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1.
Differentiation ; 99: 51-61, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29309986

RESUMO

Maintenance of the intestinal mucosa is driven by local signals that coordinate epithelial proliferation, differentiation, and turnover in order to separate antigenic luminal contents from the host's immune system. Breaches in this barrier promote gastrointestinal pathologies ranging from inflammatory bowel disease to cancer. The ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders, particularly in the colon. However, the small intestine appears to be spared from this pathology. Here we explored the physiological mechanism that underlies the preservation of mucosal homeostasis in the small intestine in mice lacking ITCH (Itcha18H/a18H). Histological analysis of the small intestines from young adult mice revealed architectural changes in animals deficient for ITCH, including villus blunting with cell crowding, crypt expansion, and thickening of the muscularis propria relative to age-matched mice sufficient for ITCH. These differences were more prominent in the distal part of the small intestine and were not dependent upon lymphoid cells. Underlying the observed changes in the epithelium were expansion of the Ki67+ proliferating transit amplifying progenitor population and increased numbers of terminally differentiated mucus-secreting goblet and anti-microbial producing Paneth cells, which are both important in controlling local inflammation in the small intestine and are known to be dysregulated in inflammatory bowel disease. Homeostasis in the small intestine of Itcha18H/a18H animals was maintained by increased cell turnover, including accelerated migration of epithelial cells along the crypt-villus axis and increased apoptosis of epithelial cells at the crypt-villus junction. Consistent with this enhanced turnover, Itcha18H/a18H mice carrying the Min mutation (Itcha18H/a18H; ApcMin/+) displayed a 76% reduction in tumor burden as compared to ApcMin/+ littermates with normal levels of ITCH. These findings highlight the role of ITCH as an important modulator of intestinal epithelial homeostasis.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Homeostase/fisiologia , Intestino Delgado/metabolismo , Ubiquitina/metabolismo , Animais , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos C57BL
2.
J Mol Cell Cardiol ; 88: 1-13, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26386426

RESUMO

Gap junctions (GJ) are intercellular channels composed of connexin subunits that play a critical role in a diverse number of cellular processes in all tissue types. In the heart, GJs mediate electrical coupling between cardiomyocytes and display mislocalization and/or downregulation in cardiac disease (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a ß-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.


Assuntos
Arritmias Cardíacas/genética , Conexina 43/genética , Hipertrofia Ventricular Esquerda/genética , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/genética , Actinas/genética , Actinas/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Conexina 43/metabolismo , Modelos Animais de Doenças , Feminino , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Regulação da Expressão Gênica , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/patologia , Fenótipo , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Proteólise , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
3.
J Biol Chem ; 288(31): 22359-68, 2013 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-23782702

RESUMO

Itch is a ubiquitin E3 ligase that regulates protein stability. Itch(-/-) mice develop an autoimmune disease phenotype characterized by itchy skin and multiorgan inflammation. The role of Itch in the regulation of osteoclast function has not been examined. We report that Itch(-/-) bone marrow and spleen cells formed more osteoclasts than cells from WT littermates in response to receptor activator of NF-κB ligand (RANKL) and was associated with increased expression of the osteoclastogenic transcription factors c-fos and Nfatc1. Overexpression of Itch in Itch(-/-) cells rescued increased osteoclastogenesis. RANKL increased Itch expression, which can be blocked by a NF-κB inhibitor. The murine Itch promoter contains NF-κB binding sites. Overexpression of NF-κB p65 increased Itch expression, and RANKL promoted the binding of p65 onto the NF-κB binding sites in the Itch promoter. Itch(-/-) osteoclast precursors had prolonged RANKL-induced NF-κB activation and delayed TNF receptor-associated factor 6 (TRAF6) deubiquitination. In WT osteoclast precursors, Itch bound to TRAF6 and the deubiquitinating enzyme cylindromatosis. Adult Itch(-/-) mice had normal bone volume, but they had significantly increased LPS-induced osteoclastogenesis and bone resorption. Thus, Itch is a new RANKL target gene that is induced during osteoclastogenesis. Itch interacts with the deubiquitinating enzyme and is required for deubiquitination of TRAF6, thus limiting RANKL-induced osteoclast formation.


Assuntos
Osteoclastos/citologia , Fator 6 Associado a Receptor de TNF/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Animais , Sequência de Bases , Primers do DNA , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Ligante RANK/metabolismo , Transdução de Sinais , Ubiquitinação
4.
Mol Cell Biol ; 33(6): 1149-63, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23297344

RESUMO

Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , MAP Quinase Quinase Quinases/metabolismo , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular , Galinhas , Células HEK293 , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Proteólise , Receptores de Antígenos/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Ubiquitinação
5.
Skelet Muscle ; 2: 14, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22769563

RESUMO

BACKGROUND: Muscle protein turnover regulation during cancer cachexia is being rapidly defined, and skeletal muscle mitochondria function appears coupled to processes regulating muscle wasting. Skeletal muscle oxidative capacity and the expression of proteins regulating mitochondrial biogenesis and dynamics are disrupted in severely cachectic ApcMin/+ mice. It has not been determined if these changes occur at the onset of cachexia and are necessary for the progression of muscle wasting. Exercise and anti-cytokine therapies have proven effective in preventing cachexia development in tumor bearing mice, while their effect on mitochondrial content, biogenesis and dynamics is not well understood. The purposes of this study were to 1) determine IL-6 regulation on mitochondrial remodeling/dysfunction during the progression of cancer cachexia and 2) to determine if exercise training can attenuate mitochondrial dysfunction and the induction of proteolytic pathways during IL-6 induced cancer cachexia. METHODS: ApcMin/+ mice were examined during the progression of cachexia, after systemic interleukin (IL)-6r antibody treatment, or after IL-6 over-expression with or without exercise. Direct effects of IL-6 on mitochondrial remodeling were examined in cultured C2C12 myoblasts. RESULTS: Mitochondrial content was not reduced during the initial development of cachexia, while muscle PGC-1α and fusion (Mfn1, Mfn2) protein expression was repressed. With progressive weight loss mitochondrial content decreased, PGC-1α and fusion proteins were further suppressed, and fission protein (FIS1) was induced. IL-6 receptor antibody administration after the onset of cachexia improved mitochondrial content, PGC-1α, Mfn1/Mfn2 and FIS1 protein expression. IL-6 over-expression in pre-cachectic mice accelerated body weight loss and muscle wasting, without reducing mitochondrial content, while PGC-1α and Mfn1/Mfn2 protein expression was suppressed and FIS1 protein expression induced. Exercise normalized these IL-6 induced effects. C2C12 myotubes administered IL-6 had increased FIS1 protein expression, increased oxidative stress, and reduced PGC-1α gene expression without altered mitochondrial protein expression. CONCLUSIONS: Altered expression of proteins regulating mitochondrial biogenesis and fusion are early events in the initiation of cachexia regulated by IL-6, which precede the loss of muscle mitochondrial content. Furthermore, IL-6 induced mitochondrial remodeling and proteolysis can be rescued with moderate exercise training even in the presence of high circulating IL-6 levels.

7.
PLoS One ; 6(9): e24650, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21949739

RESUMO

Muscle wasting that occurs with cancer cachexia is caused by an imbalance in the rates of muscle protein synthesis and degradation. The Apc(Min/+) mouse is a model of colorectal cancer that develops cachexia that is dependent on circulating IL-6. However, the IL-6 regulation of muscle protein turnover during the initiation and progression of cachexia in the Apc(Min/+) mouse is not known. Cachexia progression was studied in Apc(Min/+) mice that were either weight stable (WS) or had initial (≤5%), intermediate (6-19%), or extreme (≥20%) body weight loss. The initiation of cachexia reduced %MPS 19% and a further ∼50% with additional weight loss. Muscle IGF-1 mRNA expression and mTOR targets were suppressed with the progression of body weight loss, while muscle AMPK phosphorylation (Thr 172), AMPK activity, and raptor phosphorylation (Ser 792) were not increased with the initiation of weight loss, but were induced as cachexia progressed. ATP dependent protein degradation increased during the initiation and progression of cachexia. However, ATP independent protein degradation was not increased until cachexia had progressed beyond the initial phase. IL-6 receptor antibody administration prevented body weight loss and suppressed muscle protein degradation, without any effect on muscle %MPS or IGF-1 associated signaling. In summary, the %MPS reduction during the initiation of cachexia is associated with IGF-1/mTOR signaling repression, while muscle AMPK activation and activation of ATP independent protein degradation occur later in the progression of cachexia. IL-6 receptor antibody treatment blocked cachexia progression through the suppression of muscle protein degradation, while not rescuing the suppression of muscle protein synthesis. Attenuation of IL-6 signaling was effective in blocking the progression of cachexia, but not sufficient to reverse the process.


Assuntos
Caquexia/complicações , Caquexia/patologia , Progressão da Doença , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Neoplasias/complicações , Neoplasias/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Adiposidade , Animais , Peso Corporal , Caquexia/sangue , Ativação Enzimática , Inflamação/complicações , Inflamação/patologia , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-6/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Miofibrilas/metabolismo , Neoplasias/sangue , Tamanho do Órgão , Fosforilação , Biossíntese de Proteínas , Proteólise , Transdução de Sinais , Serina-Treonina Quinases TOR
8.
Stem Cells ; 29(10): 1601-10, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21809421

RESUMO

Patients with chronic inflammatory disorders, such as rheumatoid arthritis, often have osteoporosis due to a combination of Tumor necrosis factor-induced increased bone resorption and reduced bone formation. To test if TNF inhibits bone formation by affecting the commitment and differentiation of mesenchymal stem cells (MSCs) into osteoblasts, we examined the osteogenic potential of MSCs from TNF transgenic (TNF-Tg) mice, a model of chronic inflammatory arthritis. MSC-enriched cells were isolated from bone marrow stromal cells using negative selection with anti-CD45 antibody coated magnetic beads. The expression profile of MSC surface markers the osteogenic, chondrogenic, and adipogenic properties of CD45(-) cells were confirmed by FACS and cell differentiation assays. MSC-enriched CD45(-) cells from TNF-Tg mice formed significantly decreased numbers of fibroblast and ALP(+) colonies and had a decreased expression of osteoblast marker genes. As TNF may upregulate ubiquitin ligases, which negatively regulate osteoblast differentiation, we examined the expression levels of several ubiquitin ligases and found that Wwp1 expression was significantly increased in MSC-enriched CD45(-) cells of TNF-Tg mice. Wwp1 knockdown rescued impaired osteoblast differentiation of TNF-Tg CD45(-) cells. Wwp1 promotes ubiquitination and degradation of JunB, an AP-1 transcription factor that positively regulates osteoblast differentiation. Injection of TNF into wild-type mice resulted in decreased osteoblast differentiation of MSCs and increased JunB ubiquitination, which was completely blocked in Wwp1(-/-) mice. Thus, Wwp1 targets JunB for ubiquitination and degradation in MSCs after chronic exposure to TNF, and inhibition of Wwp1 in MSCs could be a new mechanism to limit inflammation-mediated osteoporosis by promoting their differentiation into osteoblasts.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Células Cultivadas , Condrogênese , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Técnicas de Silenciamento de Genes , Antígenos Comuns de Leucócito/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
9.
Nat Immunol ; 12(5): 399-407, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21478879

RESUMO

Although hematopoietic stem cells (HSCs) are the most thoroughly characterized type of adult stem cell, the intricate molecular machinery that regulates their self-renewal properties remains elusive. Here we showed that the E3 ubiquitin ligase Itch negatively regulated the development and function of HSCs. Itch(-/-) mice had HSCs with enhanced frequency, competence and long-term repopulating activity. Itch-deficient HSCs showed accelerated proliferation rates and sustained progenitor properties, as well as more signaling by the transcription factor Notch1, due to more accumulation of activated Notch1. Knockdown of Notch1 in Itch-mutant HSCs resulted in reversion of the phenotype. Thus, we identify Itch as a previously unknown negative regulator of HSC homeostasis and function.


Assuntos
Células-Tronco Hematopoéticas/enzimologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Processos de Crescimento Celular/imunologia , Citometria de Fluxo , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Immunoblotting , Camundongos , Camundongos Knockout , Camundongos Transgênicos , RNA Mensageiro/química , RNA Mensageiro/genética , Receptor Notch1/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Organismos Livres de Patógenos Específicos , Transcrição Genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
10.
Am J Physiol Gastrointest Liver Physiol ; 300(6): G929-38, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21415415

RESUMO

Inflammatory bowel diseases (IBDs), mainly Crohn's disease and ulcerative colitis, are dynamic, chronic inflammatory conditions that are associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism for regulating IBD. Peptidylarginine deiminases (PADs) catalyze the posttranslational conversion of peptidylarginine to peptidylcitrulline in a calcium-dependent, irreversible reaction and mediate the effects of proinflammatory cytokines. Because PAD levels are elevated in mouse and human colitis, we hypothesized that a novel small-molecule inhibitor of the PADs, i.e., chloramidine (Cl-amidine), could suppress colitis in a dextran sulfate sodium mouse model. Results are consistent with this hypothesis, as demonstrated by the finding that Cl-amidine treatment, both prophylactic and after the onset of disease, reduced the clinical signs and symptoms of colitis, without any indication of toxic side effects. Interestingly, Cl-amidine drives apoptosis of inflammatory cells in vitro and in vivo, providing a mechanism by which Cl-amidine suppresses colitis. In total, these data help validate the PADs as therapeutic targets for the treatment of IBD and further suggest Cl-amidine as a candidate therapy for this disease.


Assuntos
Anti-Inflamatórios/farmacologia , Colite/prevenção & controle , Colo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fármacos Gastrointestinais/farmacologia , Hidrolases/antagonistas & inibidores , Ornitina/análogos & derivados , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/toxicidade , Apoptose/efeitos dos fármacos , Arginina/metabolismo , Citrulina/metabolismo , Colite/induzido quimicamente , Colite/enzimologia , Colite/patologia , Colo/enzimologia , Colo/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/toxicidade , Fármacos Gastrointestinais/administração & dosagem , Fármacos Gastrointestinais/toxicidade , Células HT29 , Humanos , Hidrolases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ornitina/administração & dosagem , Ornitina/farmacologia , Ornitina/toxicidade , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Desiminases de Arginina em Proteínas , Regulação para Cima
11.
Cancer Prev Res (Phila) ; 3(3): 339-47, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20179294

RESUMO

Ulcerative colitis is a dynamic, chronic inflammatory condition associated with an increased colon cancer risk. Inflammatory cell apoptosis is a key mechanism regulating ulcerative colitis. American ginseng (AG) is a putative antioxidant that can suppress hyperactive immune cells. We have recently shown that AG can prevent and treat mouse colitis. Because p53 levels are elevated in inflammatory cells in both mouse and human colitis, we tested the hypothesis that AG protects from colitis by driving inflammatory cell apoptosis through a p53 mechanism. We used isogenic p53(+/+) and p53(-/-) inflammatory cell lines as well as primary CD4(+)/CD25(-) effector T cells from p53(+/+) and p53(-/-) mice to show that AG drives apoptosis in a p53-dependent manner. Moreover, we used a dextran sulfate sodium (DSS) model of colitis in C57BL/6 p53(+/+) and p53(-/-) mice to test whether the protective effect of AG against colitis is p53 dependent. Data indicate that AG induces apoptosis in p53(+/+) but not in isogenic p53(-/-) cells in vitro. In vivo, C57BL/6 p53(+/+) mice are responsive to the protective effects of AG against DSS-induced colitis, whereas AG fails to protect from colitis in p53(-/-) mice. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of inflammatory cells within the colonic mesenteric lymph nodes is elevated in p53(+/+) mice consuming DSS + AG but not in p53(-/-) mice consuming DSS + AG. Results are consistent with our in vitro data and with the hypothesis that AG drives inflammatory cell apoptosis in vivo, providing a mechanism by which AG protects from colitis in this DSS mouse model.


Assuntos
Apoptose/efeitos dos fármacos , Colite/prevenção & controle , Inflamação/tratamento farmacológico , Panax , Fitoterapia , Proteína Supressora de Tumor p53/metabolismo , Animais , Sulfato de Dextrana/toxicidade , Humanos , Linfócitos/imunologia , Linfócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
12.
Curr Biol ; 19(15): 1255-63, 2009 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-19592251

RESUMO

BACKGROUND: The inability to coordinate the signaling pathways that lead to proper cytokine responses characterizes the pathogenesis of inflammatory diseases such as Crohn's disease. The Crohn's disease susceptibility protein, NOD2, helps coordinate cytokine responses upon intracellular exposure to bacteria, and this signal coordination by NOD2 is accomplished, in part, through K63-linked polyubiquitin chains that create binding surfaces for the scaffolding of signaling complexes. RESULTS: In this work, we show that the NOD2 signaling partner, RIP2, is directly K63-polyubiquitinated by ITCH, an E3 ubiquitin ligase that when lost genetically causes widespread inflammatory disease at mucosal surfaces. We show that ITCH is responsible for RIP2 polyubiquitination in response to infection with listeria monocytogenes. We also show that NOD2 can bind polyubiquitinated RIP2 and that whereas ITCH E3 ligase activity is required for optimal NOD2:RIP2-induced p38 and JNK activation, ITCH inhibits NOD2:RIP2-induced nuclear factor kappa B (NFkappaB) activation. This effect can be seen independently at the whole-genome level by microarray analysis of muramyl dipeptide (MDP)-treated Itch(-/-) primary macrophages. CONCLUSIONS: These findings suggest that ITCH helps regulate NOD2-dependent signal transduction pathways and, as such, may be involved in the pathogenesis of NOD2-mediated inflammatory disease.


Assuntos
Doença de Crohn/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Acetilmuramil-Alanil-Isoglutamina , Linhagem Celular , Humanos , Immunoblotting , Imunoprecipitação , Listeria monocytogenes , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , RNA Interferente Pequeno/genética , Ubiquitinação
13.
Carcinogenesis ; 29(12): 2351-9, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18802031

RESUMO

Ulcerative colitis (UC) is a dynamic, idiopathic, chronic inflammatory condition associated with a high colon cancer risk. American ginseng has antioxidant properties and targets many of the players in inflammation. The aim of this study was to test whether American ginseng extract prevents and treats colitis. Colitis in mice was induced by the presence of 1% dextran sulfate sodium (DSS) in the drinking water or by 1% oxazolone rectally. American ginseng extract was mixed in the chow at levels consistent with that currently consumed by humans as a supplement (75 p.p.m., equivalent to 58 mg daily). To test prevention of colitis, American ginseng extract was given prior to colitis induction. To test treatment of colitis, American ginseng extract was given after the onset of colitis. In vitro studies were performed to examine mechanisms. Results indicate that American ginseng extract not only prevents but it also treats colitis. Inducible nitric oxide synthase and cyclooxygenase-2 (markers of inflammation) and p53 (induced by inflammatory stress) are also downregulated by American ginseng. Mucosal and DNA damage associated with colitis is at least in part a result of an oxidative burst from overactive leukocytes. We therefore tested the hypothesis that American ginseng extract can inhibit leukocyte activation and subsequent epithelial cell DNA damage in vitro and in vivo. Results are consistent with this hypothesis. The use of American ginseng extract represents a novel therapeutic approach for the prevention and treatment of UC.


Assuntos
Colite/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Inflamação/tratamento farmacológico , Panax , Fitoterapia , Extratos Vegetais/uso terapêutico , Animais , Western Blotting , Linfócitos T CD4-Positivos/efeitos dos fármacos , Colite/induzido quimicamente , Ensaio Cometa , Ciclo-Oxigenase 2/efeitos dos fármacos , Sulfato de Dextrana/toxicidade , Imunofluorescência , Células HT29 , Humanos , Imuno-Histoquímica , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Fitoterapia/métodos , Explosão Respiratória/efeitos dos fármacos , Proteína Supressora de Tumor p53/efeitos dos fármacos
14.
Carcinogenesis ; 29(9): 1799-806, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18567620

RESUMO

Ulcerative colitis is a dynamic, chronic inflammatory condition of the colon associated with an increased colon cancer risk. Ginkgo biloba is a putative antioxidant and has been used for thousands of years to treat a variety of ailments. The aim of this study was to test whether the standardized G.biloba extract, EGb 761, is an antioxidant that can be used to prevent and treat colitis in mice. Here, we show that EGb 761 suppresses the activation of macrophages and can be used to both prevent and treat mouse colitis. Markers of inflammation (iNOS, Cox-2 and tumor necrosis factor-alpha) and inflammatory stress (p53 and p53-phospho-serine 15) are also downregulated by EGb 761. Furthermore, we show that EGb 761 reduces the numbers of CD4+/CD25-/Foxp3- effector T cells in the colon. Interestingly, EGb 761 drives CD4+ effector T cell apoptosis in vitro and in vivo, providing a mechanistic explanation to the reduction in numbers of this cell type in the colon. This current study is in agreement with previous studies supporting a use of EGb 761 as a complementary and alternative strategy to abate colitis and associated colon cancer.


Assuntos
Antioxidantes/uso terapêutico , Apoptose/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Colite/prevenção & controle , Fitoterapia , Extratos Vegetais/uso terapêutico , Linfócitos T Reguladores/imunologia , Animais , Apoptose/fisiologia , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Colite/imunologia , Colite/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citometria de Fluxo , Imunofluorescência , Fatores de Transcrição Forkhead/metabolismo , Ginkgo biloba/metabolismo , Técnicas Imunoenzimáticas , Inflamação , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia
15.
Nat Immunol ; 9(3): 254-62, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18246070

RESUMO

The ubiquitin-editing enzyme A20 is a critical negative regulator of inflammation and cytokine-mediated activation of the transcription factor NF-kappaB; however, little is known about the mechanisms of A20-mediated inactivation of signaling intermediates such as RIP1. Here we demonstrate that the regulatory molecule TAX1BP1 recruited the E3 ligase Itch to A20 via two 'PPXY' motifs. Itch was essential for the termination of tumor necrosis factor receptor signaling by controlling A20-mediated recruitment and inactivation of RIP1. Furthermore, the Tax oncoprotein of human T cell leukemia virus type I targeted this complex for inactivation by disrupting the interaction among TAX1BP1, A20 and Itch. Thus, our studies show a previously unappreciated complexity of A20 substrate recognition and inactivation whereby TAX1BP1 and Itch function as essential subunits of an A20 ubiquitin-editing complex.


Assuntos
Regulação para Baixo/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA , Deleção de Genes , Proteínas de Homeodomínio/genética , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Neoplasias , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
16.
Cancer Metastasis Rev ; 26(3-4): 587-604, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17726579

RESUMO

Accumulating evidence suggests that E3 ubiquitin ligases play important roles in cancer development. In this article, we provide a comprehensive summary of the roles of the Nedd4-like family of E3 ubiquitin ligases in human cancer. There are nine members of the Nedd4-like E3 family, all of which share a similar structure, including a C2 domain at the N-terminus, two to four WW domains in the middle of the protein, and a homologous to E6-AP COOH terminus domain at the C-terminus. The assertion that Nedd4-like E3s play a role in cancer is supported by the overexpression of Smurf2 in esophageal squamous cell carcinoma, WWP1 in prostate and breast cancer, Nedd4 in prostate and bladder cancer, and Smurf1 in pancreatic cancer. Because Nedd4-like E3s regulate ubiquitin-mediated trafficking, lysosomal or proteasomal degradation, and nuclear translocation of multiple proteins, they modulate important signaling pathways involved in tumorigenesis like TGFbeta, EGF, IGF, VEGF, SDF-1, and TNFalpha. Additionally, several Nedd4-like E3s directly regulate various cancer-related transcription factors from the Smad, p53, KLF, RUNX, and Jun families. Interestingly, multiple Nedd4-like E3s show ligase independent function. Furthermore, Nedd4-like E3s themselves are frequently regulated by phosphorylation, ubiquitination, translocation, and transcription in cancer cells. Because the regulation and biological output of these E3s is such a complex process, study of the role of these E3s in cancer development poses some challenges. However, understanding the oncogenic potential of these E3s may facilitate the identification and development of biomarkers and drug targets in human cancer.


Assuntos
Neoplasias/etiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte , Humanos , Ubiquitina-Proteína Ligases Nedd4 , Neoplasias/enzimologia , Fosforilação , Receptores de Fatores de Crescimento/metabolismo , Proteínas Smad/metabolismo , Transcrição Genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitinação
17.
Hum Mol Genet ; 15(24): 3485-97, 2006 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-17095521

RESUMO

Homozygous itchy mice develop a fatal, late-onset autoimmune-like disease due to a loss of function mutation in an ubiquitin protein ligase. Phylogenetic and in vitro analyses suggest that Itch is a negative regulator of Notch signaling. Since Notch proteins have many important functions in the immune system, we determined whether Itch regulates Notch signaling in vivo. This was accomplished by breeding homozygous itch mice to mice carrying an activated Notch1 transgene that was specifically overexpressed in developing thymocytes. Interestingly, all itch mice carrying this transgene were smaller than their littermates and died by 12 weeks of age. These mice had a similar autoimmune disease to that seen in itch animals. However, the lesions were more severe with a much earlier age of onset, supporting the assertion that these mutations genetically interact. In addition, the combination of these mutations produced novel phenotypes including a perturbation in T cell development, with a reduction in the number of double-positive (DP) and an increase in the number of double-negative and single-positive T cells. TUNEL staining showed reduced apoptosis in the thymus of itch animals that carry the Notch1 transgene. Antibody staining displayed increased levels of full-length Notch1 and phospho-AKT specifically in DP thymocytes but no change in other signaling pathways including MAPK, p38 and JNK. These results provide the first direct demonstration that increased AKT-mediated Notch1 signaling results in autoimmunity and may provide insight into the treatment of a group of diseases that affect a significant proportion of the population.


Assuntos
Doenças Autoimunes/genética , Receptor Notch1/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Linfoma/genética , Linfoma/metabolismo , Linfoma/patologia , Masculino , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Filogenia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/patologia , Timo/metabolismo , Timo/patologia , Ubiquitina-Proteína Ligases/genética
18.
Proc Natl Acad Sci U S A ; 101(48): 16831-6, 2004 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-15550542

RESUMO

MYO5A is a major actin-based vesicle transport motor that binds to one of its cargos, the melanosome, by means of a RAB27A/MLPH receptor. When one of the members of this receptor-motor complex is mutated, the melanosomes clump in the perinuclear region of the melanocyte and are transferred unevenly to the developing hair, leading to a dilution of coat color. Mutation of a fourth gene, dilute suppressor (dsu), suppresses this coat color dilution. MYO5A is required for the peripheral accumulation of melanosomes in melanocytes, but its role in melanosome transfer to neighboring keratinocytes and the hair is unknown. Here, we show that MYO5A is nonessential for melanosome transfer, although pigment incorporation into the hair in MYO5A-deficient mice is uneven, probably due to the clumping of melanosomes that occurs in the perinuclear region of mutant melanocytes. We also show that dsu is caused by a loss-of-function mutation in a unique vertebrate-specific protein that appears to function in an MYO5A-independent pathway to alter pigment incorporation into the hair. Therefore, dsu identifies a unique protein involved in pigmentation of the mammalian hair.


Assuntos
Cor de Cabelo/genética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo V/fisiologia , Animais , Western Blotting , Cromossomos Bacterianos , Teste de Complementação Genética , Camundongos , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética
19.
Nat Cell Biol ; 4(4): 271-8, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11887186

RESUMO

Little is known about how molecular motors bind to their vesicular cargo. Here we show that myosin-Va, an actin-based vesicle motor, binds to one of its cargoes, the melanosome, by interacting with a receptor-protein complex containing Rab27a and melanophilin, a postulated Rab27a effector. Rab27a binds to the melanosome first and then recruits melanophilin, which in turn recruits myosin-Va. Melanophilin creates this link by binding to Rab27a in a GTP-dependent fashion through its amino terminus, and to myosin-Va through its carboxy terminus. Moreover, this latter interaction, similar to the ability of myosin-Va to colocalize with melanosomes and influence their distribution in vivo, is absolutely dependent on the presence of exon-F, an alternatively spliced exon in the myosin-Va tail. These results provide the first molecular description of an organelle receptor for an actin-based motor, illustrate how alternate exon usage can be used to specify cargo, and further expand the functional repertoire of Rab GTPases and their effectors.


Assuntos
Proteínas de Transporte/química , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/metabolismo , Células Cultivadas , Éxons , Proteínas de Fluorescência Verde , Cinética , Proteínas Luminescentes/metabolismo , Melanócitos/metabolismo , Melanossomas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Modelos Genéticos , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas rab de Ligação ao GTP/química , Proteínas rab27 de Ligação ao GTP , Proteína rab3A de Ligação ao GTP/metabolismo
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