RESUMO
Vitamin D is implicated in the etiology of cancers of the gastrointestinal tract, usually characterized by alteration in the APC/ß-catenin/TCF tumor suppressor pathway. The vitamin D receptor (VDR) is also implicated in cardiovascular and skin diseases as well as in immunity. Activated VDR can indirectly alter ß-catenin nuclear localization and directly suppress ß-catenin/TCF mediated transcriptional activity. We treated VDR null mice with the carcinogen azoxymethane (AOM) and generated mice bearing a mutated APC (hypomorph) on a VDR null background (Apc1638N/+Vdr-/-). VDR null mice do not develop GI or extra-colonic tumors but loss of VDR decreased intestinal tumor latency and increased progression to adenocarcinoma in both models. AOM treatment of VDR null mice also caused squamous cell carcinoma of the anus. Although levels and distribution of total or activated ß-catenin in the epithelial component of tumors were unaffected by loss of VDR, ß-catenin dependent cyclin D1 expression was affected suggesting a direct VDR effect on ß-catenin co-activator activity. Extra-colonic mucosa manifestations in Apc1638N/+Vdr-/- animals included increased nuclear ß-catenin in submucosal stromal cells, spleno- and cardiomegaly and large epidermoid cysts characteristic of the FAP variant, Gardner's syndrome. Consistent with this, SNPs in the VDR, vitamin D binding protein and CYP24 as well as mutations in APC distal to codon 850 were strongly associated with Gardners syndrome in a cohort of 457 FAP patients, This work suggests that alterations in the vitamin D/VDR axis are important in Gardner's syndrome, as well as in the etiology of anal cancer.
Assuntos
Adenocarcinoma/metabolismo , Polipose Adenomatosa do Colo/metabolismo , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Adenocarcinoma/patologia , Polipose Adenomatosa do Colo/induzido quimicamente , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Animais , Azoximetano , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Progressão da Doença , Síndrome de Gardner/genética , Genes APC , Predisposição Genética para Doença , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Polimorfismo de Nucleotídeo Único , Receptores de Calcitriol/deficiência , Receptores de Calcitriol/genética , Fatores de Risco , Fatores de Tempo , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Vitamin D and its analogs are potent inhibitors of colorectal cancer growth and metastasis. A number of recent studies have defined the intersections between the ß-catenin-TCF pathway (a known contributor to colorectal cancer progression) and the vitamin D receptor (VDR) pathway, shedding light on the underlying mechanisms. Vitamin D also regulates the innate immune response, and as such influences susceptibility to inflammatory bowel disease, a predisposing factor in colorectal cancer. Understanding the role of vitamin D in these different contexts will enable development of next generation vitamin D analogs that will serve as both chemopreventatives and cancer therapeutics, without the accompanying side effects of hypercalcemia usually associated with high vitamin D intake. This review summarizes the mechanisms of action of vitamin D and the VDR in the context of the gastrointestinal tract and colorectal carcinogenesis.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/prevenção & controle , Receptores de Calcitriol/metabolismo , Vitamina D/metabolismo , Animais , Humanos , beta Catenina/metabolismoRESUMO
Ephrin signaling is involved in various morphogenetic events, such as axon guidance, hindbrain segmentation, and angiogenesis. We conducted a yeast two-hybrid screen using the intracellular domain (ICD) of EphrinB1 to gain biochemical insightinto the function of the EphrinB1 ICD. We identified the transcriptional co-repressor xTLE1/Groucho as an EphrinB1 interacting protein. Whole-mount in situ hybridization of Xenopus embryos confirmed the co-localization of EphrinB1 and a Xenopus counterpart to TLE1, xTLE4, during various stages of development. The EphrinB1/xTLE4 interaction was confirmed by co-immunoprecipitation experiments. Further characterization of the interaction revealed that the carboxy-terminal PDZ binding motif of EphrinB1 and the SP domain of xTLE4 are required for binding. Additionally, phosphorylation of EphrinB1 by a constitutively activated fibroblast growth factor receptor resulted in loss of the interaction, suggesting that the interaction is modulated by tyrosine phosphorylation of the EphrinB1 ICD.
Assuntos
Proteínas Correpressoras/metabolismo , Efrina-B1/metabolismo , Proteínas de Xenopus/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Correpressoras/genética , Efrina-B1/genética , Feminino , Humanos , Dados de Sequência Molecular , Oócitos/fisiologia , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/genética , Xenopus laevis/anatomia & histologia , Xenopus laevis/embriologia , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
BACKGROUND: The cell-cell adhesion molecule cadherin-11 is important in embryogenesis and bone morphogenesis, invasion of cancer cells, lymphangiogenesis, homing of cancer cells to bone, and rheumatoid arthritis. However, very little is known about the regulation of cadherin-11 expression. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that cell density and GSK-3beta regulate cadherin-11 levels in cancer cells. Inactivation of GSK3beta with lithium chloride or the GSK3 inhibitor BIO and GSK3beta knockdown with siRNA repressed cadherin-11 mRNA and protein levels. RNA Polymerase II chromatin immunoprecipitation experiments showed that inhibition of GSK3 does not affect cadherin-11 gene transcription. Although the cadherin-11 3'UTR contains putative microRNA target sites and is regulated by Dicer, its stability is not regulated by GSK3 inhibition or density. Our data show that GSK3beta regulates cadherin-11 expression in two ways: first a beta-catenin-independent regulation of cadherin-11 steady state mRNA levels, and second a beta-catenin-dependent effect on cadherin-11 3'UTR stability and protein translation. CONCLUSIONS: Cadherin-11 mRNA and protein levels are regulated by the activity of GSK3beta and a significant degree of this regulation is exerted by the GSK3 target, beta-catenin, at the level of the cadherin-11 3'UTR.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase/fisiologia , Neoplasias da Próstata/genética , beta Catenina/fisiologia , Regiões 3' não Traduzidas , Feminino , Humanos , Masculino , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/análiseRESUMO
A body of evidence is emerging that shows a requirement for ephrin ligands in the proper migration of cells, and the formation of cell and tissue boundaries. These processes are dependent on the cell-cell adhesion system, which plays a crucial role in normal morphogenetic processes during development, as well as in invasion and metastasis. Although ephrinB ligands are bi-directional signalling molecules, the precise mechanism by which ephrinB1 signals through its intracellular domain to regulate cell-cell adhesion in epithelial cells remains unclear. Here, we present evidence that ephrinB1 associates with the Par polarity complex protein Par-6 (a scaffold protein required for establishing tight junctions) and can compete with the small GTPase Cdc42 for association with Par-6. This competition causes inactivation of the Par complex, resulting in the loss of tight junctions. Moreover, the interaction between ephrinB1 and Par-6 is disrupted by tyrosine phosphorylation of the intracellular domain of ephrinB1. Thus, we have identified a mechanism by which ephrinB1 signalling regulates cell-cell junctions in epithelial cells, and this may influence how we devise therapeutic interventions regarding these molecules in metastatic disease.
Assuntos
Efrina-B1/fisiologia , Junções Intercelulares , Proteínas de Xenopus/fisiologia , Animais , Embrião não Mamífero , Efrina-B1/metabolismo , Fosforilação , Ligação Proteica , Junções Íntimas , Xenopus laevisRESUMO
The Eph (erythropoietin-producing hepatoma) family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. The transmembrane ephrinB (Eph receptor interactor B) protein is a bidirectional signaling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase, residing on another cell. A reverse signal can be transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Although some insight has been gained regarding how ephrinB may send signals affecting cytoskeletal components, little is known about how ephrinB1 reverse signaling affects transcriptional processes. Here we report that signal transducer and activator of transcription 3 (STAT3) can interact with ephrinB1 in a phosphorylation-dependent manner that leads to enhanced activation of STAT3 transcriptional activity. This activity depends on the tyrosine kinase Jak2, and two tyrosines within the intracellular domain of ephrinB1 are critical for the association with STAT3 and its activation. The recruitment of STAT3 to ephrinB1, and its resulting Jak2-dependent activation and transcription of reporter targets, reveals a signaling pathway from ephrinB1 to the nucleus.
Assuntos
Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Efrina-B1/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , DNA/metabolismo , Efrina-B1/química , Efrina-B1/genética , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Dados de Sequência Molecular , Fosforilação , Fosfotirosina/metabolismo , Ligação Proteica , Ratos , Fator de Transcrição STAT3/genética , Transcrição Gênica/genética , Xenopus laevisRESUMO
We have shown previously that either Grb2- or Shc-mediated signaling from the oncogenic Met receptor Tpr-Met is sufficient to trigger cell cycle progression in Xenopus oocytes. However, direct binding of these adaptors to Tpr-Met is dispensable, implying that another Met binding partner mediates these responses. In this study, we show that overexpression of Grb2-associated binder 1 (Gab1) promotes cell cycle progression when Tpr-Met is expressed at suboptimal levels. This response requires that Gab1 possess an intact Met-binding motif, the pleckstrin homology domain, and the binding sites for phosphatidylinositol 3-kinase and tyrosine phosphatase SHP-2, but not the Grb2 and CrkII/phospholipase Cgamma binding sites. Importantly, we establish that Gab1-mediated signals are critical for cell cycle transition promoted by the oncogenic Met and fibroblast growth factor receptors, but not by progesterone, the natural inducer of cell cycle transition in Xenopus oocytes. Moreover, Gab1 is essential for Tpr-Met-mediated morphological transformation and proliferation of fibroblasts. This study provides the first evidence that Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation. This study identifies Gab1 and its associated signaling partners as potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor.
Assuntos
Ciclo Celular , Transformação Celular Neoplásica , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proliferação de Células , Células Cultivadas , Sequência Conservada , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteína Oncogênica tpr-met/farmacologia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/química , Progesterona/farmacologia , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismoRESUMO
Biological responses of hepatocyte growth factor (HGF) are mediated by the Met receptor tyrosine kinase. Although HGF is a potent mitogen for a variety of cells, the signals required for cell-cycle progression by the Met/HGF receptor are poorly defined. In this study, we have used the Xenopus oocyte system to define the role of various Met proximal-binding partners and downstream signaling pathways in cell-cycle regulation. We show that cell-cycle progression and activation of MAPK and JNK mediated by the oncogenic Met receptor, Tpr-Met, are dependent on its kinase activity and the presence of the twin phosphotyrosine (Y482 & Y489) residues in its C-terminus, but that the recruitment of Grb2 and Shc adaptor proteins is dispensable, implicating other signaling molecules. However, using Met receptor oncoproteins engineered to recruit specific signaling proteins, we demonstrate that recruitment of Grb2 or Shc adaptor proteins is sufficient to induce cell-cycle progression and activation of MAPK and JNK, while the binding of phospholipase-Cgamma or phosphatidylinositol 3-kinase alone fails to elicit these responses. Using various means to block phosphatidylinositol 3-kinase, phospholipase-Cgamma, MEK, JNK, Mos, and Raf1 activity, we show that unlike the fibroblast growth factor receptor, MEK-dependent and independent signaling contribute to Met receptor-mediated cell-cycle progression, but phospholipase-Cgamma or JNK activity and Mos synthesis are not critical. Notably, we demonstrate that Raf1 and phosphatidylinositol 3-kinase signaling are required for cell-cycle progression initiated by the Met receptor, a protein frequently deregulated in human tumors.
Assuntos
Ciclo Celular/fisiologia , Oócitos/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/fisiologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteína Adaptadora GRB2/genética , Proteína Adaptadora GRB2/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Morfolinas/farmacologia , Mutação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosforilação , Progesterona/farmacologia , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Proteínas Proto-Oncogênicas c-mos/genética , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Proto-Oncogênicas c-raf/genética , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Transfecção , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
An important step in retinal development is the positioning of progenitors within the eye field where they receive the local environmental signals that will direct their ultimate fate. Recent evidence indicates that ephrinB1 functions in retinal progenitor movement, but the signalling pathway is unclear. We present evidence that ephrinB1 signals through its intracellular domain to control retinal progenitor movement into the eye field by interacting with Xenopus Dishevelled (Xdsh), and by using the planar cell polarity (PCP) pathway. Blocking Xdsh translation prevents retinal progeny from entering the eye field, similarly to the morpholino-mediated loss of ephrinB1 (ref. 2). Overexpression of Xdsh can rescue the phenotype induced by loss of ephrinB1, and this rescue (as well as a physical association between Xdsh and ephrinB1) is completely dependent on the DEP (Dishevelled, Egl-10, Pleckstrin) domain of Xdsh. Similar gain- and loss-of-function experiments suggest that Xdsh associates with ephrinB1 and mediates ephrinB1 signalling through downstream members of the PCP pathway during eye field formation.
Assuntos
Movimento Celular , Efrina-B1/metabolismo , Fosfoproteínas/metabolismo , Retina/embriologia , Xenopus laevis/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Polaridade Celular , Proteínas Desgrenhadas , Retina/citologia , Retina/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Proteínas de Xenopus , Xenopus laevis/metabolismoRESUMO
In Xenopus oocytes, induction of the G2/M transition by progesterone is a complex process that is promoted by a network of signaling molecules whose cumulative effect results in the activation of maturation promoting factor (MPF) and germinal vesicle breakdown (GVBD). We examined the role of Mos, Mek, PI-3 kinase and c-Jun N-terminal kinase (JNK) in progesterone stimulation of GVBD. Expression of an activated form of JNK neither induced nor enhanced progesterone-mediated GVBD in oocytes, suggesting a limited role in cell-cycle progression. We blocked Mek, Mos and PI-3 kinase activities by a variety of means that included expression of dominant-negative kinase suppressor of Ras (DnKSR), expression of a dominant-negative PI-3 kinase (DnPI3K), treatment of oocytes with a Mek inhibitor (U1026) or PI-3 kinase (LY294002) inhibitor, and introduction of Mos antisense morpholinos. Inhibition of any one pathway alone failed to block GVBD induced by either high or low concentrations of progesterone. In contrast, inhibiting Mos or Mek function in addition to abrogating PI-3 kinase activity effectively blocked oocyte maturation. Furthermore, by expressing suboptimal amounts of Mos in conjunction with an activated form of Mek and an activated form of the p110 catalytic subunit of PI-3 kinase, we show cooperation among these signaling molecules toward the induction of GVBD. Moreover, expression of optimal amounts of these three proteins in conjunction with inhibitors of Mos, Mek or PI-3 kinase demonstrated that activated Mek-induced GVBD is independent of Mos or PI-3 kinase activity. In addition, Mos-induced GVBD is dependent upon Mek activity, but does not require PI-3 kinase activity. Finally, Mos appears to be a major contributor to GVBD induced by activated PI-3 kinase, while Mek is a minor contributor to this process.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase Quinases/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Transdução de Sinais/fisiologia , Xenopus laevis/metabolismo , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , MAP Quinase Quinase 4 , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Oócitos/metabolismo , Progesterona/farmacologia , Proteínas Proto-Oncogênicas c-mos/genética , Transdução de Sinais/genéticaRESUMO
The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Receptor EphA4/fisiologia , Motivos de Aminoácidos , Animais , Western Blotting , Catálise , Adesão Celular , Membrana Celular/metabolismo , Clonagem Molecular , Fator 8 de Crescimento de Fibroblasto , Hibridização In Situ , Camundongos , Mutação , Fenótipo , Fosforilação , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , RNA/química , RNA/metabolismo , RNA Mensageiro/metabolismo , Receptor EphA4/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Tirosina/química , Xenopus , Domínios de Homologia de srcRESUMO
The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, are thought to play a role in the regulation of cell adhesion and migration during development by mediating cell-to-cell signalling events. The transmembrane ephrinB protein is a bidirectional signalling molecule that sends a forward signal through the activation of its cognate receptor tyrosine kinase residing on another cell. The reverse signal is transduced into the ephrinB-expressing cell via tyrosine phosphorylation of its conserved C-terminal cytoplasmic domain. Previous work from our laboratory has implicated the activated FGFR1 (fibroblast growth factor receptor 1) as a regulator of a de-adhesion signal that results from overexpression of ephrinB1. In the present study, we report the isolation of Xenopus Grb4 (growth-factor-receptor-bound protein 4), an ephrinB1-interacting protein, and we show that when expressed in Xenopus oocytes, ephrinB1 interacts with Grb4 in the presence of an activated FGFR1. Amino acid substitutions were generated in Grb4, and the resulting mutants were expressed along with ephrinB1 and an activated FGFR in Xenopus oocytes. Co-immunoprecipitation analysis shows that the FLVR motif within the Src homology 2 domain of Xenopus Grb4 is vital for this phosphorylation-dependent interaction with ephrinB1. More importantly, using deletion and substitution analysis we identify the tyrosine residue at position 298 of ephrinB1 as being required for the physical interaction with Grb4, whereas Tyr-305 and Tyr-310 are dispensable. Moreover, we show that the region between amino acids 301 and 304 of ephrinB1 is also required for this critical tyrosine-phosphorylation-dependent event.