the mammalian homologue of Bombina variegata peptide 8 (Bv8), are two highly related endothelial cell mitogens and chemotactic factors with restricted expression profiles and selective endothelial cell activity. These peptides share two cognate G-protein coupled receptors. The expression of human EG-VEGF occurs predominantly in steroidogenic glands. Consistent with such an expression pattern, the human EG-VEGF gene promoter has a potential binding site for steroidogenic factor (SF)-1, a pivotal element for steroidogenic-specific transcription. In the human ovary, the expression of EG-VEGF is temporally and spatially complementary to the expression of VEGF-A, both in the follicular and in the luteal phase, suggesting complementary and coordinated roles of these molecules in ovarian angiogenesis. Also, EG-VEGF expression correlates with vascularity in the polycystic ovary syndrome, a leading cause of infertility. Bv8 expression is mainly restricted to the testis. The identification of these tissue-selective angiogenic factors raises the possibility that other secreted molecules with selectivity for the endothelium of other organs exist.