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1.
Clin Exp Rheumatol ; 37(6): 937-945, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31025930

RESUMO

OBJECTIVES: To evaluate early and late responses in biological-naïve patients with rheumatoid arthritis (RA) initiating tocilizumab and early tocilizumab non-responders who switched to rituximab. METHODS: In this open-label, non-randomised phase 3 study, RA patients with inadequate response to conventional synthetic DMARDs received tocilizumab 8 mg/kg intravenously at study begin and weeks 4, 8 and 12. After evaluation at week 16, early responders (Disease Activity Score based on 28 joints-erythrocyte sedimentation rate [DAS28-ESR] <2.6) completed the study; partial responders (DAS28-ESR decrease >1.2 or DAS28-ESR ≥2.6-≤3.2) were to continue tocilizumab through week 28; non-responders (DAS28-ESR decrease ≤1.2) switched to rituximab (1000 mg, weeks 16 and 18) with safety follow-up through week 66. RESULTS: Of 519 patients, 222 (42.8%) achieved early DAS28-ESR remission at week 16; 240 patients continued treatment, 213 (41.0%) received tocilizumab, and 27 (5.2%) switched to rituximab. At week 32 DAS28-ESR remission was achieved by 117/213 patients (54.9%) who continued tocilizumab and 4/27 patients (14.8%) who switched to rituximab; good EULAR response was achieved by 66.7% and 25.9% and CDAI remission by 19.2% and 14.8% of patients, respectively. Serious adverse events occurred through week 32 in 45/490 patients (9.2%) who received tocilizumab (serious infections, 2.7%) and through week 66 in 8/27 patients (29.6%) who switched to rituximab. CONCLUSIONS: Early response to tocilizumab was observed in 42.8% of patients. Half of early partial responders benefitted from continuing tocilizumab. Switching non-responders to rituximab seems feasible. No new safety signals were observed in patients treated with tocilizumab or switched to rituximab.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos , Artrite Reumatoide , Rituximab/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Método Duplo-Cego , Humanos , Indução de Remissão , Resultado do Tratamento
2.
Rheumatology (Oxford) ; 55(4): 624-35, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26515959

RESUMO

OBJECTIVE: To evaluate the tolerability, effectiveness and utilization of tocilizumab for the treatment of RA in a usual care setting. METHODS: ROUTINE was a prospective, non-interventional, observational 52-week study performed at 174 sites throughout Germany. RA patients were selected and treated according to label. Study objectives included the targeted documentation of infections, other adverse events, and various effectiveness outcomes (e.g. DAS28, clinical disease activity). Statistical analyses were performed primarily based on the data as observed. RESULTS: A total of 850 patients (75% women, mean age: 56 ± 13 years, mean RA duration: 10.3 ± 8.6 years) were enrolled. Most patients (79%) were pretreated with TNF-inhibitors, whereas 21% were pretreated with conventional DMARDs only. Most common DMARD pretreatments were MTX (79%), LEF (68%), adalimumab (53%) and etanercept (50%). At baseline, 60.5% of patients received tocilizumab in combination with any other RA drugs, while 39.5% were treated in monotherapy. Mean baseline DAS28 was 5.5 ± 1.3, and this decreased to 2.6 ± 1.6 at week 52. At week 52, good EULAR response was achieved in 62.3%, low disease activity state in 66.4%, and DAS28 remission in 55.1% of patients (adjusted relative frequencies). 35.3% of patients discontinued the study prematurely; common reasons were lack of effectiveness (10.5%) and intolerability (7.3%). Any infections and severe infections occurred in 37.6% and 7.2% of patients, respectively (N = 836); serious infections were seen in 5.3% (N = 850). Event rates of any, severe and serious infections were 70.3, 9.8 and 4.4 events/100 patient-years, respectively. CONCLUSION: Tocilizumab administered in a real-life setting showed clinically meaningful improvements and a safety profile that was consistent with data reported from pre-approval Phase III studies.


Assuntos
Anticorpos Monoclonais Humanizados/efeitos adversos , Antirreumáticos/efeitos adversos , Artrite Reumatoide/tratamento farmacológico , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Antirreumáticos/administração & dosagem , Antirreumáticos/uso terapêutico , Quimioterapia Combinada , Uso de Medicamentos/estatística & dados numéricos , Feminino , Alemanha , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Infecções Oportunistas/induzido quimicamente , Estudos Prospectivos , Índice de Gravidade de Doença , Resultado do Tratamento
3.
Mol Biol Cell ; 24(19): 3085-96, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23904271

RESUMO

The X-linked gene Rnf12 encodes the ubiquitin ligase really interesting new gene (RING) finger LIM domain-interacting protein (RLIM)/RING finger protein 12 (Rnf12), which serves as a major sex-specific epigenetic regulator of female mouse nurturing tissues. Early during embryogenesis, RLIM/Rnf12 expressed from the maternal allele is crucial for the development of extraembryonic trophoblast cells. In contrast, in mammary glands of pregnant and lactating adult females RLIM/Rnf12 expressed from the paternal allele functions as a critical survival factor for milk-producing alveolar cells. Although RLIM/Rnf12 is detected mostly in the nucleus, little is known about how and in which cellular compartment(s) RLIM/Rnf12 mediates its biological functions. Here we demonstrate that RLIM/Rnf12 protein shuttles between nucleus and cytoplasm and this is regulated by phosphorylation of serine S214 located within its nuclear localization sequence. We show that shuttling is important for RLIM to exert its biological functions, as alveolar cell survival activity is inhibited in cells expressing shuttling-deficient nuclear or cytoplasmic RLIM/Rnf12. Thus regulated nucleocytoplasmic shuttling of RLIM/Rnf12 coordinates cellular compartments during mammary alveolar cell survival.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Sobrevivência Celular/genética , Glândulas Mamárias Animais/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Núcleo Celular/genética , Desenvolvimento Embrionário , Epigênese Genética/genética , Feminino , Células HeLa , Humanos , Proteínas com Domínio LIM/genética , Proteínas com Domínio LIM/metabolismo , Camundongos , Fosforilação , Gravidez , Processos de Determinação Sexual , Ubiquitina-Proteína Ligases/genética
4.
Ann Rheum Dis ; 72(11): 1874-81, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23417988

RESUMO

OBJECTIVE: Based on previous data that have linked the small ubiquitin-like modifier-1 (SUMO-1) to the pathogenesis of rheumatoid arthritis (RA), we have investigated the expression of the highly homologous SUMO family members SUMO-2/3 in human RA and in the human tumour necrosis factor α transgenic (hTNFtg) mouse model of RA and studied their role in regulating disease specific matrixmetalloproteinases (MMPs). METHODS: Synovial tissue was obtained from RA and osteoarthritis (OA) patients and used for histological analyses as well as for the isolation of synovial fibroblasts (SFs). The expression of SUMO-2/3 in RA and OA patients as well as in hTNFtg and wild type mice was studied by PCR, western blot and immunostaining. SUMO-2/3 was knocked down using small interfering RNA in SFs, and TNF-α induced MMP production was determined by ELISA. Activation of nuclear factor-κB (NF-κB) was determined by a luciferase activity assay and a transcription factor assay in the presence of the NF-κB inhibitor BAY 11-7082. RESULTS: Expression of SUMO-2 and to a lesser extent of SUMO-3 was higher in RA tissues and RASFs compared with OA controls. Similarly, there was increased expression of SUMO-2 in the synovium and in SFs of hTNFtg mice compared with wild type animals. In vitro, the expression of SUMO-2 but not of SUMO-3 was induced by TNF-α. The knockdown of SUMO-2/3 significantly increased the TNF-α and interleukin (IL)-1ß induced expression of MMP-3 and MMP-13, accompanied by increased NF-κB activity. Induction of MMP-3 and MMP-13 was inhibited by blockade of the NF-κB pathway. TNF-α and IL-1ß mediated MMP-1 expression was not regulated by SUMO-2/3. CONCLUSIONS: Collectively, we show that despite their high homology, SUMO-2/3 are differentially regulated by TNF-α and selectively control TNF-α mediated MMP expression via the NF-κB pathway. Therefore, we hypothesise that SUMO-2 contributes to the specific activation of RASF.


Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , NF-kappa B/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Humanos , Camundongos , Camundongos Transgênicos , Osteoartrite/metabolismo , Transdução de Sinais , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo , Fator de Necrose Tumoral alfa/genética , Ubiquitinas/fisiologia
5.
J Biol Chem ; 287(34): 28378-85, 2012 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-22767598

RESUMO

Adipokines such as adiponectin and visfatin/pre-B-cell colony-enhancing factor (PBEF) have been recently shown to contribute to synovial inflammation in rheumatoid arthritis (RA). In this study, we evaluated the pathophysiological implication of visfatin/PBEF in the molecular patterns of RA synovial tissue, focusing on RA synovial fibroblasts (RASFs), key players in RA synovium. Expression of visfatin/PBEF in synovial fluid and tissue of RA patients was detected by immunoassays and immunohistochemistry. RASFs were stimulated with different concentrations of visfatin/PBEF over varying time intervals, and changes in gene expression were evaluated at the RNA and protein levels using Affymetrix array, real-time PCR, and immunoassays. The signaling pathways involved were identified. The influence of visfatin/PBEF on fibroblast motility and migration was analyzed. In RA synovium, visfatin/PBEF was predominantly expressed in the lining layer, lymphoid aggregates, and interstitial vessels. In RASFs, visfatin/PBEF induced high amounts of chemokines such as IL-8 and MCP-1, proinflammatory cytokines such as IL-6, and matrix metalloproteinases such as MMP-3. Phosphorylation of p38 MAPK was observed after visfatin/PBEF stimulation, and inhibition of p38 MAPK showed strong reduction of visfatin-induced effects. Directed as well as general fibroblast motility was increased by visfatin/PBEF-induced factors. The results of this study indicate that visfatin/PBEF is involved in synovial fibroblast activation by triggering fibroblast motility and promoting cytokine synthesis at central sites in RA synovium.


Assuntos
Artrite Reumatoide/enzimologia , Movimento Celular , Citocinas/biossíntese , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica , Mediadores da Inflamação/metabolismo , Nicotinamida Fosforribosiltransferase/biossíntese , Membrana Sinovial/enzimologia , Artrite Reumatoide/patologia , Quimiocina CCL2/biossíntese , Feminino , Fibroblastos/patologia , Perfilação da Expressão Gênica , Humanos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Masculino , Metaloproteinase 3 da Matriz/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Membrana Sinovial/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
6.
Arthritis Rheum ; 64(5): 1359-68, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22083543

RESUMO

OBJECTIVE: Integrin α2ß1 functions as a major receptor for type I collagen on different cell types, including fibroblasts and inflammatory cells. Although in vitro data suggest a role for α2ß1 integrin in regulating both cell attachment and expression of matrix-degrading enzymes such as matrix metalloproteinases (MMPs), mice that lack the α2 integrin subunit (Itga2(-/-) mice) develop normally and are fertile. We undertook this study to investigate the effect of Itga2 deficiency in 2 different mouse models of destructive arthritis: the antigen-induced arthritis (AIA) mouse model and the human tumor necrosis factor α (TNFα)-transgenic mouse model. METHODS: AIA was induced in the knee joints of Itga2(-/-) mice and wild-type controls. Human TNF-transgenic mice were crossed with Itga2(-/-) mice and were assessed clinically and histopathologically for signs of arthritis, inflammation, bone erosion, and cartilage damage. MMP expression, proliferation, fibroblast attachment, and ERK activation were determined. RESULTS: Under arthritic conditions, Itga2 deficiency led to decreased severity of joint pathology. Specifically, Itga2(-/-) mice showed less severe clinical symptoms and dramatically reduced pannus formation and cartilage erosion. Mice lacking α2ß1 integrin exhibited reduced MMP-3 expression, both in their sera and in fibroblast-like synoviocytes (FLS), due to impaired ERK activation. Further, both the proliferation and attachment of FLS to cartilage were partially dependent on α2ß1 integrin in vitro and in vivo. CONCLUSION: Our findings suggest that α2ß1 integrin contributes significantly to inflammatory cartilage destruction by promoting fibroblast proliferation and attachment and MMP expression.


Assuntos
Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Cartilagem Articular/patologia , Integrina alfa2beta1/deficiência , Sinovite/metabolismo , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Cartilagem Articular/metabolismo , Adesão Celular , Proliferação de Células , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Feminino , Fibroblastos/enzimologia , Fibroblastos/patologia , Humanos , Endogamia , Integrina alfa2beta1/genética , Masculino , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Joelho de Quadrúpedes/patologia , Sinovite/genética , Sinovite/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
7.
FASEB J ; 23(12): 4288-98, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19734303

RESUMO

The gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) has been viewed as restricted to leukocytes mediating the regulation of chemokine-induced migration and recruitment of neutrophils, monocytes, and macrophages. In line with the observation that PI3Kgamma-deficient mice display defects in adaptive immunity, inhibition of PI3Kgamma reduces synovial inflammation in the collagen-induced arthritis mouse model of inflammatory arthritis [rheumatoid arthritis (RA)], which has been attributed to reduced influx of inflammatory cells. Challenging the concept of leukocyte-restricted PI3Kgamma function, we report here a novel, nonredundant function of PI3Kgamma as an important regulator of fibroblast-induced cartilage destruction during chronic destructive arthritis. We show that in human tumor necrosis factor transgenic mice, the loss of PI3Kgamma leads to a milder inflammatory arthritis. Interestingly, PI3Kgamma deficiency does not alter the recruitment of inflammatory cells, but significantly reduces cartilage damage through reduced expression of matrix metalloproteinases in fibroblasts and chondrocytes. In vitro analyses demonstrate that the decreased invasiveness of fibroblasts is mediated by reduced phosphorylation of Akt and extracellular signal-regulated kinase. Using a PI3Kgamma specific inhibitor, these data are confirmed in human synovial fibroblasts from patients with RA who exhibit a disease-specific up-regulation of PI3Kgamma. Our data indicate that in addition to mediating the recruitment of inflammatory cells, PI3Kgamma is an important regulator of fibroblast-mediated joint destruction in RA and suggest that specific inhibitors of PI3Kgamma will interfere with the activation of RA synovial fibroblasts and reduce cartilage destruction in RA.


Assuntos
Artrite/metabolismo , Cartilagem/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Artrite/genética , Artrite/patologia , Artrite Reumatoide/metabolismo , Condrócitos/metabolismo , Classe Ib de Fosfatidilinositol 3-Quinase , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica/fisiologia , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Metaloproteases/metabolismo , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Membrana Sinovial/citologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismo
8.
Arthritis Rheum ; 60(7): 2065-70, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19565496

RESUMO

OBJECTIVE: To study the expression of small ubiquitin-like modifier 1 (SUMO-1) in aseptic loosening of prosthesis implants and to investigate its role in regulating the susceptibility of prosthesis-loosening fibroblast-like synoviocytes (FLS) to Fas-induced apoptosis. METHODS: Specimens of aseptically loosened tissue were obtained at revision surgery, and the expression of SUMO-1 was analyzed by in situ hybridization. SUMO-1 levels in FLS were determined by quantitative polymerase chain reaction and Western blot analysis. Immunohistochemistry and confocal microscopy were used to study the subcellular localization of SUMO-1. The functional role of SUMO-1 in Fas-induced apoptosis of prosthesis-loosening FLS was investigated by small interfering RNA-mediated knockdown of SUMO-1 and by gene transfer of the nuclear SUMO-specific protease SENP1. RESULTS: SUMO-1 was expressed strongly in aseptically loosened tissue and was found prominently at sites adjacent to bone. Prosthesis-loosening FLS expressed levels of SUMO-1 similar to the levels expressed by rheumatoid arthritis (RA) FLS, with SUMO-1 being found mainly in promyelocytic leukemia protein nuclear bodies. Knockdown of SUMO-1 had no effect on spontaneous apoptosis but significantly increased the susceptibility of prosthesis-loosening FLS to Fas-induced apoptosis. Gene transfer of the nuclear SUMO-specific protease SENP1 reverted the apoptosis-inhibiting effects of SUMO-1. CONCLUSION: These data suggest that SUMO-1 is involved in the activation of both RA FLS and prosthesis-loosening FLS by preventing these cells from undergoing apoptosis. Modification of nuclear proteins by SUMO-1 contributes to the antiapoptotic effects of SUMO-1 in prosthesis-loosening FLS, providing evidence for the specific activation of sumoylation during their differentiation. Therefore, SUMO-1 may be an interesting target for novel strategies to prevent aseptic prosthesis loosening.


Assuntos
Apoptose/fisiologia , Falha de Prótese , Proteína SUMO-1/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , Receptor fas/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Cisteína Endopeptidases , Endopeptidases/genética , Endopeptidases/metabolismo , Técnicas de Silenciamento de Genes , Articulação do Quadril/metabolismo , Articulação do Quadril/patologia , Humanos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteína SUMO-1/efeitos dos fármacos , Proteína SUMO-1/genética
9.
Arthritis Res Ther ; 11(1): R16, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19196465

RESUMO

INTRODUCTION: The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro. METHODS: Synovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 microg/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISAPlus) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 microg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis. RESULTS: Freshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G2/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G0/G1 phase. CONCLUSIONS: Our data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.


Assuntos
Apoptose/fisiologia , Artrite Reumatoide/patologia , Ciclo Celular/fisiologia , Fibroblastos/patologia , Membrana Sinovial/patologia , Artrite Reumatoide/metabolismo , Proliferação de Células , Proteína Ligante Fas/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Membrana Sinovial/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
10.
Arthritis Res Ther ; 10(5): 219, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18947370

RESUMO

Over the last decades, genetic factors for rheumatoid diseases like the HLA haplotypes have been studied extensively. However, during the past years of research, it has become more and more evident that the influence of epigenetic processes on the development of rheumatic diseases is probably as strong as the genetic background of a patient. Epigenetic processes are heritable changes in gene expression without alteration of the nucleotide sequence. Such modifications include chromatin methylation and post-translational modification of histones or other chromatin-associated proteins. The latter comprise the addition of methyl, acetyl, and phosphoryl groups or even larger moieties such as binding of ubiquitin or small ubiquitin-like modifier. The combinatory nature of these processes forms a complex network of epigenetic modifications that regulate gene expression through activation or silencing of genes. This review provides insight into the role of epigenetic alterations in the pathogenesis of rheumatoid arthritis and points out how a better understanding of such mechanisms may lead to novel therapeutic strategies.


Assuntos
Artrite Reumatoide/genética , Epigênese Genética , Regulação da Expressão Gênica , Animais , Humanos
11.
Proc Natl Acad Sci U S A ; 104(12): 5073-8, 2007 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-17360386

RESUMO

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.


Assuntos
Apoptose/efeitos dos fármacos , Artrite Reumatoide/patologia , Núcleo Celular/metabolismo , Proteína Ligante Fas/farmacologia , Fibroblastos/patologia , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Proteínas Correpressoras , Cisteína Endopeptidases , Endopeptidases/genética , Endopeptidases/metabolismo , Fibroblastos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Corpos de Inclusão Intranuclear/efeitos dos fármacos , Chaperonas Moleculares , Proteína da Leucemia Promielocítica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Líquido Sinovial/citologia , Líquido Sinovial/efeitos dos fármacos
12.
Genes Dev ; 19(19): 2307-19, 2005 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-16204183

RESUMO

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cones de Crescimento/enzimologia , Hipocampo/embriologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Quinases/metabolismo , Actinas/metabolismo , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/genética , Hipocampo/citologia , Humanos , Quinases Lim , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Ubiquitina/metabolismo
13.
EMBO J ; 22(13): 3411-20, 2003 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-12840003

RESUMO

Histone-modifying enzymes play essential roles in physiological and aberrant gene regulation. Since histone deacetylases (HDACs) are promising targets of cancer therapy, it is important to understand the mechanisms of HDAC regulation. Selective modulators of HDAC isoenzymes could serve as efficient and well-tolerated drugs. We show that HDAC2 undergoes basal turnover by the ubiquitin-proteasome pathway. Valproic acid (VPA), in addition to selectively inhibiting the catalytic activity of class I HDACs, induces proteasomal degradation of HDAC2, in contrast to other inhibitors such as trichostatin A (TSA). Basal and VPA-induced HDAC2 turnover critically depend on the E2 ubiquitin conjugase Ubc8 and the E3 ubiquitin ligase RLIM. Ubc8 gene expression is induced by both VPA and TSA, whereas only TSA simultaneously reduces RLIM protein levels and therefore fails to induce HDAC2 degradation. Thus, poly-ubiquitination and proteasomal degradation provide an isoenzyme-selective mechanism for downregulation of HDAC2.


Assuntos
Cisteína Endopeptidases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Complexos Multienzimáticos/metabolismo , Ácido Valproico/farmacologia , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Feminino , Hidrólise , Ácidos Hidroxâmicos/farmacologia , Camundongos , Complexo de Endopeptidases do Proteassoma , Ubiquitina/metabolismo
14.
Methods Cell Sci ; 25(1-2): 85-9, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14739592

RESUMO

The stabilities of many key proteins are regulated, e.g. via ubiquitination and proteasomal degradation, with important biological consequences. We present a convenient method that allows the analysis and comparison of protein stabilities during embryogenesis using early zebrafish development as a model system. Basically, this method involves ectopic overexpression of epitope-tagged proteins via mRNA injections in one-to-four-cell stage embryos and subsequent protein detection after various time points. Indeed, the protein stability of the ubiquitin ligase RLIM, which is able to autoubiquitinate and target itself for proteasomal degradation, was much shorter when compared to a protein consisting of a Myc epitope-tag and a nuclear localization domain. Thus, this method may be used more widely for the study of developmental protein stability.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Peixe-Zebra/metabolismo , Animais , Clonagem Molecular , Embrião não Mamífero/metabolismo , Microinjeções , Desnaturação Proteica , Peixe-Zebra/embriologia
15.
Nature ; 416(6876): 99-103, 2002 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-11882901

RESUMO

The interactions of distinct cofactor complexes with transcription factors are decisive determinants for the regulation of gene expression. Depending on the bound cofactor, transcription factors can have either repressing or transactivating activities. To allow a switch between these different states, regulated cofactor exchange has been proposed; however, little is known about the molecular mechanisms that are involved in this process. LIM homeodomain (LIM-HD) transcription factors associate with RLIM (RING finger LIM domain-binding protein) and with CLIM (cofactor of LIM-HD proteins; also known as NLI, Ldb and Chip) cofactors. The co-repressor RLIM inhibits the function of LIM-HD transcription factors, whereas interaction with CLIM proteins is important for the exertion of the biological activity conferred by LIM-HD transcription-factors. Here we identify RLIM as a ubiquitin protein ligase that is able to target CLIM cofactors for degradation through the 26S proteasome pathway. Furthermore, we demonstrate a ubiquitination-dependent association of RLIM with LIM-HD proteins in the presence of CLIM cofactors. Our data provide a mechanistic basis for cofactor exchange on DNA-bound transcription factors, and probably represent a general mechanism of transcriptional regulation.


Assuntos
Proteínas de Homeodomínio/metabolismo , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células CHO , Linhagem Celular , Cricetinae , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Proteínas com Domínio LIM , Ligases/metabolismo , Metaloproteínas/metabolismo , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas , Transfecção , Ubiquitina-Proteína Ligases
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