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1.
Methods Mol Biol ; 2043: 13-24, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463899

RESUMO

The continuous improvement of gene editing tools has allowed a major revolution in biological sciences. Although a variety of gain and loss-of-function approaches have been widely used for the last decades, some limitations arose from non-specific targeting or lack of complete inhibition of the gene of interest. CRISPR/Cas9 editing technology introduced new and significant advantages because it can directly modify the gene of interest and completely blocks its expression.In the context of cancer studies, the heterogeneity of the tumor microenvironment requires comprehensive approaches to unveil the contribution of multiple genes. For example, a deeper understanding of the biology of proteases such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) will improve our perspective of complex phenomena affected by extracellular matrix remodeling, including embryonic development, angiogenesis, immune infiltration, metastasis, and tumor plasticity. Here, we present a method using CRISPR/Cas9 technology to inhibit the expression of the representative ADAMTS1 in cancer cells. Following the first steps of gene edition, we pursue further selection of silenced cells and provide a detailed description of sequence analysis and validation assays. This method leads to inactivation of ADAMTS1 in cancer cells, providing a relevant biological tool that will allow subsequent in vivo and in vitro ADAMTS1 functional analysis.

2.
Sci Rep ; 8(1): 13103, 2018 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-30166561

RESUMO

Recent advances have emphasized the relevance of studying the extracellular microenvironment given its main contribution to tissue homeostasis and disease. Within this complex scenario, we have studied the extracellular protease ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motif 1), implicated in vascularization and development, with reported anti- and pro-tumorigenic activities. In this work we performed a detailed study of the vasculature and substrates in adult organs of wild type and Adamts1-deficient mice. In addition to the expected alterations of organs like kidney, heart and aorta, we found that the lack of ADAMTS1 differently affects lymphocyte and myeloid populations in the spleen and bone marrow. The study of the substrate versican also revealed its alteration in the absence of the protease. With such premises, we challenged our mice with subcutaneous B16F1 syngeneic tumours and closely evaluated the immune repertoire in the tumours but also in the distant spleen and bone marrow. Our results confirmed a pro-inflammatory landscape in the absence of ADAMTS1, correlating with tumour blockade, supporting its novel role as a modulator of the immune cell response.

3.
Leukemia ; 32(10): 2306, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30218009

RESUMO

The original version of this Article contained an error in the spelling of the author Juan Carlos Rodriguez-Manzaneque, which was incorrectly given as J Carlos Rodríguez-Manzaneque. This has now been corrected in both the PDF and HTML versions of the Article.

4.
Methods Mol Biol ; 1731: 179-192, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29318554

RESUMO

The relevance of tumor vasculature has been extensively recognized, and it is still the focus of numerous lines of research for basic, translational, and clinical scientists. Indeed, the knowledge of some of its regulatory mechanisms has provoked the generation of ongoing cancer therapies. Within the context of the tumor microenvironment, the information that the analysis of the vasculature provides is very valuable, and it might reveal not just its quality and the response against a specific therapy but also its close relationship with neighboring stromal and tumor players.Studies during last decades already supported the contribution of extracellular proteases in neovascularization events, including ADAMTS. However, deeper analyses are still required to better understand the modulation of their proteolytic activity in the tumor microenvironment. Future studies will clearly benefit from existing and ongoing genetically modified mouse models.Here we emphasize the use of syngeneic models to study the vasculature during tumor progression, supported by their intact immunocompetent capacities and also by the range of possibilities to play with engineered mice and with modified tumor cells. Although various high-tech and sophisticated approaches have already been reported to evaluate tumor neovascularization, here we describe a simple and easily reproduced methodology based in the immunofluorescence detection of vascular-specific molecules. A final in silico analysis guarantees an unbiased quantification of tumor vasculature under different conditions.

5.
Oncotarget ; 7(23): 34507-19, 2016 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-27120788

RESUMO

The matrix metalloprotease ADAMTS1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats 1) has been involved in tumorigenesis although its contributions appeared ambiguous. To understand the multifaceted actions of this protease, it is still required a deeper knowledge of its implication in heterogeneous tumor-stroma interactions. Using a syngeneic B16F1 melanoma model in wild type and ADAMTS1 knockout mice we found distinct stroma versus tumor functions for this protease. Genetic deletion of ADAMTS1 in the host microenvironment resulted in a drastic decrease of tumor growth and metastasis. However, the downregulation of tumor ADAMTS1 did not uncover relevant effects. Reduced tumors in ADAMTS1 KO mice displayed a paradoxical increase in vascular density and vascular-related genes; a detailed characterization revealed an impaired vasculature, along with a minor infiltration of macrophages. In addition, ex-vivo assays supported a chief role for ADAMTS1 in vascular sprouting, and melanoma xenografts showed a relevant induction of its expression in stroma compartments. These findings provide the first genetic evidence that supports the pro-tumorigenic role of stromal ADAMTS1.


Assuntos
Proteína ADAMTS1/genética , Melanoma Experimental/patologia , Melanoma/patologia , Neovascularização Patológica/patologia , Neoplasias Uveais/patologia , Proteína ADAMTS1/metabolismo , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação para Baixo , Deleção de Genes , Células HEK293 , Humanos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
PLoS Comput Biol ; 11(8): e1004436, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26248210

RESUMO

Sprouting angiogenesis, where new blood vessels grow from pre-existing ones, is a complex process where biochemical and mechanical signals regulate endothelial cell proliferation and movement. Therefore, a mathematical description of sprouting angiogenesis has to take into consideration biological signals as well as relevant physical processes, in particular the mechanical interplay between adjacent endothelial cells and the extracellular microenvironment. In this work, we introduce the first phase-field continuous model of sprouting angiogenesis capable of predicting sprout morphology as a function of the elastic properties of the tissues and the traction forces exerted by the cells. The model is very compact, only consisting of three coupled partial differential equations, and has the clear advantage of a reduced number of parameters. This model allows us to describe sprout growth as a function of the cell-cell adhesion forces and the traction force exerted by the sprout tip cell. In the absence of proliferation, we observe that the sprout either achieves a maximum length or, when the traction and adhesion are very large, it breaks. Endothelial cell proliferation alters significantly sprout morphology, and we explore how different types of endothelial cell proliferation regulation are able to determine the shape of the growing sprout. The largest region in parameter space with well formed long and straight sprouts is obtained always when the proliferation is triggered by endothelial cell strain and its rate grows with angiogenic factor concentration. We conclude that in this scenario the tip cell has the role of creating a tension in the cells that follow its lead. On those first stalk cells, this tension produces strain and/or empty spaces, inevitably triggering cell proliferation. The new cells occupy the space behind the tip, the tension decreases, and the process restarts. Our results highlight the ability of mathematical models to suggest relevant hypotheses with respect to the role of forces in sprouting, hence underlining the necessary collaboration between modelling and molecular biology techniques to improve the current state-of-the-art.


Assuntos
Vasos Sanguíneos/crescimento & desenvolvimento , Proliferação de Células/fisiologia , Modelos Cardiovasculares , Neovascularização Fisiológica/fisiologia , Animais , Biologia Computacional , Humanos , Camundongos , Neovascularização Patológica/fisiopatologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Matrix Biol ; 44-46: 38-45, 2015 May-Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25698314

RESUMO

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteases comprise the most recently discovered branch of the extracellular metalloenzymes. Research during the last 15years, uncovered their association with a variety of physiological and pathological processes including blood coagulation, tissue repair, fertility, arthritis and cancer. Importantly, a frequent feature of ADAMTS enzymes relates to their effects on vascular-related phenomena, including angiogenesis. Their specific roles in vascular biology have been clarified by information on their expression profiles and substrate specificity. Through their catalytic activity, ADAMTS proteases modify rather than degrade extracellular proteins. They predominantly target proteoglycans and glycoproteins abundant in the basement membrane, therefore their broad contributions to the vasculature should not come as a surprise. Furthermore, in addition to their proteolytic functions, non-enzymatic roles for ADAMTS have also been identified expanding our understanding on the multiple activities of these enzymes in vascular-related processes.


Assuntos
Proteínas ADAM/química , Proteínas ADAM/metabolismo , Neovascularização Patológica , Neovascularização Fisiológica , Animais , Coagulação Sanguínea , Glicoproteínas/metabolismo , Humanos , Estrutura Terciária de Proteína , Proteoglicanas/metabolismo , Especificidade por Substrato
8.
Int J Nanomedicine ; 9: 4277-91, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25246785

RESUMO

The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH-SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH-SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH-SWCNTs' use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 µg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies.


Assuntos
Materiais Biocompatíveis , Diferenciação Celular/efeitos dos fármacos , Nanotubos de Carbono , Osteogênese/efeitos dos fármacos , Aloenxertos , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/toxicidade , Linhagem Celular , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidade , Osteoblastos/efeitos dos fármacos , Ratos , Ratos Nus
9.
Oncotarget ; 5(12): 4295-304, 2014 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-24962328

RESUMO

Expression of IGFBP2 (Insulin-like Growth Factor Binding Protein 2) has been positively correlated with glioma progression. Although the proteolysis of IGFBP2 has been widely recognized, with consequences as a major modulator of IGFII signaling, the relevance of this post-translational modification has not been well studied in tumors. Using an in vivo proteomic approach by Isotope-Coded Protein Label (ICPL), we identified IGFBP2 as a target of the extracellular protease ADAMTS1 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 1). Notably, the proteolytic pattern of IGFBP2 was also detected in human glioma culture cells and, more importantly, in all glioma samples evaluated. In addition, high expression of ADAMTS1 correlates with higher levels of cleaved IGFBP2 in glioblastoma multiforme cases. Using gene expression public databases, we confirmed that IGFBP2 is a poor prognosis marker for gliomas, and we also observed an important contribution of ADAMTS1.Finally, we showed the impact of ADAMTS1 on IGFII-mediated IGF1R phosphorylation and cellular migration. Our results support a functional interaction between IGFBP2 and ADAMTS1 and suggest the need to evaluate post-translational modifications of IGFBP2 in glioma, in order to approach new therapies.


Assuntos
Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Neoplasias Encefálicas/genética , Glioma/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína ADAMTS1 , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Fosforilação , Proteólise , Transdução de Sinais , Transfecção
10.
PLoS Genet ; 9(6): e1003531, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23785295

RESUMO

PARP inhibition can induce anti-neoplastic effects when used as monotherapy or in combination with chemo- or radiotherapy in various tumor settings; however, the basis for the anti-metastasic activities resulting from PARP inhibition remains unknown. PARP inhibitors may also act as modulators of tumor angiogenesis. Proteomic analysis of endothelial cells revealed that vimentin, an intermediary filament involved in angiogenesis and a specific hallmark of EndoMT (endothelial to mesenchymal transition) transformation, was down-regulated following loss of PARP-1 function in endothelial cells. VE-cadherin, an endothelial marker of vascular normalization, was up-regulated in HUVEC treated with PARP inhibitors or following PARP-1 silencing; vimentin over-expression was sufficient to drive to an EndoMT phenotype. In melanoma cells, PARP inhibition reduced pro-metastatic markers, including vasculogenic mimicry. We also demonstrated that vimentin expression was sufficient to induce increased mesenchymal/pro-metastasic phenotypic changes in melanoma cells, including ILK/GSK3-ß-dependent E-cadherin down-regulation, Snail1 activation and increased cell motility and migration. In a murine model of metastatic melanoma, PARP inhibition counteracted the ability of melanoma cells to metastasize to the lung. These results suggest that inhibition of PARP interferes with key metastasis-promoting processes, leading to suppression of invasion and colonization of distal organs by aggressive metastatic cells.


Assuntos
Neoplasias da Mama/genética , Transformação Celular Neoplásica/genética , Melanoma Experimental/genética , Poli(ADP-Ribose) Polimerases/genética , Vimentina , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Neoplasias da Mama/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Cães , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Melanoma Experimental/patologia , Camundongos , Invasividade Neoplásica/genética , Metástase Neoplásica , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Vimentina/genética , Vimentina/metabolismo
11.
Int J Cancer ; 133(10): 2315-24, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23681936

RESUMO

The extracellular protease ADAMTS1 (A disintegrin and metalloprotease with thrombospondin repeats 1) has been described as an anti-angiogenic molecule and its role as a putative tumor protective molecule has also been suggested. Here, we have used a tumor xenograft model to determine the role of ADAMTS1 in tumor growth and angiogenesis. Increasing levels of the protease led to the complete inhibition of tumor growth. In an attempt to elucidate the mechanism of action of this protease, we focused our attention on its proteolytic activity on nidogens, one of the main components of the vascular basement membrane. The increased expression of ADAMTS1 was accompanied by increased proteolysis of nidogen-1 and -2 and their almost complete removal from vascular structures, together with major morphological alterations of tumor blood vessels and a decreased vessel density. The clinical relevance of this work is supported by our observations that ADAMTS1 expression is decreased in breast tumor specimens when compared with healthy tissue. Our studies also reveal that the cleavage of nidogen-1 and -2 is partially inhibited in human tumor samples. Moreover, the deposition of both nidogens surrounding vascular structures is drastically altered, implying a possible reduction in the maintenance of vessel integrity. Our studies reflect the requirement to explore the functional interactions between proteases and specific substrates in cancer biology.


Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/genética , Moléculas de Adesão Celular/metabolismo , Genes Supressores de Tumor , Glicoproteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Membrana Basal/metabolismo , Membrana Basal/patologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Moléculas de Adesão Celular/genética , Linhagem Celular , Regulação para Baixo , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/fisiologia , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteólise , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
12.
PLoS One ; 6(5): e19989, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21637756

RESUMO

Understanding tumor induced angiogenesis is a challenging problem with important consequences for diagnosis and treatment of cancer. Recently, strong evidences suggest the dual role of endothelial cells on the migrating tips and on the proliferating body of blood vessels, in consonance with further events behind lumen formation and vascular patterning. In this paper we present a multi-scale phase-field model that combines the benefits of continuum physics description and the capability of tracking individual cells. The model allows us to discuss the role of the endothelial cells' chemotactic response and proliferation rate as key factors that tailor the neovascular network. Importantly, we also test the predictions of our theoretical model against relevant experimental approaches in mice that displayed distinctive vascular patterns. The model reproduces the in vivo patterns of newly formed vascular networks, providing quantitative and qualitative results for branch density and vessel diameter on the order of the ones measured experimentally in mouse retinas. Our results highlight the ability of mathematical models to suggest relevant hypotheses with respect to the role of different parameters in this process, hence underlining the necessary collaboration between mathematical modeling, in vivo imaging and molecular biology techniques to improve current diagnostic and therapeutic tools.


Assuntos
Capilares/crescimento & desenvolvimento , Capilares/patologia , Modelos Biológicos , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica/patologia , Organogênese , Indutores da Angiogênese/metabolismo , Animais , Proliferação de Células , Quimiotaxia , Difusão , Camundongos
13.
Nature ; 465(7299): 813-7, 2010 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-20535211

RESUMO

Down's syndrome (DS) is a genetic disorder caused by full or partial trisomy of human chromosome 21 and presents with many clinical phenotypes including a reduced incidence of solid tumours. Recent work with the Ts65Dn model of DS, which has orthologues of about 50% of the genes on chromosome 21 (Hsa21), has indicated that three copies of the ETS2 (ref. 3) or DS candidate region 1 (DSCR1) genes (a previously known suppressor of angiogenesis) is sufficient to inhibit tumour growth. Here we use the Tc1 transchromosomic mouse model of DS to dissect the contribution of extra copies of genes on Hsa21 to tumour angiogenesis. This mouse expresses roughly 81% of Hsa21 genes but not the human DSCR1 region. We transplanted B16F0 and Lewis lung carcinoma tumour cells into Tc1 mice and showed that growth of these tumours was substantially reduced compared with wild-type littermate controls. Furthermore, tumour angiogenesis was significantly repressed in Tc1 mice. In particular, in vitro and in vivo angiogenic responses to vascular endothelial growth factor (VEGF) were inhibited. Examination of the genes on the segment of Hsa21 in Tc1 mice identified putative anti-angiogenic genes (ADAMTS1and ERG) and novel endothelial cell-specific genes, never previously shown to be involved in angiogenesis (JAM-B and PTTG1IP), that, when overexpressed, are responsible for inhibiting angiogenic responses to VEGF. Three copies of these genes within the stromal compartment reduced tumour angiogenesis, explaining the reduced tumour growth in DS. Furthermore, we expect that, in addition to the candidate genes that we show to be involved in the repression of angiogenesis, the Tc1 mouse model of DS will permit the identification of other endothelium-specific anti-angiogenic targets relevant to a broad spectrum of cancer patients.


Assuntos
Carcinoma Pulmonar de Lewis/irrigação sanguínea , Modelos Animais de Doenças , Síndrome de Down/genética , Dosagem de Genes/genética , Melanoma Experimental/irrigação sanguínea , Neovascularização Patológica/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS1 , Animais , Carcinoma Pulmonar de Lewis/complicações , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Cromossomos de Mamíferos/genética , Síndrome de Down/complicações , Síndrome de Down/fisiopatologia , Feminino , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Masculino , Melanoma Experimental/complicações , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Transplante de Neoplasias , Neovascularização Patológica/patologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteína Proto-Oncogênica c-ets-2/genética , Proteína Proto-Oncogênica c-ets-2/metabolismo , Fatores de Transcrição , Regulador Transcricional ERG , Trissomia/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
14.
Cancer Res ; 70(11): 4676-86, 2010 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-20484033

RESUMO

Cancer stem cells have been hypothesized to explain tumor plasticity, including the capability to adopt distinct differentiation commitments. Among the mechanisms of tumor neovascularization, the ability of some malignant cells to mimic an endothelial phenotype has been recognized by a capacity to form matrix-enriched pseudovascular structures. In addition to the expression of genes associated with an endothelial nature, the molecular dynamism of specific microenvironments may also be critical. Here, we report the identification of the extracellular protease ADAMTS1 as a critical molecule for tumor cells to acquire endothelial-like properties. In a fibrosarcoma model, ADAMTS1 increased tumor growth rate in an angiogenesis-independent manner, influencing the tumor cells to display an exclusive endothelial-like gene signature. We documented the relevant expression of ADAMTS1 in aggressive and highly plastic melanoma and Ewing sarcoma cells. Notably, inhibiting ADAMTS1 action compromised the endothelial mimetic attributes observed in this setting. Our findings provide insights into how the tumor microenvironment can elicit endothelial mimicry by tumor cells.


Assuntos
Proteínas ADAM/biossíntese , Melanoma/enzimologia , Melanoma/patologia , Sarcoma de Ewing/enzimologia , Sarcoma de Ewing/patologia , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/metabolismo , Proteína ADAMTS1 , Animais , Linhagem Celular Tumoral , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Fibrossarcoma/enzimologia , Fibrossarcoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fenótipo , Transplante Heterólogo
15.
J Biol Chem ; 285(4): 2463-73, 2010 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-19915008

RESUMO

Metastasis is a sequential process that allows cells to move from the primary tumor and grow elsewhere. Because of their ability to cleave a variety of extracellular signaling and adhesion molecules, metalloproteases have been long considered key components of the metastatic program. However, the function of certain metalloproteases, such as ADAMTS1, is not clear and seems to depend on the cellular environment and/or the stage of tumor progression. To characterize the function of ADAMTS1, we performed two alternative proteomic approaches, difference gel electrophoresis and stable isotope labeling by amino acids in cell culture, to identify novel substrates of the metalloprotease. Both techniques showed that overexpression of ADAMTS1 leads to the release of semaphorin 3C from the extracellular matrix. Although semaphorins are well known regulators of axon guidance, accumulating evidence shows that they may also participate in tumor progression. Here, we show that the cleavage of semaphorin 3C induced by ADAMTS1 promotes the migration of breast cancer cells, indicating that the co-expression of these molecules in tumors may contribute to the metastatic program.


Assuntos
Proteínas ADAM/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/secundário , Movimento Celular/fisiologia , Semaforinas/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Linhagem Celular Tumoral , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Humanos , Metástase Neoplásica , Proteômica , Semaforinas/genética , Especificidade por Substrato , Transfecção , Veias Umbilicais/citologia
16.
Int J Biochem Cell Biol ; 41(4): 800-10, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18775505

RESUMO

Syndecan-4 is a membrane-bound heparan sulfate proteoglycan that participates in cell-cell and cell-matrix interactions and modulates adhesion and migration of many cell types. Through its extracellular domain, syndecan-4 cooperates with adhesion molecules and binds matrix components relevant for cell migration. Importantly, syndecan-4 is a substrate of extracellular proteases, however the biological significance of this cleavage has not been elucidated. Here, we show that the secreted metalloprotease ADAMTS1, involved in angiogenesis and inflammatory processes, cleaves the ectodomain of syndecan-4. We further showed that this cleavage results in altered distribution of cytoskeleton components, functional loss of adhesion, and gain of migratory capacities. Using syndecan-4 null cells, we observed that ADAMTS1 proteolytic action mimics the outcome of genetic deletion of this proteoglycan with regards to focal adhesion. Our findings suggest that the shedding of syndecan-4 by ADAMTS1 disrupts cell adhesion and promotes cell migration.


Assuntos
Proteínas ADAM/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanas/metabolismo , Sindecana-4/metabolismo , Proteína ADAMTS1 , Proteína ADAMTS4 , Actinas/metabolismo , Animais , Sítios de Ligação , Células CHO , Adesão Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Cricetinae , Cricetulus , Adesões Focais/metabolismo , Glicosaminoglicanos/metabolismo , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Pró-Colágeno N-Endopeptidase/metabolismo , Transfecção
17.
Proteomics ; 6 Suppl 1: S28-35, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16511810

RESUMO

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.


Assuntos
Proteínas ADAM/química , Proteínas ADAM/metabolismo , Líquido Extracelular/enzimologia , Proteômica , Proteína ADAMTS1 , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Meios de Cultivo Condicionados , Eletroforese em Gel Bidimensional , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Especificidade por Substrato
18.
J Biol Chem ; 280(41): 34796-804, 2005 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-16061471

RESUMO

ADAMTS-1 is a metalloprotease that has been implicated in the inhibition of angiogenesis and is a mediator of proteolytic cleavage of the hyaluronan binding proteoglycans, aggrecan and versican. In an attempt to further understand the biological function of ADAMTS-1, a yeast two-hybrid screen was performed using the carboxyl-terminal region of ADAMTS-1 as bait. As a result, the extracellular matrix protein fibulin-1 was identified as a potential interacting molecule. Through a series of analyses that included ligand affinity chromatography, co-immunoprecipitation, pulldown assays, and enzyme-linked immunosorbent assay, the ability of these two proteins to interact was substantiated. Additional studies showed that ADAMTS-1 and fibulin-1 colocalized in vivo. Furthermore, fibulin-1 was found to enhance the capacity of ADAMTS-1 to cleave aggrecan, a proteoglycan known to bind to fibulin-1. We confirmed that fibulin-1 was not a proteolytic substrate for ADAMTS-1. Together, these findings indicate that fibulin-1 is a new regulator of ADAMTS-1-mediated proteoglycan proteolysis and thus may play an important role in proteoglycan turnover in tissues where there is overlapping expression.


Assuntos
Proteínas ADAM/fisiologia , Proteínas de Ligação ao Cálcio/química , Proteínas ADAM/química , Proteína ADAMTS1 , Animais , Northern Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Catálise , Cromatografia , Cromatografia de Afinidade , Meios de Cultivo Condicionados/farmacologia , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Genótipo , Humanos , Immunoblotting , Imunoprecipitação , Rim/embriologia , Ligantes , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Reação em Cadeia da Polimerase , Ligação Proteica , Estrutura Terciária de Proteína , Proteoglicanas/química , RNA/química , Técnicas do Sistema de Duplo-Híbrido
19.
Biochem Biophys Res Commun ; 293(1): 501-8, 2002 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-12054629

RESUMO

ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Proteoglicanas/metabolismo , Proteínas ADAMTS , Agrecanas , Substituição de Aminoácidos , Animais , Sítios de Ligação , Cartilagem/metabolismo , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Condrossarcoma/metabolismo , Primers do DNA , Proteínas da Matriz Extracelular/antagonistas & inibidores , Proteínas da Matriz Extracelular/química , Humanos , Lectinas Tipo C , Metaloendopeptidases/química , Mutagênese Sítio-Dirigida , Mutação Puntual , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
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