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1.
Methods Mol Biol ; 1489: 361-376, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27734389

RESUMO

The embryonic lethality of mice with conventional global knockout of Ccn1 (Cyr61) precludes analysis of Ccn1 functions in late embryonic development or in adulthood. To circumvent this limitation, we have generated conditional knockout mice that allow cell type-specific deletion of Ccn1, and constructed an allelic series of Ccn1 knockin mice that express CCN1 defective for binding specific integrins in lieu of the wild type protein. Here we describe the construction of these mice and discuss how analysis of these animals can provide unique insights into Ccn1 functions mediated through specific integrin receptors. It is anticipated that future analysis of mice carrying specific mutations in genes of the Ccn family will be greatly facilitated by application of the CRISPR/Cas9 gene editing methodology.


Assuntos
Alelos , Proteína Rica em Cisteína 61/genética , Animais , Proteína Rica em Cisteína 61/metabolismo , Células-Tronco Embrionárias , Fibroblastos , Ordem dos Genes , Marcação de Genes , Loci Gênicos , Camundongos , Camundongos Transgênicos , Mutação , Plasmídeos/genética
2.
Mol Cell Biol ; 33(10): 2078-90, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23508104

RESUMO

Liver fibrosis occurs as a wound-healing response to chronic hepatic injuries irrespective of the underlying etiology and may progress to life-threatening cirrhosis. Here we show that CCN1, a matricellular protein of the CCN (CYR61/CTGF/NOV) family, is accumulated in hepatocytes of human cirrhotic livers. CCN1 is not required for liver development or regeneration, since these processes are normal in mice with hepatocyte-specific Ccn1 deletion. However, Ccn1 expression is upregulated upon liver injuries and functions to inhibit liver fibrogenesis induced by either carbon tetrachloride intoxication or bile duct ligation and promote fibrosis regression. CCN1 acts by triggering cellular senescence in activated hepatic stellate cells and portal fibroblasts by engaging integrin α6ß1 to induce reactive oxygen species accumulation through the RAC1-NADPH oxidase 1 enzyme complex, whereupon the senescent cells express an antifibrosis genetic program. Mice with hepatocyte-specific Ccn1 deletion suffer exacerbated fibrosis with a concomitant deficit in cellular senescence, whereas overexpression of hepatic Ccn1 reduces liver fibrosis with enhanced senescence. Furthermore, tail vein delivery of purified CCN1 protein accelerates fibrosis regression in mice with established fibrosis. These findings reveal a novel integrin-dependent mechanism of fibrosis resolution in chronic liver injury and identify the CCN1 signaling pathway as a potential target for therapeutic intervention.


Assuntos
Senescência Celular , Proteína Rica em Cisteína 61/metabolismo , Cirrose Hepática/metabolismo , Miofibroblastos/metabolismo , Animais , Sítios de Ligação , Tetracloreto de Carbono , Células Cultivadas , Colestase/complicações , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/fisiologia , Feminino , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/fisiologia , Humanos , Integrina alfa6beta1/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miofibroblastos/fisiologia , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , Neuropeptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Análise Serial de Tecidos , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP
3.
Mol Cancer Res ; 7(7): 1045-55, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19584265

RESUMO

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plays an important role in immune surveillance and preferentially induces apoptosis in cancer cells over normal cells, suggesting its potential in cancer therapy. However, the molecular basis for its selective killing of cancer cells is not well understood. Recent studies have identified the CCN family of integrin-binding matricellular proteins as important regulators of cell behavior, including cell adhesion, proliferation, migration, differentiation, and survival. We show here that CCN1 (CYR61) supports the adhesion of prostatic carcinoma cells as an adhesion substrate through integrins and heparan sulfate proteoglycans. Knockdown of CCN1 expression in PC-3 and DU-145 androgen-independent prostate cancer cells strongly inhibited their proliferation without causing apoptosis, indicating that CCN1 promotes their growth. However, CCN1 also significantly enhances TRAIL-induced apoptosis through interaction with integrins alphavbeta3 and alpha6beta4 and the cell-surface heparan sulfate proteoglycan syndecan-4, acting through a protein kinase Calpha-dependent mechanism without requiring de novo protein synthesis. Knockdown of CCN1 expression in PC-3, DU-145, and LNCaP cells severely blunted their sensitivity to TRAIL, an effect that was reversed by exogenously added CCN1 protein. These findings reveal a functional dichotomy for CCN1 in prostate carcinoma cells, because it contributes to both cell proliferation and TRAIL-induced cell death and suggest that CCN1 expression status may be an important parameter in assessing the efficacy of TRAIL-dependent cancer therapy.


Assuntos
Apoptose/fisiologia , Proteína Rica em Cisteína 61/metabolismo , Neoplasias da Próstata/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Proteína Rica em Cisteína 61/genética , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Integrinas/metabolismo , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteína Quinase C-alfa/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
4.
EMBO J ; 26(5): 1257-67, 2007 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-17318182

RESUMO

Cytokines of the tumor necrosis factor (TNF) family regulate inflammation and immunity, and a subset of this family can also induce cell death in a context-dependent manner. Although TNFalpha is cytotoxic to certain tumor cell lines, it induces apoptosis in normal cells only when NFkappaB signaling is blocked. Here we show that the matricellular protein CCN1/CYR61 can unmask the cytotoxic potential of TNFalpha without perturbation of NFkappaB signaling or de novo protein synthesis, leading to rapid apoptosis in the otherwise resistant primary human fibroblasts. CCN1 acts through binding to integrins alpha(v)beta(5), alpha(6)beta(1), and syndecan-4, triggering the generation of reactive oxygen species (ROS) through a Rac1-dependent mechanism via 5-lipoxygenase and the mitochondria, leading to the biphasic activation of JNK necessary for apoptosis. Mice with the genomic Ccn1 locus replaced with an apoptosis-defective Ccn1 allele are substantially resistant to TNFalpha-induced apoptosis in vivo. These results indicate that CCN1 may act as a physiologic regulator of TNFalpha cytotoxicity, providing the contextual cues from the extracellular matrix for TNFalpha-mediated cell death.


Assuntos
Apoptose/efeitos dos fármacos , Integrinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Caspase 3/genética , Caspase 3/metabolismo , Células Cultivadas , Proteína Rica em Cisteína 61 , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Recém-Nascido , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Mutação , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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