RESUMO
Symplekin is a ubiquitously expressed protein involved in cytoplasmic RNA polyadenylation and transcriptional regulation and is localized at tight junctions (TJs) in epithelial cells. Nuclear symplekin cooperates with the Y-box transcription factor zonula occludens 1-associated nucleic acid-binding protein (ZONAB) to increase the transcription of cell cycle-related genes and also inhibits differentiation of intestinal cells. We detected high levels of nuclear symplekin in 8 of 12 human colorectal cancer (CRC) samples. shRNA-mediated reduction of symplekin expression was sufficient to decrease significantly the anchorage-independent growth and proliferation of HT-29 CRC cells as well as their tumorigenicity when injected into immunodeficient animals. Symplekin down-regulation also was found to alter ion transport through TJs, to promote the localization of ZONAB in the membrane rather than the nucleus, and strongly to enhance cell polarization in a 3D matrix, leading to the formation of spheroids organized around a central lumen. Claudin-2 expression was reduced following symplekin down-regulation, an effect mimicked when ZONAB expression was down-regulated using selective siRNA. Virus-mediated restoration of claudin-2 expression was found to restore nuclear expression of ZONAB in HT29DeltaSym cells and to rescue the phenotypic alterations induced by symplekin down-regulation of cell polarity, paracellular transport, ZONAB localization, cyclin D1 expression, proliferation, and anchorage-independent growth. Finally, siRNA-mediated claudin-2 down-regulation increased the transepithelial resistance and decreased cyclin D1 expression and ZONAB nuclear localization, similar to observations in symplekin-depleted cells. Our results suggest that nuclear overexpression of symplekin promotes tumorigenesis in the human colon and that the regulation of claudin-2 expression is instrumental in this effect.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Proteínas Nucleares/genética , Animais , Western Blotting , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proliferação de Células , Claudinas , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclina D1/genética , Ciclina D1/metabolismo , Imunofluorescência , Células HT29 , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Proteínas Nucleares/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Carga Tumoral , Regulação para CimaRESUMO
Cartilage degradation is mediated by matrix metalloproteinases (MMPs) and their inhibitors, tissue metalloproteinases (TIMPs), which are transcriptionally regulated by a variety of growth factors and cytokines. The levels of various MMPs as well as TIMPs have been shown to increase in response to certain cytokines. These include leukaemia inhibitory factor (LIF) and Oncostatin M (OSM), both of which have been detected in the synovial fluids of patients with rheumatoid arthritis (RA). However, the role of LIF and OSM in the regulation of various MMPs and TIMPs is still incompletely understood. The aims of this study were to examine the effects of LIF and OSM on MMP-1, MMP-3, and TIMP-1 production. In addition, the capacity of the LIF antagonist, MH35-BD, to block LIF and OSM induced MMP expression was examined. Primary chondrocytes, isolated from porcine metacarpophalangeal cartilage, were cultured in the presence and absence of LIF and OSM, with and without a predetermined concentration of the LIF antagonist. We analysed the levels of MMP-1, MMP-3 and TIMP-1 expression using qRT-PCR, Northern blot, and ELISA assays. The results indicate that LIF and OSM increase the expression of MMP-1, MMP-3, and TIMP-1 several fold. Furthermore their expression is reduced to basal levels in the presence of the LIF antagonist MH35-BD.
Assuntos
Cartilagem Articular/citologia , Condrócitos/fisiologia , Fator Inibidor de Leucemia , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Oncostatina M/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Animais , Condrócitos/citologia , Humanos , Fator Inibidor de Leucemia/antagonistas & inibidores , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Suínos , Inibidor Tecidual de Metaloproteinase-1/genéticaRESUMO
The mouse model of 2,4,6-Trinitrobenzene Sulfonic Acid (TNBS)-induced intestinal fibrosis allows for detailed study of the extracellular matrix changes that complicate Crohn's disease. Indomethacin induces intestinal fibrosis, while retinoic acid (RA) reduces liver fibrosis. Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix-modifying agent, may potentially link these opposing effects. Our aim was to determine the effects of indomethacin and RA and to evaluate their correlation to SPARC expression in the TNBS mouse model. CD-1 mice were randomised to TNBS enemas weekly for 2 or 8 weeks with or without indomethacin (0.2 mg/kg per day) or RA (100 microg/kg per day). At 2 weeks, indomethacin/TNBS enhanced and RA reduced inflammation, tissue destruction and fibrosis. The expression of SPARC was inversely related to fibrosis, but not to inflammation, in the TNBS-alone groups at 2 weeks; these differences were lost by 8 weeks. The results demonstrate that indomethacin increases TNBS-induced fibrosis in mice, while RA reduces it, and that SPARC may link these opposing effects.
Assuntos
Doença de Crohn/tratamento farmacológico , Indometacina/farmacologia , Osteonectina/metabolismo , Tretinoína/farmacologia , Animais , Doença de Crohn/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Técnicas Imunoenzimáticas , Camundongos , RNA/análise , Distribuição Aleatória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Ácido TrinitrobenzenossulfônicoRESUMO
Leukemia inhibitory factor (LIF) and oncostatin M (OSM) are found in appreciable concentrations in synovial fluid from patients with rheumatoid arthritis (RA) but not osteoarthritis. Accordingly, both are potential therapeutic targets in inflammatory diseases of the joints. Several LIF antagonists have been developed. They have the capacity to inhibit the biologic activities of not only LIF but also other interleukin-6 (IL-6) subfamily cytokines, including OSM. Both LIF and OSM share the same receptor, which is part of a cytokine receptor super family in which the glycoprotein 130 (gp130) subunit is a common constituent. The aim of this study was to evaluate the antagonistic potentials of two LIF mutants, LIF05 and MH35-BD. Both are mutant forms of human LIF with reduced affinity for gp130 and greater LIF receptor (LIFR) binding affinity. The results, using Ba/F3 cell proliferation assay, acute-phase protein (haptoglobin) induction analysis in HepG2 human hepatoma cells, a porcine cartilage glycosaminoglycan release assessment for proteoglycan degradation, and a collagen release assay, show that these antagonists inhibit relevant LIF, OSM, and other IL-6 subfamily cytokines in vitro albeit with differential potencies and have, therefore, therapeutic potential for treatment of RA and perhaps other diseases.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/terapia , Receptores de OSM-LIF/antagonistas & inibidores , Animais , Linhagem Celular , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/imunologia , Glicosaminoglicanos/metabolismo , Humanos , Hidroxiprolina/metabolismo , Fator Inibidor de Leucemia/genética , Fator Inibidor de Leucemia/imunologia , Oncostatina M/imunologia , Receptores de OSM-LIF/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SuínosRESUMO
The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.
Assuntos
Diferenciação Celular/fisiologia , Dopamina/metabolismo , Células-Tronco Embrionárias/fisiologia , Neurônios/metabolismo , Acetilcolina/metabolismo , Trifosfato de Adenosina/metabolismo , Agonistas alfa-Adrenérgicos/metabolismo , Animais , Atropina/metabolismo , Cálcio/metabolismo , Células Cultivadas , Colinérgicos/metabolismo , Cocaína/metabolismo , Antagonistas de Dopamina/metabolismo , Inibidores da Captação de Dopamina/metabolismo , Células-Tronco Embrionárias/citologia , Corantes Fluorescentes/metabolismo , Ácido Glutâmico/metabolismo , Haloperidol/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Antagonistas Muscarínicos/metabolismo , Neurônios/citologia , Norepinefrina/metabolismo , Fenótipo , Tetrodotoxina/metabolismo , Trítio/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Two leukaemia inhibitory factor (LIF) mutants, designated MH35-BD and LIF05, have been shown to have a capacity to inhibit the biological activities of not only human LIF (hLIF) but also other interleukin-6 (IL-6) subfamily cytokines such as human oncostatin M (hOSM). These cytokines share the same receptor complex in which the glycoprotein 130 (gp130) subunit is a common constituent. However, at low concentrations and in their monomeric forms, such molecules have a relatively short plasma half-life due to rapid clearance from the kidneys. Here, to prolong their serum half-lives, we have used a multi-step polymerase chain reaction (PCR) to fuse each of the LIF05 and MH35-BD cDNA fragments to a sequence encoding the Fc portion, and the hinge region, of the human immunoglobulin G (hIgG) heavy chain. The linking was achieved through an oligomer encoding a thrombin-sensitive peptide linker thus generating MH35-BD:Fc and LIF05:Fc, respectively. Both Fc fusion constructs were expressed in insect cell Sf21 and the proteins were purified by two successive affinity chromatography steps using nickel-nitrilotriacetic acid (Ni-NTA) agarose and protein A beads. The Ba/F3 cell-based proliferation assay was used to confirm that the proteins were biologically active. In addition, preliminary pharmacokinetics indicates that the Fc fusion constructs have a longer serum half-life compared to their non-fusion counterparts.