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1.
Dev Cell ; 36(5): 550-61, 2016 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-26954549

RESUMO

For muscles to function, myofibers have to stretch and anchor at the myotendinous junction (MTJ), a region rich in extracellular matrix (ECM). Integrin signaling is required for MTJ formation, and mutations affecting the cascade lead to muscular dystrophies in mice and humans. Underlying mechanisms for integrin activation at the MTJ and ECM modifications regulating its signaling are unclear. We show that lysyl oxidase-like 3 (LoxL3) is a key regulator of integrin signaling that ensures localized control of the cascade. In LoxL3 mutants, myofibers anchor prematurely or overshoot to adjacent somites, and are loose and lack tension. We find that LoxL3 complexes with and directly oxidizes Fibronectin (FN), an ECM scaffold protein and integrin ligand enriched at the MTJ. We identify a mechanism whereby localized LoxL3 secretion from myofiber termini oxidizes FN, enabling enhanced integrin activation at the tips of myofibers and ensuring correct positioning and anchoring of myofibers along the MTJ.


Assuntos
Aminoácido Oxirredutases/metabolismo , Adesão Celular/fisiologia , Fibronectinas/metabolismo , Integrinas/metabolismo , Músculos/metabolismo , Animais , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Miofibrilas/metabolismo , Oxirredução , Somitos/metabolismo , Tendões/metabolismo
2.
PLoS Genet ; 9(8): e1003603, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23966864

RESUMO

The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of ß-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to ß-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of ß-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of ß-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to ß-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the ß-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of ß-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.


Assuntos
Fator 2 Ativador da Transcrição/genética , Carcinogênese/genética , Fator 1-alfa Nuclear de Hepatócito/genética , Neoplasias/genética , beta Catenina/genética , Fator 2 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica no Desenvolvimento , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias/patologia , Regiões Promotoras Genéticas , Transdução de Sinais , Ativação Transcricional/genética , Via de Sinalização Wnt/genética , Xenopus laevis
3.
Ann Surg ; 257(3): 548-54, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23011390

RESUMO

OBJECTIVE: To determine the incidence of Wnt pathway activation in patients with stage I NSCLC and its influence on lung cancer recurrence. BACKGROUND: Despite resection, the 5-year recurrence with localized stage I nonsmall cell lung cancer (NSCLC) is 18.4%-24%. Aberrant Wnt signaling activation plays an important role in a wide variety of tumor types. However, there is not much known about the role the Wnt pathway plays in patients with stage I lung cancer. METHODS: Tumor and normal lung tissues from 55 patients following resection for stage I NSCLC were subjected to glutathione S-transferase (GST) E-cadherin pulldown and immunoblot analysis to assess levels of uncomplexed ß-catenin, a reliable measure of Wnt signaling activation. The ß-catenin gene was also screened for oncogenic mutations in tumors with activated Wnt signaling. Cancer recurrence rates were correlated in a blinded manner in patients with Wnt pathway-positive and -negative tumors. RESULTS: Tumors in 20 patients (36.4%) scored as Wnt positive, with only 1 exhibiting a ß-catenin oncogenic mutation. Patients with Wnt-positive tumors experienced a significantly higher rate of overall cancer recurrence than those with Wnt-negative tumors (30.0% vs. 5.7%, P = 0.02), with 25.0% exhibiting distal tumor recurrence compared with 2.9% in the Wnt-negative group (P = 0.02). CONCLUSIONS: Wnt pathway activation occurred in a substantial fraction of Stage I NSCLCs, which was rarely due to mutations. Moreover, Wnt pathway activation was associated with a significantly higher rate of tumor recurrence. These findings suggest that Wnt pathway activation reflects a more aggressive tumor phenotype and identifies patients who may benefit from more aggressive therapy in addition to resection.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Recidiva Local de Neoplasia/genética , Estadiamento de Neoplasias , Proteínas Wnt/genética , Idoso , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Feminino , Humanos , Incidência , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Mutação , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/metabolismo , Estudos Retrospectivos , Transdução de Sinais/genética , Estados Unidos/epidemiologia , Proteínas Wnt/metabolismo
4.
Cancer Cell ; 19(5): 601-12, 2011 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-21575861

RESUMO

Wnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n = 45) and 65% of sarcoma cell lines (n = 23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Furthermore, in Wnt autocrine cell lines, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 coreceptors and epigenetic silencing of different cell surface Wnt antagonists. Mutations in adenomatous polyposis coli (APC) gene were observed in two nonautocrine Wnt-positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A.


Assuntos
Proliferação de Células , Sarcoma/metabolismo , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Fosfatases cdc25/metabolismo , Animais , Comunicação Autócrina , Linhagem Celular Tumoral , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes APC , Genes Reporter , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Mutação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sarcoma/genética , Sarcoma/patologia , Fatores de Transcrição TCF/genética , Fatores de Tempo , Transfecção , Carga Tumoral , Proteínas Wnt/genética , beta Catenina/genética , Fosfatases cdc25/genética
5.
J Cell Biol ; 185(1): 67-75, 2009 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-19349579

RESUMO

Genetic evidence indicates that Wnt signaling is critically involved in bone homeostasis. In this study, we investigated the functions of canonical Wnts on differentiation of adult multipotent human mesenchymal stem cells (hMSCs) in vitro and in vivo. We observe differential sensitivities of hMSCs to Wnt inhibition of osteogenesis versus adipogenesis, which favors osteoblastic commitment under binary in vitro differentiation conditions. Wnt inhibition of osteogenesis is associated with decreased expression of osteoblastic transcription factors and inhibition of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation, which are involved in osteogenic differentiation. An hMSC subpopulation exhibits high endogenous Wnt signaling, the inhibition of which enhances osteogenic and adipogenic differentiation in vitro. In an in vivo bone formation model, high levels of Wnt signaling inhibit de novo bone formation by hMSCs. However, hMSCs with exogenous expression of Wnt1 but not stabilized beta-catenin markedly stimulate bone formation by naive hMSCs, arguing for an important role of a canonical Wnt gradient in hMSC osteogenesis in vivo.


Assuntos
Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Proteína Wnt1/fisiologia , Diferenciação Celular , Humanos , Células-Tronco Mesenquimais/citologia , Modelos Biológicos , Transdução de Sinais , beta Catenina/genética , beta Catenina/fisiologia
6.
Cancer Res ; 68(17): 6922-31, 2008 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-18757406

RESUMO

Semaphorin-3B (sema3B) and semaphorin-3F (sema3F) are secreted tumor suppressors of lung cancer. Sema3F functions as an antiangiogenic factor that repels endothelial cells and compromises their proliferation/survival. However, tumor cells expressing either endogenous or recombinant sema3B fail to repel endothelial cells efficiently. Sema3B found in the conditioned medium of such cells is almost completely cleaved by furin-like pro-protein convertases, generating inactive 61- and 22-kDa fragments. We have generated a sema3B variant that was point mutated at the cleavage site (sema3B-m), thereby conferring partial resistance to cleavage. Conditioned medium from HEK293 cells expressing sema3b-m and conditioned medium of HEK293 cells expressing sema3B contained similar concentrations of semaphorin but sema3B-m was cleaved much less than sema3B. In contrast to HEK293 cells expressing native sema3B, cells expressing sema3b-m strongly repel endothelial cells. Conditioned medium from sema3B-m-expressing cells rapidly caused disassembly of focal adhesions and a collapse of the actin cytoskeleton of endothelial cells, inhibited vascular endothelial growth factor-induced phosphorylation of extracellular signal-regulated kinase 1/2, induced apoptosis of endothelial cells, and inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay more potently than conditioned medium from cells expressing sema3B. Furthermore, HEK293 cells expressing sema3B-m inhibited basic fibroblast growth factor-induced angiogenesis in vivo much more potently than cells expressing sema3B. Repulsion of human umbilical vascular endothelial cells by sema3B-m was mediated primarily by the neuropilin-1 (np1) receptor but sema3B-m was also able to transduce signals via neuropilin-2 (np2). These results suggest that up-regulation of furin-like pro-protein convertases in malignant cells may enable tumors to evade the antiangiogenic effects of sema3B.


Assuntos
Inibidores da Angiogênese/farmacologia , Furina/fisiologia , Glicoproteínas de Membrana/farmacologia , Semaforinas/farmacologia , Inibidores da Angiogênese/antagonistas & inibidores , Inibidores da Angiogênese/metabolismo , Sequência de Bases , Linhagem Celular , Meios de Cultivo Condicionados , Primers do DNA , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Humanos , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Semaforinas/antagonistas & inibidores , Semaforinas/metabolismo , Transdução de Sinais
7.
J Hepatol ; 43(3): 499-507, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16023247

RESUMO

BACKGROUND/AIMS: Lysyl-oxidases catalyze the oxidation of lysine residues in collagen and elastin thereby promoting their polymerization. We have studied here the expression of four lysyl-oxidases in normal and diseased human liver. METHODS: The expression of the different lysyl-oxidases in paraffin embedded liver sections was studied using in-situ hybridization and immunohistochemistry. The enzymatic activity of lysyl-oxidase like protein-2 (Loxl2 or LOR-1) using a previously described lysyl-oxidase assay. RESULTS: We have found that the four lysyl-oxidases which we examined are not significantly expressed in the normal liver. By contrast, Wilson's disease and primary biliary cirrhosis (PBC) patients express lysyl-oxidase (Lox) and lysyl-oxidase like protein-2 (Loxl2 or LOR-1) in hepatocytes, and the expression is accompanied by collagen deposition around the hepatocytes. Lysyl-oxidases are also expressed in additional fibrotic liver diseases such as hepatitis B and C but in these diseases the expression is confined to the fibrotic lesions and collagen does not accumulate around hepatocytes. We have found that Loxl2 is able to oxidize lysine residues of collagen, and behaves in that respect similarly to Lox. The copper chelator D-penicillamine inhibits Loxl2 induced oxidation of collagen but the Lox inhibitor beta-aminopropionitrile did not inhibit the oxidation using a BAPN concentration at which Lox activity was completely inhibited. Loxl2 also catalyzed the oxidation of cell surface proteins on HepG2 hepatoblastoma cells and inhibited their proliferation. CONCLUSIONS: Upregulation of Lox and Loxl2 in hepatocytes of Wilson's disease and PBC patients may contribute to liver damage by various mechanisms. The upregulation of Lox and Loxl2 in Wilson's disease could perhaps be utilized for diagnostic purposes since their expression is up-regulated in hepatocytes even before the onset of fibrosis.


Assuntos
Aminoácido Oxirredutases/genética , Hepatócitos/enzimologia , Degeneração Hepatolenticular/enzimologia , Proteína-Lisina 6-Oxidase/genética , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Clonagem Molecular , Endotélio Vascular/enzimologia , Hepatócitos/patologia , Degeneração Hepatolenticular/patologia , Humanos , Hibridização In Situ , Cirrose Hepática/enzimologia , Transfecção
8.
Cancer Res ; 63(7): 1657-66, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12670920

RESUMO

The lysyl oxidase gene family members function as extracellular matrix modulating enzymes. We have found that another member of this family, lysyl oxidase related protein-1 (LOR-1), is highly expressed in metastatic breast cancer-derived cell lines but not in the nonmetastatic estrogen-dependent MCF-7 cells. Furthermore, LOR-1 expression in periductal tumor cells of breast carcinomas is significantly correlated with increased tumor malignancy. MCF-7 cells expressing recombinant LOR-1 formed estrogen-dependent tumors that developed much slower than tumors derived from empty vector-transfected MCF-7 cells. The cells of these LOR-1-expressing tumors were surrounded by a high concentration of dense collagen fibers, and the tumors contained many fibrotic foci. Induction of fibrosis in vivo by lysyl oxidase-like enzymes has never been observed before and suggests that LOR-1 may function as an autonomous inducer of fibrosis. The appearance of fibrotic foci in spontaneous breast cancer tumors is correlated with poor prognosis and metastasis, and we, therefore, examined the invasiveness of the LOR-1-expressing tumors. LOR-1-expressing MCF-7 cells invaded the pseudocapsules surrounding the tumors. In contrast, vector-transfected MCF-7 cells did not invade the pseudocapsules. This observation suggests that LOR-1 enhances the malignancy of the tumors. Furthermore, the LOR-1-expressing tumor cells invaded blood vessels, nerves, and muscles adjacent to the tumor, indicating that the LOR-1-expressing MCF-7 cells acquired metastatic properties. We conclude that LOR-1 promotes tumor fibrosis and tumor invasiveness simultaneously, which indicates that these two processes may be associated.


Assuntos
Aminoácido Oxirredutases/fisiologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Animais , Neoplasias da Mama/genética , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Colágeno/metabolismo , Progressão da Doença , Feminino , Fibrose/enzimologia , Humanos , Camundongos , Camundongos Nus , Necrose , Metástase Neoplásica , Transfecção , Células Tumorais Cultivadas
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